UNIVERZITA KARLOVA V PRAZE 2. LÉKAŘSKÁ FAKULTA. Studijní obor: Fyziologie a patofyziologie člověka

UNIVERZITA KARLOVA V PRAZE 2. LÉKAŘSKÁ FAKULTA

Studijní obor: Fyziologie a patofyziologie člověka

MUDr. Věra Lachmanová

VLIV RADIKÁLOVÉHO POŠKOZENÍ V ČASNÉ FÁZI EXPOZICE HYPOXIE NA ROZVOJ HYPOXICKÉ PLICNÍ HYPERTENZE

DISERTAČNÍ PRÁCE

Vedoucí závěrečné práce: prof. MUDr. Jan Herget, DrSc.

Praha 2011

Prohlášení:

Prohlašuji, že jsem závěrečnou práci zpracovala samostatně a že jsem uvedla všechny použité informační zdroje. Současně dávám svolení k tomu, aby tato závěrečná práce byla archivována v Ústavu vědeckých informací 2. lékařské fakulty Univerzity Karlovy v Praze a zde užívána ke studijním účelům. Za předpokladu, že každý, kdo tuto práci použije pro svou přednáškovou nebo publikační aktivitu, se zavazuje, že bude tento zdroj informací řádně citovat. Souhlasím se zpřístupněním elektronické verze mé práce v Digitálním repozitáři Univerzity Karlovy v Praze (http://repozitar.cuni.cz). Práce je zpřístupněna pouze v rámci Univerzity Karlovy v Praze

Souhlasím.

V Praze, 15. 02. 2011

MUDr. Věra Lachmanová

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PODĚKOVÁNÍ Na tomto místě bych chtěla poděkovat svému školiteli prof. MUDr. Janu Hergetovi, DrSc. za odborné vedení v průběhu mého postgraduálního studia a za pomoc při zpracování publikací a disertační práce. Děkuji také prof. RNDr. Václavu Hamplovi, DrSc. za pomoc a cenné rady při zpracování publikací během mého postgraduálního studia. Ráda bych poděkovala paní Olze Hniličkové a RNDr. Daně Mikové za praktické rady a pomoc během laboratorních měření a zpracování výsledků.

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OBSAH 1. ÚVOD……………………………………………………..………………….……………9 2. LITERÁRNÍ PŘEHLED………………….…………..……………………….……......10 2.1. Plicní cévy a hypoxie………………………………………………………….…....10 2.1.1. Hypoxická plicní vazokonstrikce………………………………………......10 2.1.2. Hypoxická plicní hypertenze…………………………………..…..……….15 2.2. Reaktivní sloučeniny kyslíku (ROS) a hypoxie………...…………………....…..18 2.2.1. ROS…………………………………………………………………...……...18 2.2.2. Zdroj ROS………………………………………………..……………......…20 2.2.3. Důsledky působení ROS………………………………………………...….21 2.3. Oxid dusnatý …………………….…………..…………………………………..….24 2.3.1. Oxid dusnatý v plicním řečišti…………………………………………....…24 3. CÍLE DISERTAČNÍ PRÁCE…………...…………………………………………..….27

4. METODY……………………………...……………………………..…………..……...29 4.1. Hypoxická komora………………………………………………………………….29 4.2. 5–denní hypoxie………………………………………….……………………….29 . 4.2.1. Izolované perfundované plíce laboratorního potkana………….….……29 4.2.2. Experimentální protokol………………………...………………………….30 4.2.2.1. Experimentální skupiny……………………………………….…30 4.2.2.2. Měření reaktivity plicních cév…………………………….……..30 4.2.2.3. Měření mechanických vlastností (rezistence) plicních cév….31 4.2.3. Statistické hodnocení……….………………………..……………………..31 4.3.

4-týdenní hypoxie……………………………………………………………....32 5

4.3.1. Experimentální protokol…………………………………………………….32 4.3.2. Měření tlaku v a. pulmonalis………………………..……..……………….32 4.3.3. Měření systémového arteriálního tlaku…………………...……………….33 4.3.4. Měření srdečního výdeje………………… ………………..……….……..33 4.3.5. Analýza váhy jednotlivých srdečních oddílů…………………...………….33 4.3.6. Stanovení hematokritu………..………………….……………….…………33 4.3.7. Statistické zpracování……………………………..…..………..……………34 4.4.

Výdej NO po 4 dnech hypoxie……………..…………………………………34

4.4.1. Experimentální protokol…………………...……………………………….34 4.4.2. Stanovení celkové produkce NO…………………..………………………34 4.4.3.

Stanovení produkce NO dolními cestami dýchacími……………..…….35

4.4.4. Stanovení produkce NO horními cestami dýchacími…..………………..35 4.4.5. Statistické zpracování……………………………………………………….35 5. VÝSLEDKY……………………………………………………………………………..36 5.1. Vliv časného podávání NAC na odporové vlastnosti plicního řečiště………..36 5.2. Vliv časného podávání NAC na reaktivitu plicních cév…………...……….….36 5.3. Vliv časného podávání NAC na plicní hypertenzi……...…...…………………40 5.3.1. Tlak v a. pulmonalis……………….………………………………………..40 5.3.2. Systémový arteriální tlak…………………………….………………..……42 5.3.3. Srdeční výdej………………………………….……...……………………..42 5.3.4. Hypertrofie pravé komory…………….……………………………...…….42 5.3.5. Hematokrit…………………………………………………………….…….42 5.4.

Měření produkce NO z plic a horních cest dýchacích……………….…….44

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6. DISKUZE A ZÁVĚRY……………………….…………………………………………45 6.1. Úloha volných kyslíkových radikálů v časné fázi expozice hypoxii……..……46 6.2. Úloha oxidu dusnatého v časné fázi expozice hypoxii…………………..…….47 6.3. Peroxynitrit……………………………………………….……………..………….48 6.4. Změny tvorby radikálů v časovém průběhu hypoxie……….………………….49

7. OBECNÉ ZÁVĚRY VYPLÝVAJÍCÍ Z DISERTACE………………….……………..51 8. LITERATURA……………………………………………..…….………………………52 9. VLASTNÍ PUBLIKACE AUTORA………………………….………………………….67 10. PŘÍLOHY…………………………….…………………………………………………68

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SEZNAM ZKRATEK POUŢITÝCH V TEXTU

ARDS

syndrom akutní dechové tísně dospělých, acute respiratory distress syndrom

CHOPN

chronická obstrukční choroba bronchopulmonální

GSH

redukovaný glutathion

GSSG

oxidovaný glutathion

NAC

N – acetyl -L-cystein

HPH

hypoxická plicní hypertenze

HPV

hypoxická plicní vazokonstrikce

IL-1

interleukin 1

INF-

interferon

L-NAME

NG – nitro-L – arginin methyl ester

MMP

matrixová metaloproteináza

NADPH oxidáza

nikotinamidadenindinukleotidfosfát oxidáza

NOS

NO syntáza

eNOS

endoteliální NO syntáza

iNOS

inducibilní NO syntáza

nNOS

neuronální NO syntáza

PAP

tlak v arteria pulmonalis

ROS

reaktivní sloučeniny kyslíku (reactive oxygen species)

SAP

systémový střední arteriální tlak

SOD

superoxiddismutáza

TNF

tumor necrosis factor

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1. ÚVOD U dospělých jedinců je plicní řečiště vzhledem k řečišti systémovému nízkotlaké. Hemodynamický odpor a tlak krve jsou v plicním oběhu přibližně pět- až šestkrát nižší než v systémové cirkulaci (Harris a Heath 1977, Fishman 1985). Médie periferních plicních cév je méně muskularizovaná než v systémových cévách (Hislop a Reid 1978). Plicním řečištěm protéká celý srdeční výdej na rozdíl od jednotlivých orgánových řečišť. Díky velké poddajnosti plicních cév a možnosti otevřít část cévního řečiště, která v klidu není perfundovaná, zůstává řečiště plic nízkotlaké i při významném zvýšení srdečního výdeje.

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2. LITERÁRNÍ PŘEHLED 2.1. Plicní cévy a hypoxie Plicní cévy reagují na hypoxii 2 různými ději, které se liší mechanismem vzniku. Jsou to hypoxická plicní vazokonstrikce (HPV) a hypoxická plicní hypertenze (HPH). Tyto děje se liší mechanismem vzniku, ale je pravděpodobné, že u obou hraje důležitou roli při jejich vzniku zvýšená tvorba volných kyslíkových radikálů (ROS) a změna v produkci oxidu dusnatého (NO). V minulosti se předpokládalo, že HPV je reakcí plicního řečiště na akutní hypoxii a expozice chronické hypoxii vede ke vzniku HPH na podkladě remodelace cévní stěny. Před několika lety bylo prokázáno, že inhibitory Rho-kinázového systému lze snížit plicní rezistenci u již rozvinuté HPH (Stenmark a McMurtry 2005). V současnosti přepokládáme, že HPH se při chronické hypoxii rozvíjí na podkladě vazokonstrikce (Crossno, Garat et al. 2007) a remodelace periferních plicních cév (Reid 1986).

2.1.1. Hypoxická plicní vazokonstrikce

HPV je klíčový regulační mechanismus, který zajišťuje optimální okysličení krve v organismu. Jedná se o lokální plicní reakci, která není zprostředkována nervově nebo cirkulujícími mediátory.

Systémové cévy se v některých oblastech (např.

renální cévy) v hypoxii spíše dilatují.

Při hypoxii v části plic dochází k lokální

vazokonstrikci, čímž se zajišťuje přednostní perfúze dobře ventilovaných alveolů a omezuje se průtok ve špatně ventilovaných alveolech. Při globální hypoxii, tzn., že postihuje většinu nebo všechny plicní alveoly (např. pobyt ve vysoké nadmořské

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výšce) je vazokonstrikce přítomná v celém plicním řečišti a působí zvýšení tlaku krve v plicnici, které je reverzibilní při návratu k normoxii. Hypoxická plicní vazokonstrikce se objevuje za několik desítek sekund od počátku působení hypoxie. Maximální odpovědi je dosaženo během 5 – 15 minut. Velikost odpovědi na hypoxii záleží na stupni hypoxie. U člověka má hypoxická plicní vazokonstrikce nejvyšší účinnost pro udržení příznivého poměru ventilace a perfúze při arteriálním parciálním tlaku kyslíku kolem 60 mm Hg (Melot, Naeije et al. 1987). Po ukončení akutní hypoxie vazokonstrikce ustupuje podobně rychle, jako nastupuje. V izolovaných plicních artériích in vitro má většinou vazokonstrikční odpověď na hypoxii bifázický průběh (Bennie, Packer et al. 1991). První fáze (fáze I) dosahuje maxima během 5 minut, pak vazokonstrikce poněkud ustupuje, avšak obvykle nedojde k úplné relaxaci na klidovou úroveň a je přechodná. Potom se pomaleji (po 10 – 15 minutách trvání hypoxie) objevuje druhá fáze konstrikce (fáze II), která přetrvává po dobu hypoxie. Během obou fází se zvyšuje cytoplazmatická koncentrace vápníku. Tento nárůst se uskutečňuje různými mechanismy. Fáze I alespoň částečně souvisí s napětím řízeným vstupem vápníku do buněk, ve fázi II se uplatňují jiné, napěťově nezávislé cesty influxu vápníku do cytoplazmy buněk cévního hladkého svalu (Robertson, Hague et al. 2000). Bifázická vazokonstrikční odpověď na hypoxii byla ojediněle pozorována i na izolovaných perfundovaných plicích laboratorního potkana. Fáze I pravděpodobně souvisí s počáteční rychlou vazokonstrikční odpovědí na hypoxii, která je známa in vivo, a s okamžitou regulací poměru plicní ventilace a perfúze. In vivo má hypoxická plicní vazokonstrikce jen jednu fázi – reverzibilní zvýšení plicního cévního odporu.

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Senzor hypoxie Dříve se předpokládalo, že pro vyvolání hypoxické plicní vazokonstrikce by mohla být potřebná souhra více buněk různých tkání. Novější výsledky ukazují, že se celý děj – od rozpoznání hypoxie až po vlastní svalovou kontrakci – odehrává pouze v hladkém svalu plicních cév. Nicméně řada působků z jiných buněčných typů (např. endotel, ve kterém jsou tvořeny NO, endoteliny, prostaglainy) hypoxickou vazokonstrikci významně moduluje. Plnohodnotnou hypoxickou plicní vazokonstrikci lze vyvolat i po denervaci cév, proto je nutné hledat i senzor hypoxie v plicích. U člověka potvrzuje tuto skutečnost přítomnost hypoxické plicní vazokonstrikce u pacientů po transplantaci plic v období, kdy transplantát ještě nemohl být reinervován (Robin, Theodore et al. 1987). Hypoxická kontrakce byla demonstrována i na izolovaných buňkách hladkých svalů plicních cév. Při akutní hypoxii je kontrakce nejvýrazněji přítomná na arteriální straně plicního řečiště. Nejvíce se kontrahují prekapilární (alveolární) cévy, větší tepny vodivé části plicního řečiště na změny parciálního tlaku kyslíku reagují s menší intenzitou, mění se však jejich poddajnost. Názory na hypoxickou reaktivitu plicních žil jsou dosud nejednotné, ale velmi pravděpodobně není příliš významná. Mechanismus zjišťování hypoxie hladkým svalem plicních cév není jasný. Do úvahy může připadat podíl některého z následujících dějů:

1. Inhibice respiračního řetězce (Yuan, Tod et al. 1994; Bright, Salvaterra et al. 1995) 2. Změna redoxního stavu buňky (Archer, Will et al. 1986; Franco-Obregon a LopezBarneo 1996)

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3. Aktivace mitochondriální NAD(P)H oxidázy a zvýšení produkce superoxidu (Marshall, Mamary et al. 1996; Waypa, Chandel et al. 2001) 4. Inhibice cytochromu P 450 (Sylvester a McGowan 1978; Knoblauch, Sybert et al. 1981; Chang, Dutton et al. 1992) 5. Snížení intracelulárního pH (Berger, Vandier et al. 1998; Madden, Keller et al. 2000)

Efektor hypoxické plicní vazokonstrikce Kontraktilní odpověď cévního hladkého svalu na hypoxii je vyvolána navázáním vápníku na kalmodulin. Komplex kalmodulin – kaldesmon ovlivňuje aktivitu různých proteinů v buňce a mezi nimi skupinu proteinkináz, známou jako kalmodulin dependentní proteinkinázy. Z nich hraje hlavní roli v regulaci kontrakce hladkého svalu enzym kináza lehkého řetězce myozinu. Výsledkem jeho aktivace je fosforylace lehkého řetězce myozinu, která je podmínkou interakce myozinu a aktinu, tedy kontrakce cévního hladkého svalu. Je tedy zřejmé, že součástí hypoxické plicní vazokonstrikce musí být buď zvýšení koncentrace vápenatých iontů v kompartmentu kontraktilního aparátu hladkého svalu, nebo zvýšení citlivosti kinázy lehkého řetězce myozinu k vápníku. Zvýšení citlivosti k vápníku není moc probádané, ale objevují se úvahy, že se na vazokonstrikci podílí (Rhoades, Jin et al. 1990). Intracelulární koncentrace vápenatých iontů zajišťuje rovnováhu mezi vstupem vápníku do cytoplazmy z extracelulárního prostoru (specifickými kanály) a z intracelulárních zásob (zejména sarkoplazmatického retikula, a snad i z mitochondrií a plasmalemy); řídí i aktivní odčerpávání ven z buňky nebo zpět do sarkoplazmatického retikula. Užitím fluorescenčních mikroskopických technik bylo prokázáno, že hypoxie zvyšuje

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koncentraci cytoplazmatického vápníku. Při absenci extracelulárního vápníku je tento nárůst malý, ale protože je přítomen, lze předpokládat využití vápníku uvolněného z intracelulárních zásob (Gelband a Gelband 1997).

Klinický význam HPV Udržení optimálního parciálního tlaku kyslíku v krvi je velmi důležitý úkol v anesteziologické klinické praxi. Při celkové anestezii se snižuje alveolární ventilace některých oblastí plic a zvyšuje se zde periferní cévní rezistence. Na těchto změnách se významně podílí poloha pacienta (poloha na zádech, na boku, pronační poloha). Dalším faktorem ovlivňujícím alveolární ventilaci a plicní perfúzi je řízená ventilace. Selektivní ventilace jedné plíce, která je využívána v hrudní chirurgii, by byla velmi problematická při absenci HPV. V neventilované plíci se zvyšuje periferní cévní rezistence a krev přednostně perfunduje plíci ventilovanou. Tím je zajištěno snížení venózní příměsi z neventilované plíce v arteriální krvi. U pacientů s chronickým plicním onemocněním bývá hypoxická plicní vazokonstrikce oslabená. Při selektivní ventilaci jedné plíce u těchto pacientů je zvýšení venózní příměsi z neventilované plíce jedním z důvodů poklesu oxygenace arteriální krve. HPV ovlivňují i anestetika. Volatilní anestetika (halotan, izofluran, sevofluran) HPV oslabují. Toto oslabení obvykle nevede k významnému poklesu saturace arteriální krve kyslíkem ani u pacientů s chronickým onemocněním plic. Zdá se, že alespoň některá anestetika snižují HPV prostřednictvím draslíkových kanálů (Liu, Ueda et al. 2001). Intravenózní anestetika velikost HPV neovlivňují (thiopental, ketamin) nebo ji mohou i zesilovat (propofol) (Nakayama a Murray 1999).

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2.1.2. Hypoxická plicní hypertenze

Plicní hypertenze obecně je syndrom charakterizovaný zvýšením středního tlaku v plicnici nad 25 mmHg v klidu nebo 30 mmHg při zátěži. Různé formy plicní hypertenze jsou poměrně časté a jsou spojeny se značnou morbiditou a mortalitou. Patofyziologicky lze rozdělit podle mechanismu vzniku na prekapilární, postkapilární a hyperkinetickou (Riedel 2002). V roce 1973 byla rozdělena na primární (neznámá příčina) a sekundární (známá příčina). Klasifikace WHO z roku 2003 ji dělí do 5 kategorií (Simonneau 2003; Galie, Torbicki et al. 2004): plicní arteriální hypertenze, plicní žilní hypertenze, plicní hypertenze při hypoxémii, plicní hypertenze při chronické trombotické nebo embolické nemoci a plicní hypertenze z jiných příčin. Tato klasifikace byla v roce 2008 opět upravena a rozšířena (tabulka 1) (Simonneau, Robbins et al. 2009). Důsledkem plicní hypertenze je ztluštění plicních artérií, tlakové přetížení pravé komory, její hypertrofie a v pozdních stadiích její selhávání (Abraham, Kay et al. 1971; Hislop a Reid 1978; Rabinovitch, Gamble et al. 1979).

Hypoxická plicní hypertenze vzniká jako následek dlouhodobého pobytu ve vysokých nadmořských výškách nebo chronického onemocnění plic, které doprovází alveolární hypoventilace (např. chronická obstrukční choroba bronchopleulární, plicní fibróza, tuberkulóza). Je přítomna i u pacientů, u kterých je hypoventilace sekundárním následkem jiného než plicního onemocnění (např. omezení plicní ventilace při kyfoskolióze, syndrom spánkové apnoe).

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U laboratorních potkanů se rozvíjí hypoxická plicní hypertenze během 10 – 14 dnů expozice hypoxii, po této době dále neprogreduje (Herget, Suggett et al. 1978) (Reeves a Herget 1984). U lidí s chronickou hypoventilací trvá její rozvoj obvykle roky.

Na vzniku hypoxické plicní hypertenze se podílí vazokonstrikce a morfologická přestavba cévní stěny. Hypoxická vazokostrikce je odpovědí cév na akutní hypoxii, působí

jako

fyziologický

regulační

mechanismus,

který

z hypoventilovaných alveolů a brání vzniku hypoxémie (viz výše).

odvádí

krev

Působením

chronické hypoxie se mění stavba cévní stěny, ale rovněž přetrvává zvýšený tonus plicních cév. Mění se distribuce a zvětšuje se množství hladkého svalu ve stěně prealveolárních plicních arterií a venul (Reid 1986). Stimuluje se metabolismus pojivových struktur a objevuje se fibróza cévní stěny (Bishop, Guerreiro et al. 1990).

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Tabulka 1. Klasifikace plicní hypertenze z roku 2008

1. PAH (Plicní arteriální hypertenze) 1.1 idiopatická 1.2 hereditární 1.2.1 BMPR2 1.2.2 ALK 1 1.2.3 neznámá 1.3 indukované léky a toxiny 1.4 sdružená: 1.4.1 se systémovým onemocněním pojivové tkáně 1.4.2 s HIV infekcí 1.4.3 s portální hypertenzí 1.4.4 s vrozenými srdečními vadami 1.4.5 s chronickou hemolytickou anemií 1.5 perzistující plicní hypertenze u dětí 1´plicní venookluzní nemoc a/nebo plicní kapilární hemangiomatóza 2. Plicní hypertenze při levostranném onemocnění srdce 3. Plicní hypertenze při onemocnění plicního parenchymu nebo hypoxická 4. Chronická tromboembolická plicní hypertenze (CTEPH) 5. Plicní hypertenze multifaktoriální 5.1 hematologické poruchy 5.2 systemické poruchy 5.3 metabolické poruchy 5.4 ostatní, tumory a jiné

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Reaktivní sloučeniny kyslíku (ROS) a hypoxie

2.2.

2.2.1. ROS Reaktivní sloučeniny kyslíku (ROS) jsou skupinou radikálů, mezi které řadíme superoxid, peroxid vodíku a hydroxylový anion. Již více než před 35 lety byla prokázána produkce ROS fagocyty (Babior, Kipnes et al. 1973). V minulých letech se ukázalo, že ROS se tvoří i v jiných buňkách (Bayraktutan, Blayney et al. 2000; Griendling, Sorescu et al. 2000; Tammariello, Quinn et al. 2000). Za fyziologických podmínek mají ROS produkované alveolárními makrofágy roli baktericidní, ale zároveň i roli druhého posla. Zdrojem ROS je mitochondriální elektronový transportní řetězec (Turrens, Beconi et al. 1991; Paraidathathu, de Groot et al. 1992). Superoxid je tvořen rovněž aktivací nikotinamidadenindinukleotidfosfát (NADPH) oxidázy – enzymový komplex vázaný na buněčné membráně. Zvýšená koncentrace ROS jako následek stimulace NADPH oxidázy je přechodná. Antioxidativní enzymy vrací koncentraci ROS na původní hodnoty. U zdravého organismu je poměr tvorby ROS a aktivity antioxidativních enzymů v rovnováze a podílí se na udržení redoxního stavu buňky. Antioxidanty lze rozdělit na: a) enzymatické – SOD, kataláza, glutathionperoxidáza b) neenzymatické tvořené in vivo – GSH, ubichinon, kyselina močová, proteiny transportující kovy (transferrin, ceruloplasmin) c) neenzymatické získané ze stravy – beta-karoten, vitamín C, vitamín E

18

Enzymy s antioxidačním účinkem jako je superoxiddismutáza (SOD) jsou tyto radikály redukovány na peroxid vodíku (H2O2). Tato redukce na stabilnější H2O2 se děje v mitochondriích (Burke a Wolin 1987; Moudgil, Michelakis et al. 2005). 2 O2.- +

2H2

SOD

H2O2

+

O2

H2O2 je následně katalýzou redukován na H2O a O2. Toto se děje za pomocí enzymu katalázy (je téměř výhradně přítomna v buněčných peroxizomech). 2 H2O2

kataláza

H2O

+

O2

Další enzym eliminující H2O2 GSH peroxidáza (selenoprotein), která se nachází v cytosolu a mitochondriích. Redukuje H2O2 pomocí glutathionu (GSH) a produkuje oxidovaný glutathion (GSSG). Tento enzym je rovněž schopný redukovat peroxynitrit. Glutathion je velmi důležitý v obraně buněk proti oxidativnímu stresu (Meister 1988). Poměr glutathiondisulfidu ke glutathionu GSSG/2GSH může sloužit jako indikátor redoxního stavu buňky (Schafer a Buettner 2001). Tento poměr vychází z kombinace počtu H2O2 odstraněného GSH peroxidázou a GSSG redukovaného GSH reduktázou, která reguluje koncentraci GSH.

H2O2

+

2GSH

glutathionperoxidáza

GSSG

Při ischemicko-reperfúzním poškození dochází k významnému poklesu thiolových skupin a GSH, naopak roste GSSG, poměr GSH/GSSG klesá.

19

2.2.2. Zdroj ROS Z mnoha studií vyplývalo, že expozice hypoxii vede k oxidativnímu poškození, které je způsobené peroxidací lipidů (Block, Patel et al. 1989; Nakanishi, Tajima et al. 1995; Wilhelm a Herget 1999; Veselá a Wilhelm 2002) a že klíčovou roli by mohl hrát H2O2 (Russell a Jackson 1994; Kinnula, Crapo et al. 1995). Také je již dlouho známo, že expozice hypoxii aktivuje leukocyty in vivo (Sobin, Tremer et al. 1983; Burghuber, Mathias et al. 1984). V současnosti máme velké množství důkazů, že v časných fázích expozice hypoxii (přibližně několik prvních dnů) se tvoří v izolovaných buňkách různých tkání ve zvýšené míře ROS (Duranteau, Chandel et al. 1998; Vanden Hoek, Becker et al. 1998; Chandel, McClintock et al. 2000; Chandel a Schumacker 2000). Mezi tyto buňky patří i buňky cévního endotelu (Ali, Schlidt et al. 1999). Zvyšuje se permeabilita alveolokapilární membrány. Tím dochází k transsudaci tekutiny a komponent krevní plazmy, které v prvních několika dnech aktivují alveolární makrofágy a dochází k nárůstu tvorby H2O2 (Duranteau, Chandel et al. 1998). Hypoxie stimuluje alveolární makrofágy k větší produkci H2O2 in vitro (Tuohy, Bain et al. 1993). Rovněž u zvířat chovaných 3 dny v hypoxických podmínkách bylo ve vydechovaném vzduchu naměřeno zvýšené množství H2O2. Hypoxická zvířata mají zvýšenou koncentraci 3-nitrotyrosinu (marker interakce superoxidového radikálu s radikály NO) v séru vzhledem ke zvířatům normoxickým. Podle současných poznatků lze předpokládat, že redoxní stav alveolárních makrofágů nebo jiných buněk plic hraje roli v regulaci plicního zánětu. Tento zánět může být vyvolán bakteriálním či jiným poškozením. Alveolární makrofágy mají kromě schopnosti fagocytózy i schopnost syntetizovat mediátory zánětu jako

20

leukotrien B4 a četné cytokiny (tumor necrosis factor α, interleukin 6, interferon ) (Gordon a Read 2002). Četné studie u zvířat s deplecí schopnosti tvořit některý z cytokinů zánětu ukázaly na nižší aktivitu makrofágů a těžší průběh plicního zánětu.

2.2.3. Důsledky působení ROS Zvýšená produkce ROS působí oxidační změny pojivových proteinů matrix cévní stěny (Bačáková, Wilhelm et al. 1997). Oxidovaný kolagen stimuluje proliferaci hladkých svalových buněk více než neoxidovaný (Bačáková, Wilhelm et al. 1997). Oxidační poškození extracelulární matrix cévní stěny je důležitý faktor zahajující remodelaci cévní stěny při hypoxii. ROS a peroxynitrit (vzniká reakcí superoxidu s NO (Rubbo, Radi et al. 1994)) aktivují metaloproteinázy (MMP) (Rajagopalan, Meng et al. 1996). Mezi MMP patří tkáňová kolagenáza, která štěpí nativní kolagen. Důležitým zdrojem

kolagenolytických

enzymů

jsou

aktivované

žírné

buňky.

Počet

perivaskulárních žírných buněk v plicích se zvyšuje u zvířat chovaných v hypoxických podmínkách (Kay, Waymire et al. 1974; Mungall 1976; Tucker, McMurtry et al. 1977; Williams, Heath et al. 1981; Migally, Tucker et al. 1983). Počet těchto žírných buněk je také zvýšený u lidí žijících ve vysokých nadmořských výškách (Heath 1992). Denaturovaný kolagen pak může být štěpen dalšími MMP a také dalšími proteinázami. Za normálních okolností jsou kolagenolytické enzymy přítomny ve stěně plicních cév v inaktivní podobě, nebo jsou účinně inhibovány tkáňovými inhibitory MMP. Aktivace MMP zahajuje kaskádu, která vede ke zvýšenému 21

metabolickému obratu kolagenu (Bishop, Guerreiro et al. 1990; Poiani, Tozzi et al. 1990). . Opakované plicní záněty s epizodami alveolární hypoxie jsou důležitou příčinou HPH u pacientů s CHOPN (Filley, Beckwitt et al. 1968; Turato, Zuin et al. 2001). Při alveolární hypoxii dochází k degranulaci žírných buněk (Nadziejko, Loud et al. 1989). Při hypoxické plicní hypertenzi je metaloproteinázová aktivita zvýšená (Novotná a Herget 1998). Metaloproteináza MMP13 hraje hlavní roli ve štěpení nativního kolagenu.

Tato metaloproteináza je produkována žírnými buňkami, které byly

izolovány z plic laboratorních potkanů žijících v hypoxických podmínkách (Maxová, Novotná et al. 2008). Rozpad kolagenu je nejvyšší v prvních dnech expozice hypoxii (Novotná a Herget 2001). Vzniklé degradační produkty štěpení metabolismus kolagenu v plicní tkáni dále stimulují (Gardi, Pacini et al. 1990; Gardi, Calzoni et al. 1994) (Bačáková, Herget et al. 2003). Akumulace nízkomolekulárních štěpů pojivových bílkovin aktivuje mezenchymové buňky (Novotná a Herget 1998). Experimentální inhibice metabolismu kolagenu vede k redukci cévní přestavby u potkanů chovaných v hypoxických podmínkách a inhibuje rozvoj HPH (Kerr, Riley et al. 1984; Kerr, Ruppert et al. 1987), (Herget, Novotná et al. 2003). Rovněž blokáda elastolytické aktivity inhibuje rozvoj HPH (Rabinovitch 1999). Po zotavování po expozici chronické hypoxii je zvýšení kolagenolytické aktivity v plicích důležité pro normalizaci stavby stěny plicních cév (Tozzi, Thakker-Varia et al. 1998).

22

Obrázek č. 1. Mechanismus vzniku hypoxické plicní hypertenze

Alveolární hypoxie

alveolární makrofágy

ROS

NO

peroxynitrit

poškození stěny plicních cév

ţírné buňky

MMP

štěpení kolagenu

přestavba stěny plicních cév (fibrotizace) 23

2.3. Oxid dusnatý

Oxid dusnatý (NO) je nestabilní radikál, který se vyskytuje přednostně v plynné formě. NO je syntetizován z L-argininu a molekulárního kyslíku za vzniku L-citrulinu (Palmer, Rees et al. 1988; Moncada, Palmer et al. 1989; Palmer a Moncada 1989) (Leone, Palmer et al. 1991; Nathan 1992; Nathan a Xie 1994). Tuto reakci katalyzuje enzym NO syntáza (Palmer a Moncada 1989; Leone, Palmer et al. 1991; Knowles a Moncada 1994). NO syntáza se vyskytuje ve 3 základních izoformách - inducibilní (iNOS), endoteliální (eNOS) a neuronální (nNOS)(Isaacson, Hampl et al. 1994). NO syntázy neuronální a endoteliální jsou exprimovány trvale. Endoteliální NOS je důležitá v regulaci cévního tonu. Tvoří menší množství oxidu dusnatého, které je přesně regulováno hlavně intracelulární koncentrací Ca2+ (Knowles a Moncada 1994; Nathan a Xie 1994). Inducibilní NOS je nezávislá na kalciu a kalmodulinu. Po stimulaci zánětlivými cytokiny (IL-1, TNF, IFN-γ) nebo lipopolysacharidem bakteriální stěny (endotoxin) je exprimována hlavně makrofágy, ale i jinými buňkami (např. chondrocyty, neutrofily, hepatocyty, hladkými svalovými buňkami) (Gaston, Drazen et al. 1994).

2.3.1. Oxid dusnatý v plicním řečišti Oxid dusnatý je velmi účinný vazodilatátor. Hraje velmi významnou úlohu v regulaci tonu systémového řečiště (Furchgott a Zawadzki 1980; Ignarro, Buga et al. 1987; Palmer, Ferrige et al. 1987; Moncada a Higgs 1993). V minulosti se v regulaci cévního tonu přepokládala analogie mezi systémovým a plicním řečištěm. Četné práce ale tuto analogii popřeli. Ukázalo se, že bazální syntéza oxidu dusnatého je ve

24

zdravých plicních cévách minimální (Isaacson, Hampl et al. 1994; Hampl, Cornfield et al. 1995). Tím se plicní cirkulace významně liší od cirkulace systémové. Oxid dusnatý se dále podílí na inhibici neutrofilů, na inhibici aktivace a agregace destiček a oslabuje proliferaci buněk hladkých svalů (Adnot, Raffestin et al. 1995). Velké množství studií prokázalo, že při expozici chronické hypoxii roste syntéza NO (Shaul, North et al. 1995; Xue a Johns 1996; Carville, Adnot et al. 1997). Tvorba

NO

v

plicích

stoupá

v prvních

dnech

expozice

hypoxii.

Ve

vydechovaném vzduchu u laboratorních potkanů Hampl, Bíbová et al. (2006) naměřili významný vzestup koncentrace NO během prvního týdne expozice chronické hypoxii, dále tvorba NO neprogredovala. Při zotavování z expozice chronické hypoxii tvorba NO klesala zpět k bazální úrovni (graf 1).

Je velmi pravděpodobné, že NO má při rozvoji hypoxické plicní hypertenze 2 protikladné role. Jako aktivní radikál je spoluodpovědný za poškození plicních cév. Vazodilatační a antiproliferativní účinky mohou naopak působit protektivně. V méně pokročilých stádiích plicní hypertenze je produkce NO zvýšená. V terminálních fázích CHOPN, kdy je přítomna těžká plicní hypertenze může být produkce NO snížena (Dinh-Xuan, Higenbottam et al. 1991). Příčinou je pravděpodobně poškození endotelu.

25

Graf 1. Koncentrace NO ve vydechovaném vzduchu je u laboratorních potkanů na začátku expozice chronické hypoxii zvýšená (Hampl, Bíbová et al. 2006).

NO (ppb)

5

4 3 2

1.51

0

5

7

14 hypoxie

19

25 1 2 3 4 5

dny

zotavení

26

3. CÍLE DISERTAČNÍ PRÁCE

Údaje o vzniku hypoxické plicní hypertenze jsou dosud velmi nedostatečné. Předchozí experimenty naznačili, že by se na vzniku hypoxické plicní hypertenze mohl podílet oxidativní stres a to hlavně v prvním týdnu expozice hypoxii. Hlavním cílem této práce bylo objasnit, zda podání antioxidantu v časné fázi expozice hypoxii ovlivní velikost plicní hypertenze více než jeho pozdní podání v období již rozvinuté nemoci. Jako antioxidační látku jsme použili N-acetyl-L-cystein (NAC). Sledovali jsme změny odporu plicního cévního řečiště, změny reaktivity plicních cév na akutní hypoxii v závislosti na procentu kyslíku ve vdechovaném vzduchu. Tato měření byla prováděna ex vivo na modelu izolovaných perfundovaných plic laboratorního potkana. V další části práce jsme sledovali vliv časného a pozdního podávání N-acetylcysteinu na tlak v a. pulmonalis, systémový arteriální tlak, srdeční výdej a hematokrit u laboratorních potkanů vystavených hypoxii na 4 týdny.

NAC je již několik desetiletí užíván jako mukolytikum (Holdiness 1991; Ruffmann a Wendel 1991). NAC působí také jako antioxidant in vitro i in vivo. In vitro je jeho antioxidační působení spojené přímo s inaktivací elektrofilních skupin volných radikálů (Aruoma, Halliwell et al. 1989). In vivo je jeho funkce antioxidatu spojena s jeho hlavním metabolitem cysteinem, který je hlavním prekurzorem v biosyntéze glutathionu (Meister a Anderson 1983; Meister 1984; Meister 1988). NAC byl již použit v mnoha studiích, které se zabývaly poškozením plic při oxidativním stresu. (Ceconi, Curello et al. 1988; Leff, Wilke et al. 1993; Hoshikawa, Ono et al. 2001).

27

Poslední část této práce se zabývá zdrojem NO při expozici chronické hypoxii. V našich pokusech bylo důležité odlišit velikost tvorby NO v paranazálních dutinách a přímo v plicích.

28

4. METODY

4.1. Hypoxická izobarická komora

Komora s uzavřeným nuceným oběhem směsi plynů. Kontinuálně se zde měřil obsah kyslíku a spotřebovaný kyslík se dodával zpětnovazebně řízenou pumpou. Rovněž se kontinuálně měřil oxid uhličitý. CO2 se adsorboval KOH a natronovým vápnem. Vlhkost vzduchu se odstraňovala průchodem směsi plynů vrstvou silikagelu a ochlazením. Ve všech našich experimentech byla zvířata vystavena koncentraci 10

O2 ve vdechovaném vzduchu. Naše hypoxická izobarická komora umožňovala

vystavit hypoxii současně až 30 laboratorních potkanů.

4.2. 5-denní hypoxie

4.2.1. Izolované perfundované plíce laboratorního potkana

Mechanické vlastnosti plicních cév a jejich reaktivitu jsme studovali na preparátu izolovaných perfundovaných ventilovaných plic (McMurtry, Davidson et al. 1976). V anestezii thiopentalem (40 mg/kg t.hm. i.p.) jsme provedli tracheostomii a laboratoní potkany ventilovali normoxickou směsí plynů (21

O2, 5

CO2, 74

N2)

50 dechy /min. Otevřeli jsme hrudní dutinu ve stření čáře. Vtokovou kanylu jsme umístili do plicnice nastřiženým otvorem ve stěně pravé komory a výtokovou kanylu do levé komory. Z hrudní dutiny jsme vypreparovali společně plíce se srdcem a umístili jsme je do vyhřívané (38 C) a zvlhčované komůrky. Plíce jsme perfundovali 29

solným roztokem (NaCl 6,95 g, KCl 0,35 g, MgSO4 bezvodý 0,14g, NaHCO3 1,43g, KH2PO4 0,16g, dextrosa 0,99g, CaCl2 0,47g/na 1 litr destilované vody) se 4 albuminem, L-NAME (50

M) a meklofenamátem (17

M, všechny chemikálie

vyrobila firma Sigma-Aldrich). Výtoková kanyla byla připojena k reservoáru perfúzního roztoku tak, aby perfuzát mohl recirkulovat.

4.2.2. Experimentální protokol

4.2.2.1. Experimentální skupiny Dospělé samce laboratorních potkanů kmene Wistar jsme rozdělili do 6 skupin označených A až F. Zvířata ve skupině A (n=5) byla na vzduchu (FiO 2 0,21) a pila pouze vodu, zvířata ve skupině B (n=5) byla na vzduchu a pila roztok 2% NAC ve vodě, zvířata ve skupině C (n=6) byla 5 dní vystavena hypoxii a během celého experimentu pila pouze vodu, zvířata ve skupině D (n=5) byla 5 dní v hypoxii a NAC pila pouze v období expozice hypoxii. Zvířata ve skupině E (n=6) pila NAC 5 dní před obdobím hypoxie, v hypoxii pila pouze vodu. Zvířata ve skupině F (n=6) pila roztok NAC jak před hypoxií, tak i v období hypoxie.

4.2.2.2. Měření reaktivity plicních cév Preparát izolovaných plic jsme nechali 15 minut stabilizovat při konstantním průtoku (0,04 ml/g t. hm./min) a při ventilaci normoxickou směsí plynů (vrcholový inspirační tlak 10 cm H2O, pozitivní tlak na konci výdechu 2,5 cm H2O). Měřili jsme vazokonstrikční odpověď na podání bolusu angiotenzinu II a na akutní hypoxii. První dvě hypoxické odpovědi (0

O2 + 5

CO2) jsme použili jako priming, následovně

30

jsme měřili vazokonstrikční odpověď v závislosti na procentu zastoupení kyslíku ve vdechované směsi (10, 5, 3, 0

O2).

4.2.2.3. Měření mechanických vlastností (rezistence) plicních cév Připravili

jsme

preparát

izolovaných

perfundovaných

plic.

Plíce

jsme

stabilizovali při konstantním průtoku (0,04 ml/g t. hm./min). Využili jsme vztahu mezi perfúzním tlakem a průtokem (P/Q). Perfúzní pumpu jsme na 30 s zastavili, následovně jsme zvyšovali perfúzní průtok v 30 s krocích (0 2 2,25 7,5 13 18 ml/min). Perfúzní tlak byl zaznamenáván. Nakonec byl obnoven původní perfúzní průtok 0,04 ml/g t.hm./min. Pro každé plíce byla konstruována regresní přímka. Tato přímka byla hodnocena pomocí dvou parametrů: 1. sklon (směrnice P/Q), která informuje o cévní poddajnosti a odporu a 2. pomocí průsečíku s tlakovou osou (Ro), hypotetický tlak při nulovém průtoku, kritický otevírací tlak. Tato veličina nás informuje o velikosti bazálního tonu alveolárních plicních cév. Pro každou pokusnou skupinu byla spočítána průměrná hodnota sklonu a průměrná hodnota průsečíku.

4.2.3. Statistické hodnocení

Pro hodnocení vztahu tlak-průtok jsme užili lineární regresní analýzu. Křivku vztahu tlak-průtok jsme interpretovali pomocí parametrů slope a intercept tlakové osy. Tradičně je užíván Starlingův model rezistoru. V našem modelu se intercept tlakové osy interpretuje jako kritický uzavírací tlak pro kolabovatelné úseky plicních cév. Slope odpovídá odporovým vlastnostem cév vzhůru od částí schopných kolabovat (Mitzner a Huang, 1987). Výsledky jsou prezentovány jako průměr

SEM.

Křivka tlak-průtok a dose response byla analyzována pomocí opakovaných měření

31

ANOVA. Užili jsme Fischerův post-hoc test. Za statisticky signifikantní jsme považovali p

4.3.

0,05.

4-týdenní hypoxie

4.3.1.

Experimentální protokol

Dospělí samci laboratorního potkana kmene Wistar byli vystaveni hypoxii 3 - 4 týdny. NAC byl podáván v pitné vodě v průběhu 7 dní před expozicí hypoxii a dále v průběhu prvního týdne hypoxie (n

9), nebo až v průběhu druhých dvou týdnů

hypoxie. Měli jsme kontrolní skupinu zvířat žijících na vzduchu (n v hypoxických podmínkách bez podávání NAC (n

9) a

9). Na konci pokusu jsme měřili

tlak v a. pulmonalis, systémový arteriální tlak, srdeční výdej hematokrit a váhu pravé komory a levé komory se septem.

4.3.2. Měření tlaku v a. pulmonalis Plicní artérii jsme kanylovali přes vypreparovanou v. jugularis interna při zavřeném hrudníku u spontánně ventilujících zvířat (Herget a Paleček 1972). Polyethylenový katetr je připojen k tlakovému snímači a monitoru. O lokalizaci katetru nás informuje tvar křivky na monitoru. Tlak změřený v a. pulmonalis jsme zaznamenávali, střední arteriální tlak byl získáván elektronickou integrací.

32

4.3.3. Měření systémového arteriálního tlaku Tlak jsme měřili přímou metodou přes kanylu zavedenou do a. carotis. Polyethylenový katetr byl připojen k tlakovému snímači a monitoru.

4.3.4. Měření srdečního výdeje Zvířatům

jsme

provedli

tracheostomii

a

ventilovali

jsme

je

přes

tracheostomickou kanylu 50 dechy/min. Provedli jsme sternotomii s výraznou snahou minimalizovat krvácení. Ultrasonografickou sondu (2,5 mm SS-series with J reflector, Transonic Systems, Ithaca, NY, USA) k měření jsme přiložili na ascendentní aortu a měřili jsme průtok krve ultrazvukovým průtokoměrem (T 106 flowmeter, Transonic System).

4.3.5. Analýza váhy jednotlivých srdečních oddílů O přítomnosti chronické plicní hypertenze vypovídá míra hypertrofie pravé srdeční komory. Po provedení všech plánovaných měření jsme srdce vyjmuli z hrudníku. Oddělili jsme pravou srdeční komoru a levou srdeční komoru se septem (Fulton, Hutchinson et al. 1952). Jednotlivé části jsme zvážili. Poměrem váhy pravé komory k levé komoře se septem lze vyjádřit míru hypertrofie pravé komory při plicní hypertenzi.

4.3.6. Stanovení hematokritu Hematokrit jsme stanovovali kapilární metodou z krve odebrané z a. carotis. Krev odebranou do kapiláry jsme stáčeli v centrifuze (Mikro 20, Hettich Zentrifugen).

33

4.3.7. Statistické zpracování Hodnoty jsou vyjádřeny jako průměr

SEM a statisticky významný rozdíl mezi

jednotlivými skupinami byl stanovován pomocí one-way ANOVA testu. Užili jsme Scheffého post hoc test. Při P

0,05 byl rozdíl považován za statisticky významný.

4.4. Výdej NO po 4 dnech hypoxie

4.4.1. Experimentální protokol Dospělí samci laboratorního potkana kmene Wistar byli vystaveni hypoxii 4 dny (n = 6). Kontrolní skupina zvířat žila na vzduchu (n

7). Obě skupiny měly volný

přístup k vodě a stravě stejné kvality. Na konci pokusu jsme v celkové anestezii thiopentalem (40 mg/kg i.p.) všem zvířatům provedli tracheostomii, tím jsme vyloučili horní cesty dýchací z ventilace. K měření jsme použili chemiluminiscenční NO analyzátor CLD 77 AM, EcoPhysis, Duernten, Švýcarsko.

4.4.2. Stanovení celkové produkce NO Zvířata jsme jednotlivě umístili do pletysmografu (V = 2,1 l), který byl před umístěním zvířat propláchnut a naplněn vzduchem z tlakové nádoby s jasně definovanou směsí plynů, která neobsahovala NO. Po 10 minutách jsme měřili koncentraci NO ve vzduchu v pletysmografu.

34

4.4.3. Stanovení produkce NO dolními cestami dýchacími K měření jsme použili latexový balónek (V = 250 ml) nasazený přímo na tracheostomickou kanylu pomocí dvojcestného ventilu. Zvířata vydechovala vzduch do balónku po dobu 2 minut.

4.4.4. Stanovení produkce NO horními cestami dýchacími Tvorba NO dýchacími cestami představuje rozdíl mezi celkovou produkcí NO (vzduch z pletysmografu) a produkcí NO dolními cestami dýchacími (vzduch z latexového balónku). Tento výpočet jsme provedli jednotlivě pro každé zvíře.

4.4.5. Statistické zpracování Hodnoty jsou vyjádřeny jako průměr

SEM a statisticky významný rozdíl mezi

jednotlivými skupinami byl stanovován pomocí one-way ANOVA testu. Užili jsme Fischerův post hoc test. Při P

0,05 byl rozdíl považován za statisticky významný.

35

5. VÝSLEDKY

5.1. Vliv časného podávání NAC na odporové vlastnosti plicního řečiště

Hypoxická zvířata měla významně vyšší základní perfúzní tlak před měřením závislosti tlak-průtok. Hodnoty interceptu tlakové osy se mezi jednotlivými skupinami nelišily (tabulka 2). Slope byl signifikantně vyšší u zvířat, která byla v hypoxii a pila pouze vodu. Zvířata, která byla v hypoxii a NAC jim byl podáván před obdobím hypoxie a dále i během expozice hypoxii měla slope významně nižší než zvířata hypoxická, a jeho hodnota se téměř neliší od normoxických zvířat. Zvířata, kterým byl NAC podáván pouze v období hypoxie, měla P/Q slope rovněž nižší než zvířata hypoxická, která pila pouze vodu (graf 2).

5.2. Vliv časného podávání NAC na reaktivitu plicních cév

Reaktivita na akutní hypoxii byla zvýšena u zvířat, která byla 5 dní v hypoxických podmínkách a pila pouze vodu. Podání NAC neovlivnilo reaktivitu plicních cév na akutní hypoxii u zvířat, která byla vystavena chronické hypoxii (tabulka 3).

36

Tabulka 2. Základní perfúzní tlak, intercept.

Skupina

Normoxie Normoxie+ NAC Hypoxie

Základní n perfúzní tlak SEM mm Hg 5 9,8 1,2 8,3 0,9 5 6

Hypoxie+ NACprevent 5 . Hypoxie+ 6 NACterap Hypoxie+ NAC obojí 6

Intercept SEM

1,6 0,6

13,2 2,8

2,8 0,6

10,9 0,4

3,4 0,3

9,5 0,6

3,1 0,6

6,8 1,6

Hodnoty jsou uvedeny jako průměr p

mm Hg 2,2 0,7

2,8 0,8

SEM.

0,05: zvířata, která pila roztok NAC na vzduchu a v hypoxii před i během

expozice měla statisticky významně nižší bazální perfúzní tlak než zvířata, která pila vodu a byla vystavena hypoxii. Intercept se statisticky významně nelišil.

37

Graf 2. P/Q slope

1,4 1,2

P/Q slope

1

normoxie normoxie+NAC

0,8

hypoxie hypoxie+NACpozdně

0,6

hypoxie+NACčasně hypoxie+NACobojí

0,4 0,2 0

p

skupina

0,02: P/Q slope (rezistence plicních cév) zvířat, která pila pouze vodu a byla

vystavena hypoxii, byl statisticky významně vyšší než u všech zvířat, která byla na vzduchu. Také byl statisticky významně vyšší než u zvířat, která byla v hypoxii a pila NAC časně (skupina hypoxie+NAC časně) anebo před i během expozice hypoxii (skupina hypoxie+NAC obojí).

..

38

Tabulka 3. Základní perfúzní tlak izolovaných plic laboratorního potkana, změny perfúzního tlaku při ventilaci plic hypoxickými směsmi s FiO2 10%, 5%, 3% a 0%. Základní Základní ∆ perfúzní perfúzní perfúzního tlak po tlaku při Skupina n tlak před primingem primingu FiO2 10% SEM SEM SEM mm Hg mm Hg mm Hg 11,4 1,3 Normoxie 5 8,1 0,8 0,4 0,5 Normoxie + NAC Hypoxie Hypoxie+ NAC prevent Hypoxie+ NACterap Hypoxie+ NAC obojí

∆ ∆ perfúzního perfúzního tlaku při tlaku při FiO2 3% FiO2 0% SEM SEM mm Hg mm Hg

3,8 0,9

4,8 0,7

6,0 1,6 4,3 0,9

5

7,8 1,0

9,4 1,2

0,1 0,1

2,7 1,2

3,6 1,0

6

7,6 0,8

10,7 0,8

-0,1 0,2

2,3 0,4

4,8 1,0

5

7,9 0,8

-0,1 0,4

2,7 1,3

3,7 1,6

6,5 1,5

6

8,2 0,6

10,4 0,4

-0,3 0,1

1,5 0,5

2,3 0,9

4,8 1,5

6

7,7 0,4

8,5 0,5

0,3 0,1

0,8 0,3

2,5 0,9

4,9 1,9

11,6 0,5

Hodnoty jsou uvedeny jako průměr p

∆ perfúzního tlaku při FiO2 5% SEM mm Hg

9,2 2,0

SEM.

0,05: zvířata, která byla vystavena chronické hypoxii a pila pouze vodu, měla

statisticky významně zvýšenou reaktivitu plicních cév na akutní hypoxii. Podání NAC neovlivnilo reaktivitu plicních cév na akutní hypoxii u zvířat, která byla vystavena 4 dní hypoxii chronické. p

0,02: u skupin zvířat s tímto označením byl statisticky významně vyšší perfúzní

tlak po primingu.

39

5.3.

Vliv časného podávání NAC na plicní hypertenzi

Na začátku experimentu se zvířata v jednotlivých skupinách významně nelišila tělesnou váhou. Na konci pokusu všechna zvířata vystavená hypoxii vážila významně méně než zvířata žijící v normoxických podmínkách. Během expozice hypoxii zemřela dvě zvířata ze skupiny, které byl podáván NAC až ve druhé polovině hypoxie.

5.3.1. Tlak v a. pulmonalis

Chronická hypoxie vedla u zvířat k plicní hypertenzi se signifikantním zvýšením středního tlaku v plicnici a hypertrofií pravé komory. Všechna zvířata vystavená hypoxii měla významně vyšší tlak v plicnici než zvířata normoxická. PAP byl významně nižší u hypoxických zvířat, kterým byl NAC podáván časně v porovnání s hypoxickými zvířaty, která NAC dostávala až ve druhé polovině hypoxie, v období rozvinuté plicní hypertenze (graf 3).

40

Graf 3. Střední tlak v a. pulmonalis.

40 35

PAP (mm Hg)

30 normoxie

25

hypoxie

20

hypoxie+NACčasně hypoxie+NACpozdně

15 10 5 0 skupina

p

0,05: zvířata, která pila NAC před a časně během expozice hypoxii, měla

statisticky

významně

nižší

plicní

hypertenzi

než

ostatní

zvířata

chovaná

v hypoxických podmínkách a také než zvířata pijící NAC až v době rozvinuté plicní hypertenze. p

0,001: u všech zvířat vystavených hypoxii se rozvinula plicní hypertenze.

41

5.3.2. Systémový arteriální tlak

Zvířata, kterým byl NAC podáván časně, měla významně nižší systémový arteriální tlak než všechna ostatní zvířata (tabulka 4).

5.3.3. Srdeční výdej

Srdeční výdej byl významně nižší u všech hypoxických zvířat. To není překvapující vzhledem k tomu, že všechna hypoxická zvířata vážila na konci experimentu významně méně než zvířata normoxická. Srdeční index se mezi jednotlivými skupinami nelišil (tabulka 4).

5.3.4. Hypertrofie pravé komory

Podávání NAC až ve druhé polovině expozice hypoxii neovlivnilo hypoxií indukovanou hypertrofii pravé komory (tabulka 4).

5.3.5. Hematokrit

Všechna hypoxická zvířata měla významně vyšší hematokrit než kontrolní normoxická zvířata (tabulka 4).

42

Tabulka 4. Tělesná váha, srdeční výdej, systémový arteriální tlak, hematokrit, poměr vlhké váhy pravé komory k levé komoře se septem.

Skupina

n

Tělesná hmotnost

g 9 315 4 9 197 7 9 202 9

Normoxie Hypoxie Hypoxie+ NAC časně Hypoxie+ 6 186 5 NAC pozdně

Srdeční výdej

0,05,

p

0,02,

Hematokrit

Váha pravé komory/ váha levé komory + septum

ml/min 34,1 2,7 25,1 1,2

mm Hg 106 2 109 3

48,5 0,4 78,3 1,2

0,276 0,010 0,510 0,042

23,3 2,4

94 4

78,4 3,2

0,430 0,026

25,4 1,8

103 6

80,5 2,1

0,530 0,031

Hodnoty jsou uvedeny jako průměr p

Systémový arteriální tlak

p

SEM.

0,001: hypoxické skupiny se statisticky významně liší

od normoxické skupiny. p

0,02: skupina zvířat, kterým byl NAC podáván před a v časném období hypoxie

se statisticky významně liší od všech ostatních skupin. p

0,05: zvířata, kterým byl NAC podáván před a v časném období hypoxie se

statisticky významně liší od zvířat, která NAC pila až ve druhé polovině expozice hypoxii.

43

Měření produkce NO z plic a horních cest dýchacích

5.4.

Na začátku experimentu se zvířata významně nelišila tělesnou váhou. Na konci pokusu měla všechna zvířata vystavená hypoxii významně nižší tělesnou váhu, než zvířata žijící v normoxických podmínkách. Expozice 4-denní hypoxii statisticky významně zvýšila produkci NO. Statisticky významně je zvýšená tvorba NO v dolních cestách dýchacích. Horní cesty dýchací se na zvýšení tvorby NO během hypoxie významně nepodílí (tabulka 5).

Tabulka 5. Produkce NO dýchacími cestami, produkce NO dolními dýchacími cestami a produkce NO horními dýchacími cestami.

Skupina

Normoxie 4 dny Hypoxie 4 dny

g

NO NO NO produkované produkované produkované dolními horními celkem cestami cestami dýchacími dýchacími SEM SEM SEM ppb ppb ppb

338 9

1,431 0,160 0,551 0,072 0,988 0,216

251 4

2,122 0,203 1,133 0,116

Hmotnost SEM

n

7 6

Hodnoty jsou uvedeny jako průměr p

0,02,

p

0,880 0,120

SEM.

0,001

Hypoxické skupiny se statisticky významně liší od normoxických ve všech sledovaných parametrech s výjimkou NO produkovaném horními cestami dýchacími – zde se nepodařilo najít statistickou závislost.

44

6. DISKUSE A ZÁVĚRY

Hypoxická plicní hypertenze vzniká ve dvou fázích. Počáteční fáze je charakterizovaná poškozením stěn periferních plicních artérií, které stimuluje proliferaci buněk cévních hladkých svalů a fibroblastů. K tomuto poškození přispívá zvýšená tvorba superoxidu a oxidu dusnatého v prvních dnech expozice hypoxii. Poškození je způsobeno volnými kyslíkovými radikály (Herget, Wilhelm et al. 2000; Hoshikawa, Ono et al. 2001), které se v hypoxických podmínkách tvoří v organizmu ve zvýšené míře. Je velmi pravděpodobné, že jejich zvýšená tvorba je lokalizována v mitochondriích. Mitochondrie jsou tedy senzorem hypoxie i místem zvýšené tvorby ROS v hypoxických podmínkách (Duranteau, Chandel et al. 1998; Chandel, Mc Clintock et al. 2000; Chandel a Schumacker 2000) (Ali, Schlidt et al. 1999; Waypa, Chandel et al. 2001). Vlivem radikálového poškození je zvýšená migrace leukocytů do perivaskulárního prostoru a rovněž se zvyšuje permeabilita cévní stěny pro albumin (Wood, Johnson et al. 2000). Dochází ke zvýšení plicní cévní rezistence a prealveolární cévy ztrácejí svou poddajnost. U laboratorních potkanů je plicní hypertenze plně rozvinutá ve třetím týdnu expozice hypoxii, a dále již neprogreduje. Po tomto období rovněž klesá produkce kyslíkových radikálů i oxidu dusnatého k normálním hodnotám (Wilhelm, Sojková et al. 1996; Wilhelm, Frydrychová et al. 1999). Naše výsledky potvrdily naši hypotézu, že během několika prvních dní expozice hypoxii je za poškození stěny plicních cév a následně vznik hypoxické plicní hypertenze odpovědné radikálové poškození.

45

6.1. Úloha volných kyslíkových radikálů v časné fázi expozice hypoxii

Podávání antioxidantu NAC před a v časné fázi expozice chronické hypoxii bránilo rozvoji plicní hypertenze. Podání NAC v pozdní fázi expozice hypoxii, v období rozvinuté plicní hypertenze, bylo bez efektu. Podávání antioxidantů při plně rozvinuté plicní hypertenzi neovlivní tlak v plicnici. Vedlejším překvapujícím nálezem bylo snížení systémového arteriálního tlaku u zvířat, která dostávala NAC před expozicí hypoxii. Všechna ostatní zvířata měla systémový arteriální tlak normální. V současné době nemáme vysvětlení pro tento nález, ale mohl by také ukazovat na radikálovou nerovnováhu v období časné hypoxie. Změnu morfologie stěny plicních cév laboratorních potkanů po 3-denní expozici hypoxii pozorovali již Rabinovitch (Rabinovitch, Gamble et al. 1981). Fakt, že kritické období pro rozvoj HPH jsou první dny expozice hypoxii, potvrzují také další práce z našeho pracoviště. Tvorba ROS se po prvním týdnu expozice hypoxii vrací k normálním hodnotám (Wilhelm, Sojková et al. 1996; Wilhelm, Frydrychová et al. 1999). Po 4-denní expozici laboratorních potkanů hypoxii byl nalezen signifikantní nárůst počtu žírných buněk a jejich MMP 13 u stěn prealveolárních arterií. U potkanů, kteří byli v hypoxických podmínkách 20 dní, nebyl nárůst počtu žírných buněk v této lokalizaci zaznamenán. MMP 13 pozitivní žírné buňky se akumulovaly u konduitních artérií a subpleurálně (Bishop, Guerreiro et al. 1990; Vajner, Vytášek et al. 2006). Podání blokátoru degranulace žírných buněk v časné fázi expozice hypoxii vedlo k inhibici rozvoje HPH a k inhibici přítomnosti štěpů kolagenu ve stěně plicních artérií (Baňasová, Maxová et al. 2008).

46

6.2. Úloha oxidu dusnatého v časné fázi expozice hypoxii

Oxid dusnatý, volný radikál s velmi krátkým poločasem, je vysoce nestabilní v biologických systémech. Je tvořen z L-argininu pomocí enzymu NO syntázy (NOS). Tento enzym se vyskytuje ve formě 3 izoenzymů. Naše výsledky potvrzují, že pro rozvoj poškození plicních cév v prvních dnech expozice hypoxii hraje klíčovou roli inducibilní NO syntáza (iNOS). Exprese iNOS je významně zvyšována různými prozánětlivými mediátory. Na tonu plicních cév se podílí rovněž NO tvořené pomocí endoteliální NO syntázy (eNOS), tento izoenzym exprimovaný cévním endotelem (Mawji a Marsden 2003) se tvoří konstitutivně. Předpokádáme, že ovlivňuje tonus plicních cév v klidových podmínkách a pozdních fázích expozice hypoxii. V prvních dnech expozice hypoxii dochází ke zvýšené expresi iNOS v plicních cévách a následně k významné nadprodukci NO (Hampl, Bíbová et al. 2006). Podání specifického inhibitoru iNOS (L-NIL) v časné fázi expozice hypoxii bránilo rozvoji plicní hypertenze, jeho podání v období již rozvinuté plicní hypertenze bylo bez efektu (Hampl, Bíbová et al. 2006). Bylo prokázáno, že hlavním místem tvorby oxidu dusnatého jsou horní cesty dýchací, zejména nazální a paranazální dutiny (Dillon, Hampl et al. 1996), (Lundberg, Farkas-Szallasi et al. 1995). Nebylo jasné, zda jsou za rychlý nárůst NO na počátku hypoxie zodpovědné horní nebo dolní cesty dýchací. Náš experiment prokázal, že v časných fázích expozice hypoxii jsou stále hlavním místem tvorby NO horní cesty dýchací, ale za nárůst tvorby NO jsou zodpovědné dolní cesty dýchací. Mechanismus jakým radikálové poškození v časné fázi hypoxie vyvolá přestavbu prealveolárních cév je pravděpodobně spojen se změnou matrixových proteinů. Modifikace kolagenových proteinů volnými radikály stimuluje proliferaci

47

buněk hladkého svalu cévní stěny in vitro. Chronická hypoxie indukuje zvýšení kolagenolytické aktivity, to vede k akumulaci štěpných produktů kolagenu ve stěně periferních plicních artérií. Peroxynitrit (viz dále) aktivuje metaloproteinázy. Redukovaný glutathion tuto aktivaci potencuje (Okamoto, Akaike T. et al. 1997). Větší množství fragmentů kolagenu je přítomno v časné fázi expozice hypoxii. Bylo prokázáno, že štěpné produkty kolagenu stimulují produkci fibroblastů in vitro i in vivo.

6.3.

Peroxynitrit

Peroxynitrit je vysoce cytotoxická sloučenina, která vzniká reakcí superoxidu a NO. Za fyziologických podmínek se peroxynitrit tvoří v malém množství a oxidativní poškození je minimalizováno endogenní antioxidativní obranou (Radi, Cassina et al. 2002; Radi, Cassina et al. 2002). Peroxynitrit je mnohem reaktivnější než jeho mateřské molekuly (Beckman 1990; Beckman, Beckman et al. 1990; Beckman a Koppenol 1996). Jeho poločas je krátký, ale dostatečný k překonání buněčné membrány

(Denicola,

Souza

et

al.

1998)

a

k interakci

s nejdůležitějšími

biomolekulami (Pryor a Squadrito 1995). Způsobuje poškození DNA, inaktivaci enzymů účastnících se metabolismu, iontových kanálů a stavebních bílkovin. Také narušuje integritu buněčných membrán. Peroxynitrit má schopnost oxidativně působit na biomolekuly, proto je považován za potencionálního vyvolavatele mnoha chorob (kardiovaskulární, neurodegenerativní, zánět, autoimunitní a další). Zvýšené množství peroxynitritu nepříznivě ovlivňuje funkci cévního endotelu. Za fyziologických podmínek se cévní endotel podílí na udržování cévního tonu, na

48

rovnováze mezi pro a antikoagulačními faktory a na regulaci adheze a migraci leukocytů do tkání. Peroxynitrit potencuje aktivaci neutrofilů. Zvyšuje se syntéza IL-8, který je hlavním chemoatraktantem lidských leukocytů (Zouki, Jozsef et al. 2001; Jozsef, Zouki et al. 2002; Khreiss, Jozsef et al. 2005). Peroxynitrit rovněž zvyšuje β2integrin na povrchu leukocytů (Zouki, Zhang et al. 2001) a tím se podíli na zvýšené interakci leukocytů s endotelem.

Vzhledem k tomu, že v časné fázi hypoxie dochází v plicních cévách k nadprodukci superoxidu a oxidu dusnatého, je zvýšená i tvorba této látky. Toto podporují nálezy zvýšeného množství 3-nitrotyrosinu (marker peroxynitritu, vzniká nitrací tyrosinu nebo tyrosinových zbytků proteinů (Eiserich, Vossen et al. 1994)) v plicní tkáni po 4-denní expozici hypoxii. Během dalších 3 týdnů expozice hypoxii se množství 3-nitrotyrosinu v plicní tkáni normalizuje (Herget, Wilhelm et al. 2000). V prvních dnech expozice hypoxii se rovněž zvyšuje plazmatická hladina 3nitrotyrosinu (Mrázková, Ošťádal et al. 2000). Zvýšené množství 3-nitrotyrosinu v plicích bylo nalezeno i u pacientů se závažnou primární i sekundární plicní hypertenzí (Bowers, Cool et al. 2004).

6.4.

Změny tvorby radikálů v časovém průběhu hypoxie

Naše experimenty prokázaly, že kritickým obdobím pro vznik hypoxické plicní hypertenze je časná fáze (několik prvních dní) expozice hypoxii. V tomto kritickém období se významně zvyšuje tvorba kyslíkových radikálů i NO. Za zvýšenou tvorbu

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NO je zodpovědná zvýšená exprese enzymu iNOS. Není jasné, zda se jedná o příčinu nebo následek poškození plicních cév vedoucí k jejich remodelaci. Je možné, že NO na začátku zhoršuje hypoxickou plicní hypertenzi. Tento fakt by mohl souviset s tím, že NO nezlepšuje dlouhodobý outcome u pacientů s ARDS (Kaisers, Busch et al. 2003) a perzistující plicní hypertenzi novorozenců (Kinsella, Walsh et al. 1999; Clark, Kueser et al. 2000). V tomto období časné hypoxie převažuje funkce NO jako radikálu nad jeho vazodilatačním a antiproliferačním účinkem. V dalším průběhu expozice hypoxii exprese iNOS pravděpodobně klesá, za tvorbu NO je odpovědná eNOS. V tomto pozdním období expozice hypoxii (týdny) již převažuje vazodilatační účinek NO.

V současnosti se zdá, že celá situace radikálového poškození v časné fázi expozice hypoxii má klinicky nejblíže k akutní exacerbaci CHOPN, která se řadí mezi zánětlivá onemocnění. Předpokládá se, že oxidativní stres má centrální úlohu v patogenezi CHOPN, protože spouští ostatní faktory podílející se na vzniku tohoto onemocnění (Rahman, Skwarska et al. 1999; MacNee a Rahman 2001; MacNee 2005; Macnee 2007). Mnohé studie prokázaly nárůst markerů oxidativního stresu v plicích pacientů s CHOPN v porovnání se zdravými jedinci (kuřáky) (Kanazawa a Yoshikawa 2005; Ceylan, Kocyigit et al. 2006). Během akutní exacerbace CHOPN pacienti vydechují více H2O2 než během klidové fáze onemocnění (Gerritsen, Asin et al. 2005; Owen 2005). Jejich alveolárními makrofágy tvoří ve zvýšené míře superoxid (Owen 2005). Proto se zdá, že terapie antioxidanty u pacientů s CHOPN by redukovala poškození oxidačním stresem a následně i ostatní typy poškození.

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7. OBECNÉ ZÁVĚRY VYPLÝVAJÍCÍ Z DISERTACE

1) V patogenezi hypoxické plicní hypertenze se uplatňuje zvýšená tvorba reaktivních sloučenin kyslíku. 2) Preventivní podání antioxidantů brání hypoxickému poškození stěny plicních cév více než jejich podávání v průběhu expozice hypoxii. 3) Oxidační poškození v časných fázích expozice hypoxii určuje další rozvoj onemocnění. 4) Terapeutické podávání antioxidantů je při rozvinuté hypoxické plicní hypertenzi bez efektu. 5) Tvorba NO se během expozice chronické hypoxii zvyšuje v dolních cestách dýchacích. 6) Velikost tvorby NO v horních dýchacích cestách expozice chronické hypoxii neovlivňuje. 7) Práce podporuje názor o možném příznivém působení antioxidantů a scavengerů volných radikálů u nemocných s plicním onemocněním (CHOPN, ARDS). Podmínkou jejich dostatečné účinnosti na ovlivnění průběhu nemoci je podávání v časné fázi rozvoje onemocnění. 8) Antioxidanty a scavengery volných radikálů by bylo možné využít v předoperační přípravě pacientů v hrudní chirurgii. Pacienti s preexistujícím plicním onemocněním mají již předoperačně inadekvátní antioxidační kapacitu. Výkony, kde se využívá technika ventilace jedné plíce, jsou doprovázeny signifikantní indukcí oxidativního stresu (hlavně uvolněním superoxidu) (Cheng, Chan et al. 2006). Po přechodu zpět na ventilaci obou plic mají tito pacienti zvýšné riziko plicního edému, který je jednou z nejzávažnějších komplikací u operačních výkonů v hrudníku.

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9. VLASTNÍ PUBLIKACE AUTORA Publikace v časopise s impakt faktorem: V. Lachmanová, O. Hniličková, V. Povýšilová, V. Hampl, J. Herget. “N-acetylcysteine inhibits hypoxic pulmonary hypertension most effectively in the initial phase of chronic hypoxia“ LifeSci 2005 May 27, 77(2): 175-82 V. Hampl, J. Bíbová, A. Baňasová, J. Uhlík, D. Miková, O. Hniličková, V. Lachmanová, J. Herget. “Pulmonary vascular iNOS induction participates in the onset of chronic hypoxic pulmonary hypertension“AmJPhysiol LungCellMolPhysiol 2006 Jan, 290(1): L11-20 L. Vajner, R. Vytášek, V. Lachmanová, J. Uhlík, V. Konrádová, J. Novotná, V. Hampl, J. Herget. “Acute and chronic hypoxia as well as recovery from chronic hypoxia affects the distribution of pulmonary mast cells and their MMP-13 expression in rats“IntJExpPathol 2006 Oct, 87(5): 383-91 Publikace v časopise bez impakt faktoru: V. Lachmanová. (2003) “Hypoxická plicní vazokonstrikce.“ Anesteziologie a intenzivní medicína, 14(2): 94-97

Abstrakta: V. Lachmanová, D. Hodyc, J. Herget. (2002) “Plicní vazokonstrikce brání arteriální hypoxemii.“ Cor et Vasa, 44 (10): K 221-222 V. Lachmanová, O. Hniličková, J. Herget. (2002) “N- acetylcysteine (NAC) but not LNAME inhibits pulmonary vascular effects of 5 days hypoxia in rats.“ PhysiolRes, 51(6): P68 D. Hodyc, V. Lachmanová, J. Herget. (2002) “ Pulmonary vasoconstriction prevents arterial hypoxemia in isolated rat lungs ventilated with hypoxic gas.“ Physiol Res, 51(6): P67 V. Lachmanová, O. Hniličková, J. Herget. (2003) “Preventive use of antioxidant Nacetylcysteine (NAC) inhibits hypoxic injury of pulmonary vasculature.“ Physio lRes, 53 (2): P3 V. Hampl, V. Lachmanová, O. Hniličková, J. Herget. (2004) “Critical role of oxygen radicals in the early phase of hypoxic pulmonary hypertension development.“ FASEB Journal 18: A 1056 V. Lachmanová, J. Bíbová, D. Miková, O. Hniličková, V. Hampl, J. Herget. (2005) “Nitric oxide production into the exhaled air is elevated by 4days of hypoxia in the lower, but not the upper, respiratory tract.“ Physiol Res, 54 (1)

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Life Sciences 77 (2005) 175 – 182 www.elsevier.com/locate/lifescie

N-acetylcysteine inhibits hypoxic pulmonary hypertension most effectively in the initial phase of chronic hypoxia Veˇra Lachmanova´, Olga Hnilicˇkova´, Viera Povy´sˇilova´, Va´clav Hampl, Jan HergetT Department of Physiology, 2nd Medical School, Charles University and Center for Experimental Cardiovascular Research, Plzenˇska´ 221, Prague 5, Czech Republic Received 9 August 2004; accepted 4 November 2004

Abstract Exposure to chronic hypoxia results in hypoxic pulmonary hypertension (HPH). In rats HPH develops during the first two weeks of exposure to hypoxia, then it stabilizes and does not increase in severity. We hypothesize that free radical injury to pulmonary vascular wall is an important mechanism in the early days of the hypoxic exposure. Thus antioxidant treatment just before and at the beginning of hypoxia should be more effective in reducing HPH than antioxidant therapy of developed pulmonary hypertension. We studied adult male rats exposed for 4 weeks to isobaric hypoxia (FiO2 = 0.1) and treated with the antioxidant, N-acetylcysteine (NAC, 20 g/l in drinking water). NAC was given bearlyQ (7 days before and the first 7 days of hypoxia) or blateQ (last two weeks of hypoxic exposure). These experimental groups were compared with normoxic controls and untreated hypoxic rats (3-4 weeks hypoxia). All animals kept in hypoxia had significantly higher mean pulmonary arterial blood pressure (PAP) than normoxic animals. PAP was significantly lower in hypoxic animals with early (27.1 F 0.9 mmHg) than late NAC treatment (30.5 F 1.0 mmHg, P b 0.05; hypoxic without NAC 32.6 F 1.2 mmHg, normoxic controls 14.9 F 0.7 mmHg). Early but not late NAC treatment inhibited hypoxia-induced increase in right ventricle weight and muscularization of distal pulmonary arteries assessed by quantitative histology. We conclude that release of free oxygen radicals in early phases of exposure to hypoxia induces injury to pulmonary vessels that contributes to their structural remodeling and development of HPH. D 2005 Elsevier Inc. All rights reserved. Keywords: Hypoxic pulmonary hypertension; Oxidant stress; Vascular remodeling

T Corresponding author. Tel./fax: +420 257210995. E-mail address: [email protected] (J. Herget). 0024-3205/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2004.11.027

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Introduction Exposure to chronic hypoxia causes hypoxic pulmonary hypertension (HPH). The rise in pulmonary peripheral vascular resistance in chronic hypoxia results from vasoconstriction and from structural remodeling of the walls of peripheral pulmonary arteries (Reid, 1979). Pulmonary arterial blood pressure (PAP) increases and consequently right ventricular hypertrophy develops. In rats exposed to an environment with 10% O2 HPH develops within 10 – 14 days. Then it essentially does not progress any further and remains steady during this adaptation phase (Herget et al., 1978; Reeves and Herget, 1984). Exposure to hypoxia generates oxidative stress of lung tissue (Block et al., 1989; Chang et al., 1989; Nakanishi et al., 1995; Hoshikawa et al., 2001). We hypothesize that hypoxia-induced free radical injury to the walls of peripheral pulmonary arteries at the beginning of hypoxia is an important pathogenetic factor in the development of HPH. In the first few days of hypoxic exposure alveolar macrophages are activated and primed for an enhanced production of H2O2 (Wilhelm et al., 1996). Rats exposed to 3-days hypoxia have more H2O2 in their breath than normoxic animals. Then, after the first week of exposure, the concentration of H2O2 in exspired air gradually decreases to normal values (Wilhelm et al., 1999). The concentrations of different biochemical indicators of lung tissue oxidative stress are increased in the first week of chronic hypoxia. After a more prolonged exposure, in the steady phase of HPH, the biochemical signs of lung tissue oxidative injury disappear (Wilhelm and Herget, 1999; Herget et al., 2000; Hoshikawa et al., 2001). To test our hypothesis that oxidative injury is critical only in the initial development of HPH we compared the effects of antioxidant N-acetylcysteine (NAC) treatment, applied in rats just before and at the beginning of chronic hypoxia, with the results of its application in the fully developed HPH at steady state. It has been repeatedly reported that NAC given during the whole period of prolonged hypoxic exposure reduces the development of HPH (Hoshikawa et al., 1995; Herget et al., 1998; Hoshikawa et al., 2001).

Methods Four groups of adult male Wistar rats (Anlab, Czech Rep.) were studied. Experimental rats were exposed for 4 weeks to isobaric hypoxia (FiO2 = 0.1) (Hampl and Herget, 1990) and treated with NAC (20 g/l in drinking water). NAC was given before and at the beginning of exposure (7 days before and the first 7 days of hypoxia, n = 9, early NAC application) or in rats with developed HPH (last two weeks of hypoxic exposure, n = 8, late NAC application). The rationale for starting NAC treatment well before the beginning of the hypoxic exposure was to make sure that at the moment of onset of hypoxia, NAC had already reached an effective, stable level. Experimental groups were compared with normoxic controls (n = 9) and untreated rats exposed to hypoxia (3-4 weeks hypoxia, n = 9). Experiments were performed in accordance with the European Community and NIH guidelines for using experimental animals. All procedures were approved by our institution’s Animal Studies Committee. In rats anesthetized with thiopental (30 mg/kg b.w. i.p.) left carotid artery was cannulated to measure systemic arterial blood pressure (SAP) and to obtain a blood sample for hematocrit determination. Right jugular vein was exposed, pulmonary artery was catheterized without opening the chest, and PAP was recorded (Herget and Palecˇek, 1972) in rats spontaneously breathing atmospheric air. Than tracheal cannula was introduced and the rats were mechanically ventilated with room air at 50 breaths/min by

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positive pressure (peak inspiratory pressure 10 cm H2O, zero end-exspiratory pressure). The chest was opened by sternotomy with extra care taken to minimize bleeding. Ultrasonic flow probe (2.5 mm SSseries with J reflector, Transonic Systems, Ithaca, NY, USA) was placed at the ascending aorta to measure aortic blood flow (T 106 flowmeter, Transonic Systems) as an estimate of cardiac output (CO) (Hampl et al., 1993). After the measurements were completed the heart and lungs were removed from the chest. Right and left ventricles plus septum were separated and weighed (Fulton et al., 1952). Lungs were filled with neutral formol solution through the trachea at a pressure of 12 cm H2O and then placed in the same solution for 3 – 4 weeks. Lung sections were then cut and stained by the hematoxylin resorcin fuchsin method. Remodeling of the walls of peripheral pulmonary arteries was assessed by counting distal vessels bound to alveolar ducts or to alveoli (V 300 Am) on one slide from each rat and determining how many of them were muscularized (Hunter et al., 1974; Herget et al., 2003). Counted as muscularized were those vessels that had internal and external elastic laminae separated at least in half of the vessel circumference. All peripheral pulmonary blood vessels found in sagital sections through the hilus region of the right and left lungs were counted. All counting was performed by one person blinded to the group assignment of the slides. The number of vessels counted was 52 – 85 (range) in each rat. The result is reported as percentage of double-laminated (muscularized) peripheral vessels (%DL) (Herget et al., 1978).

Statistical analysis The results were evaluated by ANOVA with Scheffe’s post-hoc test. Values of p b 0.05 were considered significant. The results are presented as means F SEM.

Results At the beginning of treatment, the groups did not differ in body weight. Rats exposed to chronic hypoxia gained body weight more slowly than controls, and consequently, their body weight at the end of the exposure was significantly less compared to normoxic controls (Table 1). Two rats from the group with the late NAC application died; there was no mortality in the other groups.

Table 1 Body weight, cardiac output, systemic arterial pressure, hematocrit and right heart ventricle/left ventricle + septum weight ratio (RV/LV + S) Group

n

Body weight [g]

Cardiac output [ml/min]

SAP [mmHg]

Hematocrit [%]

RV/LV+S

Normoxia Hypoxia Hypoxia NAC early application Hypoxia NAC late application

9 9 9 6

315F4 197F7 *** 202F9 *** 186F5 ***

34.1F2.7 25.1F1.2 * 23.3F2.4 * 25.4F1.8 *

106F2 109F3 94F4 ## 103F6

48.5F0.4 78.3F1.2 *** 78.4F3.2 *** 80.5F2.1 ***

0.276F0.010 0.510F0.042*** 0.430F0.026** + 0.530F0.031***

Data are means F SEM, *: p b 0,05, **: p b 0,02, ***: p b 0,001 hypoxic groups differ from a normoxic group, ##: p b 0,02 early NAC treated group differs from all other groups, +: p b 0,05 NAC early NAC treated hypoxic group differs from late NAC treated hypoxic group. SAP = systemic arterial blood pressure.

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V. Lachmanova´ et al. / Life Sciences 77 (2005) 175–182 P<0.05

Pulmonary arterial mean BP [mmHg]

35 30 25 20 15 10

5 0 Normoxia

Hypoxia

Hypoxia + NAC early Hypoxia + NAC late

Fig. 1. Pulmonary mean arterial blood pressure in rats exposed to hypoxia and treated with N-acetylcysteine. Hypoxia + NAC early = rats exposed to hypoxia and treated with N-acetylcysteine one week before and during the 1st week of exposure to hypoxia. Hypoxia + NAC late = rats exposed to hypoxia and treated with N-acetylcysteine during the last two weeks of exposure to hypoxia. All groups exposed to hypoxia are significantly different from controls (Normoxia). P b 0.05 = statistical difference of groups treated early and late with N-acetylcysteine.

Chronic hypoxia induced pulmonary hypertension characterized by a significant increase in PAP (Fig. 1) and increased right to left heart ventricle + septum weight ratio (RV/LV + S, Table 1). All animals kept in hypoxia had significantly higher PAP than normoxic animals. However, PAP was significantly lower in the group of hypoxic rats with early NAC treatment than in rats with late application (Fig. 1). The same applied for the RV/LV + S ratio (Table 1). In all groups of rats exposed to hypoxia, the relative 35

P<0.05

30

DL [%]

25

20

15

10 5 0 Normoxia

Hypoxia

Hypoxia + NAC early Hypoxia + NAC late

Fig. 2. Percentage of muscularized (double laminated, DL) peripheral pulmonary arteries in rats exposed to hypoxia and treated with N-acetylcysteine. For the groups denomination see Fig. 1. All groups exposed to hypoxia are significantly different from controls (Normoxia). P b 0.05 = statistical difference of groups treated early and late with N-acetylcysteine.

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weight of right ventricle was larger than in normoxic controls. In the group exposed to hypoxia with the early NAC treatment the relative right ventricle weight was significantly lower (95.9 F 4.4 mg/100 g of b. w.) than in hypoxic rats with the late NAC application (118.2 F 5.7 mg/100 g of b. w., P b 0.01). The value in hypoxic rats with the late NAC application did not differ from that in unterated hypoxic rats. The relative weight of the left ventricle was slighly, but significantly, higher in all rats exposed to hypoxia. We did not observe any effect of NAC treatment on the left heart ventricle weight (data not shown). Chronic hypoxia induced the typical remodeling of peripheral pulmonary arteries with its characteristic feature – muscularization of walls of prealveolar vessels. The percentage of muscularized peripheral lung vessels was significantly higher in all groups of hypoxic rats than in normoxic controls. Vascular muscularization was slightly but significantly lower in rats with early NAC treatment than in hypoxic rats with late NAC treatment and in not treated hypoxic rats (Fig. 2). The animals in the group with early NAC treatment had significantly lower SAP than other groups. CO was significantly lower and hematocrit significantly higher in all hypoxic groups than in the groups kept in normoxia, but there were no differences in CO and hematocrit attributable to NAC treatment.

Discussion The main finding of our study is that the antioxidant NAC given to rats just before and during the first few days of hypoxic exposure reduces the development of HPH. It contrasts with the lack of effect of NAC applied in the late phase of exposure (3rd and 4th week of hypoxia). This observation strongly supports our hypothesis that the development of HPH proceeds in two phases. The initial phase is characterized by injury to the walls of peripheral pulmonary arteries that stimulates vascular smooth muscle and fibroblast proliferation. Structural remodeling of peripheral pulmonary arteries and increase in smooth muscle tonus result in the increase in peripheral pulmonary vascular resistance and prealveolar vessels become less compliant. While the role of free radicals in this process has been emerging during the recent years (for review, see Hampl and Herget, 2000) the inhibition of HPH only by the early NAC treatment in our present study suggests that release of free radicals participates in the pathogenesis of HPH specifically at the very onset of HPH. In the rat species the pulmonary hypertension is fully developed by the third week of hypoxia. Then it becomes stable and does not progress any further (Hunter et al., 1974; Herget and Palecˇek, 1978; Herget et al., 1978; Reeves and Herget, 1984). The release of oxygen radicals declines to normal values (Wilhelm et al., 1996; Wilhelm et al., 1999; Wilhelm and Herget, 1999) and, therefore, the antioxidant treatment did not influence the HPH in this stable stage. An important feature of the early phase of HPH is the presence of pulmonary vasoconstriction which contributes to the increase in pulmonary vascular resistance. This increase in vascular tension does not appear to be a simple extension of acute hypoxic pulmonary vasoconstriction inasmuch the latter is blunted in chronic hypoxia (McMurtry et al., 1978; Hampl and Herget, 1990). In another study in isolated rat lungs we found that NAC treatment prevented the blunting of hypoxic pulmonary vasoconstriction brought about by 5-d hypoxia (Lachmanova´ and Herget, 2002). Therefore, it is unlikely that the reduction of HPH by NAC was due to inhibition of hypoxic pulmonary vasoconstriction. The dose of NAC, which was dissolved in drinking water, depended on water intake by individual animals. We estimated water consumption roughly by weighing water bottles in each animal cage every other day. Watter consumption was lower in the group with the early NAC administration (17 ml/rat/day,

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range: 16–19 ml, 1st week of hypoxic exposure) than in rats with the late NAC treatment (27 ml/rat/day, range 20 – 31 ml, last week of exposure). Therefore the actual NAC intake was probably higher in the group where we did not observe any effect of NAC on HPH. We did not measure biochemically the effect of NAC administration on markers of oxidative stress. Hoshikawa and co-workers (Hoshikawa et al., 2001) used 1% of NAC in drinking water (50% of our dose) in a very similar experimental arrangement. They found that NAC administration significantly attenuated the increase in phosphatidylcholine hydroperoxide concentration during the first week of hypoxia. A surprising finding is the slightly but significantly reduced SAP in the group exposed to hypoxia with the early NAC treatment, especially as SAP was normal in both the untreated rats exposed to hypoxia and in the group with the late NAC treatment. In our previous study we did not observe any effect of chronic NAC treatment on SAP in normoxic rats (Herget et al., 1998) While we do not have an explanation for this finding, it seems to indicate that radicals somehow counterbalance some hypotensive effect of early hypoxia on the systemic circulation. Thus, free radicals may have an important role during the first days of hypoxia in both the pulmonary and systemic vessels. The mechanism of how the radical injury to pulmonary vascular wall initiates the remodeling of prealveolar vessels may be linked to the changes of matrix proteins. In the first week of exposure to hypoxia the peripheral pulmonary arteries are surrounded by numerous mast cells positive to immunostaining for interstitial collagenase (Vajner et al., 2003). The collagenolytic activity in the walls of peripheral pulmonary arteries is increased (Novotna´ and Herget, 1998) and it is particularly high at the beginning of exposure to hypoxia (Novotna´ and Herget, 2001). ROS and peroxynitirite (product of superoxide and nitric oxide interaction) are potent activators of collagenases (Rajagopalan et al., 1996) and NAC inhibits this effect (Tyagi, 1998). Collagen breakdown products that accummulate in the vascular wall (Novotna´ and Herget, 1998) may stimulate fibroproduction and smooth muscle proliferation (Gardi et al., 1990, 1994; Bacˇa´kova´ et al., 2003) in the walls of peripheral pulmonary arteries. Various cytokines and other regulatory molecules are involved in this cascade. Their interaction with oxidative stress induced by hypoxia is complex.

Conclusion We conclude that antioxidant, applied in the early phase of exposure to hypoxia reduces the development of HPH. We propose protection of matrix proteins of prealveolar vessels from free radicalinduced increase in collagenolysis as a possible mechanism.

Acknowledgements The study was supported by grant GACR 304/02/1348 and GAUK 53/2002C.

References Bacˇa´kova´, L., Herget, J., Novotna´, J., Eckhardt, A., Lisa´, V., 2003. Vascular smooth muscle cells in cultures on collagen I degraded by matrix metalloproteinase-13. Physiological Research 52 (23P).

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Block, E.R., Patel, J.M., Edwards, D., 1989. Mechanism of hypoxic injury to pulmonary artery endothelial cell plasma membrane. American Journal Physiology - Cell Physiology 257, C223 – C231. Chang, S.W., Selzner, T.J., Weil, J.V., Voelkel, N.F., 1989. Hypoxia increases plasma glutathione disulfide in rats. Lung 167, 269 – 272. Fulton, R.M., Hutchinson, E.C., Jones, A.M., 1952. Ventricular weight in cardiac hypertrophy. British Heart Journal 14, 413 – 420. Gardi, C., Pacini, A., de Santi, M.M., Calzoni, P., Viti, A., Corradeschi, F., Lungarella, G., 1990. Development of interstitial lung fibrosis by long-term treatment with collagen breakdown products in rabbits. Research Communications in Chemical Pathology and Pharmacology 68, 235 – 250. Gardi, D., Calzoni, P., Marcolongo, P., Vavarra, E., Vanni, L., Lungarella, G., 1994. Collagen breakdown and lung collagen metabolism: an in vitro study on fibroblast cultures. Thorax 49, 312 – 318. Hampl, V., Archer, S.L., Nelson, D.P., Weir, E.K., 1993. Chronic EDRF inhibition and hypoxia: effects on pulmonary circulation and systemic blood pressure. Journal of Applied Physiology 75, 1748 – 1757. Hampl, V., Herget, J., 1990. Perinatal hypoxia increases hypoxic pulmonary vasoconstriction in adult rats recovering from chronic exposure to hypoxia. American Review of Respiratory Diseases 142, 619 – 624. Hampl, V., Herget, J., 2000. Role of nitric oxide in the pathogenesis of chronic pulmonary hypertension. Physiological Reviews 80, 1337 – 1372. Herget, J., Bı´bova´, J., Hampl, V., 1998. Antioxidant N-acetylcysteine inhibits the development of hypoxic pulmonary hypertension in rats. Presented at INABIS 98 - 5th Internet World Congress on Biomedical Sciences at McMaster University Available at URL http://mcmasterca/inabis98/oxidative/herget0589/indexhtml. Herget, J., Novotna´, J., Bı´bova´, J., Povy´sˇilova´, V., Vanˇkova´, M., Hampl, V., 2003. Metalloproteinase inhibition by Batimastat attenuates pulmonary hypertension in chronically hypoxic rats. American Journal of Physiology – Lung Cellular and Molecular Physiology 285, L199 – L208. Herget, J., Palecˇek, F., 1972. Pulmonary arterial blood pressure in closed chest rats. Changes after catecholamines, histamine and serotonin. Archives internationales de Pharmacodynamie et de The´rapie 198, 107 – 117. Herget, J., Palecˇek, F., 1978. Experimental chronic pulmonary hypertension. International. Review of Experimental Pathology 18, 347 – 406. Herget, J., Suggett, A.J., Leach, E., Barer, G.R., 1978. Resolution of pulmonary hypertension and other features induced by chronic hypoxia in rats during complete and intermittent normoxia. Thorax 33, 468 – 473. Herget, J., Wilhelm, J., Novotna´, J., Eckhardt, A., Vyta´sˇek, R., Mra´zkova´, L., Osˇta´dal, M., 2000. A possible role of oxidant tissue injury in the development of pulmonary hypertension. Physiological Research 49, 493 – 501. Hoshikawa, Y., Ono, S., Suzuki, S., Tanita, T., Chida, M., Song, C., Noda, M., Tabata, T., Voelkel, N.F., Fujimura, S., 2001. Generation of oxidative stress contributes to the development of pulmonary hypertension induced by hypoxia. Journal of Applied Physiology 90, 1299 – 1306. Hoshikawa, Y., Ono, S., Tanita, S., Sakuma, T., Noda, M., Tabata, T., Ueda, S., Ashino, Y., Fujimura, S., 1995. Contribution of oxidative stress to pulmonary hypertension induced by chronic hypoxia. Nihon Kyobu Shikkan Gakkai Zasshi 33, 1169 – 1173. Hunter, C., Barer, G.R., Shaw, J.W., Clegg, E.J., 1974. Growth of the heart and lungs in hypoxic rodents. A model of human hypoxic disease. Clinical Science 46, 375 – 391. Lachmanova´, V., Herget, J., 2002. N-acetylcysteine (NAC) but not L-NAME inhibits pulmonary vascular effects of 5 days hypoxia in rats. Physiological Research 51 (68P). McMurtry, I.F., Petrun, M.D., Reeves, J.T., 1978. Lungs from chronically hypoxic rats have decreased pressor response to acute hypoxia. American Journal of Physiology 235, H104 – H109. Nakanishi, I., Tajima, F., Nakamura, A., Yagura, S.Y., Ookawara, T., Yamashita, H., Suzuki, K., Taniguchi, N., Ohno, H., 1995. Effects of hypobaric hypoxia on antioxidant enzymes in rats. Journal of Physiology 489, 869 – 876. Novotna´, J., Herget, J., 1998. Exposure to chronic hypoxia induces qualitative changes of collagen in the walls of peripheral pulmonary arteries. Life Sciences 62, 1 – 12. Novotna´, J., Herget, J., 2001. Small collagen cleavage fragments present in peripheral pulmonary arteries (ppa) of rats exposed to 4 days hypoxia diaspear in chronic hypoxic exposure. Physiogical Research 50, 21. Rajagopalan, S., Meng, X.P., Ramasamy, S., Harrison, D.G., Galis, Z.S., 1996. Reactive oxygen species produced by macrophage-derived foam cells regulate the activity of vascular matrix metalloproteinases in vitro - implications for atherosclerotic plaque stability. Journal of Clinical Investigation 98, 2572 – 2579.

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Reeves, J.T., Herget, J., 1984. Experimental models of pulmonary hypertension. In: Weir, E.K., Reeves, J.T. (Eds.) Pulmonary Hypertension, Futura Publ. Co, New York, pp. 361 – 391. Reid, L.M., 1979. The pulmonary circulation: remodeling in growth and disease. American Review of Respiratory Diseases 119, 531 – 546. Tyagi, S.C., 1998. Homocysteine redox receptor and regulation of extracellular matrix components in vascular cells. American Journal of Physiology – Cell Physiology 274, C396 – C405. Vajner, L., Vyta´sˇek, R., Novotna´, J., Uhlı´k, J., Konra´dova´, V., Herget, J., 2003. Redistribution of pulmonary mast cells and their MMP-13 production in hypoxic rats. Physiological Research 52 (44P). Wilhelm, J., Frydrychova´, M., Vı´zek, M., 1999. Hydrogen peroxide in the breath of rats. The effects of hypoxia and paraquat. Physiological Research 48, 445 – 449. Wilhelm, J., Herget, J., 1999. Free radicals in rat lung during and after hypoxia. Physiological Research 48 (53P). Wilhelm, J., Sojkova´, J., Herget, J., 1996. Production of hydrogen peroxide by alveolar macrophages from rats exposed to subacute and chronic hypoxia. Physiological Research 45, 185 – 1991.

Am J Physiol Lung Cell Mol Physiol 290: L11–L20, 2006. First published August 19, 2005; doi:10.1152/ajplung.00023.2005.

Pulmonary vascular iNOS induction participates in the onset of chronic hypoxic pulmonary hypertension Va´clav Hampl,1,5 Jana Bı´bova´,1,5 Alena Banˇasova´,2,5 Jirˇ´ı Uhlı´k,3 Dana Mikova´,1,5 Olga Hnilicˇkova´,1,5 Veˇra Lachmanova´,4,5 and Jan Herget1,5 Departments of 1Physiology, 2Pathophysiology, 3Histology, and 4Anesthesiology, Charles University Second Medical School; and 5Centre for Cardiovascular Research, Prague, Czech Republic Submitted 12 January 2005; accepted in final form 3 August 2005

(NO) is formed in mammalian cells as an endogenous mediator, many attempts were made to define its possible role in the pathogenesis of pulmonary hypertension (reviewed in Ref. 23). Although the capacity of lung vessels to produce NO can be reduced in terminal phases of severe pulmonary hypertension (15), possibly due to the progressive endothelial damage, less advanced stages (at least in adults) are associated with increased expression of NO synthase (NOS) and augmented NO production (reviewed in Ref. 23). This is particularly well documented in

the frequently used and clinically relevant model of pulmonary hypertension elicited by chronic hypoxia. In principle, as the actions of NO in the body are multifaceted, two main functional consequences of the elevated lung NO synthesis in chronic hypoxic pulmonary hypertension are possible. On one hand, the vasodilator and antiproliferative effects of NO may limit the extent of pulmonary vascular resistance elevation. This possibility is supported by numerous reports that acute administration of NOS blockers, such as NG-nitro-L-arginine methyl ester (L-NAME), increases perfusion pressure in lungs isolated from chronically hypoxic animals more than in normoxic controls (reviewed in Ref. 23). On the other hand, due to its radical nature, NO may contribute to the oxidative injury to the walls of the pulmonary vessels that appears to initiate their morphological remodeling (27, 28, 35). Such a hypertension-promoting effect may more or less negate the tone- and proliferation-reducing effects. Evidence is accumulating that the principal free radical insult that initiates the process of vascular remodeling occurs during the first few days of chronic hypoxia. For example, indexes of pulmonary hypertension, determined at the end of a prolonged hypoxia, are reduced in rats that were treated with antioxidants at the beginning of the exposure (28, 35). By analogy, the present study was designed to test the hypothesis that elevated lung NO production in the first few days of chronic hypoxia contributes to the development of pulmonary hypertension, presumably by contributing to oxidative vascular wall injury. We reasoned that if this hypothesis were true, then treatment with NOS inhibitor only during the first week of hypoxia would result in reduced pulmonary hypertension. Chronic hypoxia increases NOS expression in the lung vessels (reviewed in Ref. 23). Traditionally, functional role was implicitly assumed for the endothelial NOS isoform (eNOS). The possible role of the inducible NOS (iNOS, also called NOS II) is unknown, yet there are analogies from other organs that iNOS is functionally significant in situations characterized by tissue injury. Therefore, we also hypothesized that NO participating in the pathogenesis of pulmonary hypertension at the beginning of chronic hypoxia is produced mostly by iNOS. Treatment of hypoxic rats with a relatively selective iNOS inhibitor, L-N6-(1-iminoethyl)lysine (L-NIL) (21, 40), was used to test this hypothesis. In addition, iNOS expression was evaluated with immunohistochemistry at the initial and advanced stages of hypoxia (days 4 and 20, respectively). The results were reported in a preliminary form (6, 21).

Address for reprint requests and other correspondence: Va´clav Hampl, Dept. of Physiology, Charles Univ. Second Medical School, Plzenˇska 130/221, 150 00 Prague 5, Czech Republic (e-mail: [email protected]).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

pulmonary circulation; nitric oxide; rat; inducible nitric oxide synthase

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Hampl, Va´clav, Jana Bı´bova´, Alena Banˇasova´, Jirˇ´ı Uhlı´k, Dana Mikova´, Olga Hnilicˇkova´, Veˇra Lachmanova´, and Jan Herget. Pulmonary vascular iNOS induction participates in the onset of chronic hypoxic pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol 290: L11–L20, 2006. First published August 19, 2005; doi:10.1152/ajplung.00023.2005.—Pathogenesis of hypoxic pulmonary hypertension is initiated by oxidative injury to the pulmonary vascular wall. Because nitric oxide (NO) can contribute to oxidative stress and because the inducible isoform of NO synthase (iNOS) is often upregulated in association with tissue injury, we hypothesized that iNOS-derived NO participates in the pulmonary vascular wall injury at the onset of hypoxic pulmonary hypertension. An effective and selective dose of an iNOS inhibitor, L-N6-(1-iminoethyl)lysine (L-NIL), for chronic peroral treatment was first determined (8 mg/l in drinking water) by measuring exhaled NO concentration and systemic arterial pressure after LPS injection under ketamine⫹xylazine anesthesia. A separate batch of rats was then exposed to hypoxia (10% O2) and given L-NIL or a nonselective inhibitor of all NO synthases, NG-nitro-L-arginine methyl ester (L-NAME, 500 mg/l), in drinking water. Both inhibitors, applied just before and during 1-wk hypoxia, equally reduced pulmonary arterial pressure (PAP) measured under ketamine⫹xylazine anesthesia. If hypoxia continued for 2 more wk after L-NIL treatment was discontinued, PAP was still lower than in untreated hypoxic controls. Immunostaining of lung vessels showed negligible iNOS presence in control rats, striking iNOS expression after 4 days of hypoxia, and return of iNOS immunostaining toward normally low levels after 20 days of hypoxia. Lung NO production, measured as NO concentration in exhaled air, was markedly elevated as early as on the first day of hypoxia. We conclude that transient iNOS induction in the pulmonary vascular wall at the beginning of chronic hypoxia participates in the pathogenesis of pulmonary hypertension.

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METHODS

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Fig. 1. Schematic of the timing of chronic nitric oxide synthase (NOS) blockade in relation to hypoxic exposure. In one set of experiments, rats received NOS blockers [NG-nitro-L-arginine methyl ester (L-NAME) or L-N6(1-iminoethyl)lysine (L-NIL)] just before and during a 1-wk hypoxic exposure (top pair of bars). In another set, the treatment with NOS blockers was the same, but hypoxia continued after cessation of NOS blockade for 2 more wk (middle pair of bars). In the 3rd set, the hypoxic exposure was the same as in the middle set (3 wk), but NOS was blocked only during the last 10 days (bottom pair of bars). Note that the duration of the treatment with the NOS inhibitors was always the same. See METHODS for detailed description. CH, chronic hypoxia.

cardiac index (CI). The values obtained with this method are known to be lower than those in vivo due to the anesthesia and especially the thoracotomy; however, this error systematically affects all experimental groups, so that meaningful intergroup comparisons are possible. Finally, the heart was removed, and right ventricle-to-left ventricle plus septum wet weight ratio (RV/LV⫹S) was used as an index of right ventricular hypertrophy associated with pulmonary hypertension (18). NOS inhibition during the first week of a 3-wk hypoxia. To test whether the effects of NOS inhibition at the beginning of hypoxia are overcome by continued, prolonged hypoxia after discontinuation of the NOS blockade, an experiment was performed in which three groups of rats were exposed to 3-wk hypoxia (10% O2) and compared with a normoxic control group (n ⫽ 6). Of the groups exposed to hypoxia, two received NOS blocker in drinking water (L-NAME 500 mg/l, n ⫽ 7; or L-NIL 8 mg/l, n ⫽ 8) for the last 3 days before the hypoxic exposure and during the first week of hypoxia (Fig. 1). The third hypoxic group received no treatment (n ⫽ 8). We did not observe any differences among the groups in the amount of water consumed. After 3 wk of hypoxia, exhaled NO, SAP, PAP, CI, and RV/LV⫹S were measured as described above. NOS inhibition during the last 10 days of a 3-wk hypoxia. This experiment was performed to see whether chronic NOS inhibition starting after pulmonary hypertension has already developed would have effects similar to those seen with NOS inhibition during the first week of hypoxia. Rats were exposed to hypoxia for 3 wk as described above. For the last 10 days of hypoxia, the animals received either L-NIL (8 mg/l, n ⫽ 8) or L-NAME (500 mg/l, n ⫽ 10) in their drinking water (Fig. 1). Eight rats exposed to hypoxia received no treatment to serve as controls. Measurements were the same as in the 1-wk hypoxia study (exhaled NO, SAP, PAP, CI, RV/LV⫹S). iNOS immunohistochemistry. Rats exposed to hypoxia (10% O2) for 4 days (n ⫽ 10) or 20 days (n ⫽ 8) were compared with nine normoxic controls. After killing the rats by cutting their cervical vertebral column in deep chloral hydrate anesthesia (300 mg/kg body wt ip; Tamda, Olomouc, Czech Republic), we fixed the whole left lung with Baker’s fluid and embedded it in paraffin. Slides (4 – 6 ␮m 290 • JANUARY 2006 •

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The project was performed on adult male Wistar specific pathogenfree rats (Anlab, Prague, Czech Republic). The experiments conformed to the European Community and National Institutes of Health guidelines for using experimental animals and were approved by the Animal Research Committee of the Charles University Second Medical School. Unless stated otherwise, all drugs were from Sigma (Prague, Czech Republic). Dose of L-NIL for long-term, peroral use in rats. L-NIL has been reported to relatively selectively inhibit iNOS, but not other NOS isoforms, in in vitro experiments and when administered acutely to anesthetized animals (40). However, no data were available regarding effective and selective dose for a prolonged, peroral administration. To find it, we gave one group of rats L-NIL in drinking water at a dose of 3 mg/l (n ⫽ 7); another group received 8 mg/l (n ⫽ 7). The third group received plain water to serve as controls (n ⫽ 8). As a comparison with a well-described, nonselective NOS blocker, the fourth group received L-NAME (500 mg/l, n ⫽ 7). Because inhibition of eNOS is known to increase systemic arterial pressure (SAP) (47), we measured systolic arterial pressure noninvasively with a tail cuff (25) after 3 days of treatment to make sure that L-NIL at the selected doses did not inhibit eNOS. Subsequently, the rats were anesthetized with ketamine (100 mg/kg body wt ip) and xylazine (16 mg/kg im) and intubated via tracheotomy. Exhaled gas was collected via a two-way valve into a small bag (made from a condom). The groups did not differ in the time needed to fill the bag (⬃2 min). NO concentration in the collected gas was analyzed with a chemiluminescence analyzer (CLD 77 AM; EcoPhysics, Duernten, Switzerland). After the first NO measurement, the rats were injected with LPS (5 mg/kg body wt) into the jugular vein to induce iNOS expression. Exhaled gas NO analysis was then repeated every 60 min. Inhibition of NOS during 1-wk hypoxia. To test the hypothesis that NO contributes to the pathogenesis of the initial phase of pulmonary hypertension, 24 rats were exposed to 1 wk hypoxia (10% O2) in a normobaric hypoxic chamber (22). During the exposure, their drinking water was supplemented either with a nonselective inhibitor of all NOS isoforms, L-NAME (500 mg/l, n ⫽ 8), or with a selective iNOS inhibitor, L-NIL (8 mg/l, n ⫽ 9). The treatment with NOS blockers started 3 days before the hypoxic exposure to make sure that effective NOS inhibition had been achieved by the time hypoxia begun (Fig. 1). The doses were based on literature data (L-NAME) (45, 54) and our experiment described above (L-NIL). The groups treated with NOS inhibitors were compared with untreated hypoxic (10% O2, 1 wk; n ⫽ 7) and normoxic (n ⫽ 8) controls. In all experiments, the hypoxic chamber was opened for up to 30 min every 2–3 days for cleaning and feeding. Immediately after termination of the hypoxic exposure, exhaled NO was measured to confirm the effectiveness of NOS inhibition. Each rat was sealed individually in a glass jar (2.1 l) flushed with NO-free air. After 15 min, NO accumulated in the jar was measured with chemiluminescence NO analyzer. After completing the exhaled NO measurement, we anesthetized the rats (ketamine ⫹ xylazine as above). Their carotid artery was cannulated to measure mean SAP. Under oscilloscopic guidance, the pulmonary artery was then catheterized with a thermoplastically shaped polyethylene catheter (1.1 mm outer and 0.75 inner diameter) via a right jugular vein introducer, as previously described (22, 26). After we obtained a stable reading of mean pulmonary arterial pressure (PAP), the rats were intubated via tracheotomy and ventilated with room air at ⬃60 breaths/min (peak inspiratory pressure 10 cmH2O, peak expiratory pressure 0 cmH2O). The chest was opened in midline, and as much as possible extra care was taken to avoid the rats’ bleeding. Ascending aorta blood flow was measured with an ultrasonic flow meter (2.5 mm SS-series with J reflector ⫹ T106 flow meter; Transonic Systems, Ithaca, NY) as an estimate of cardiac output (20, 42). This value relative to body weight is reported as

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measure 3-nitrotyrosine concentration in soluble extract of lyophilized lung tissue of control and 4-day hypoxic rats. Nitrotyrosine is considered a suitable marker of peroxynitrite production, which originates from a very rapid reaction between NO and superoxide (5). The details of lung tissue preparation and ELISA determination, using our own mouse monoclonal antibody against nitrotyrosine and commercial anti-mouse Ig rabbit antibody conjugated with peroxidase, are described elsewhere (17). Nitrotyrosine concentration was expressed per gram of extracted protein, determined by the bicinchoninic acid method (50). Statistical analysis. Data were analyzed statistically using StatView 5.0.1 software (SAS Institute, Cary, NC) and presented as means ⫾ SE. Unpaired t-test or one-factor ANOVA followed with Fisher’s protected least-significant difference post hoc test were used for group comparisons as appropriate. P ⬍ 0.05 was considered statistically significant. Because not all measurements were successful in all animals (e.g., bleeding prevented CI measurement in some rats), the actual n for each value is given in the figures and tables. RESULTS

Dose of L-NIL for peroral administration. Before the LPS injection, the concentration of NO in the collected exhalate was close to the detection limit of the chemiluminescence NO analyzer (⬃1 ppb), and no differences among the groups could be discerned (Fig. 2). Exhaled NO did not change 1 h after LPS injection, but it was elevated by 2 h after LPS. In rats that were not treated with any NOS blocker, the exhaled NO continued to rise for the remainder of the experiment (4 h post-LPS) (Fig. 2A). The increase in exhaled NO was blunted by ⬃50% by 3 mg/l L-NIL. The higher L-NIL dose (8 mg/l) did not have any further inhibitory effect compared with 3 mg/l L-NIL, implying that maximally effective dose was reached (Fig. 2A). L-NAME reduced exhaled NO even more than L-NIL (Fig. 2A). Systolic arterial pressure was significantly elevated by L-NAME treatment (confirming eNOS inhibition), but not by either L-NIL dose (Fig. 2B). This suggests that the maximally effective L-NIL dose against iNOS (8 mg/l) did not cause any measurable nonselective inhibition of eNOS. Because IC50 of L-NIL for the third NOS isoform, nNOS (NOS I), is almost 30⫻ higher than that for iNOS (40), we surmised that the dose of L-NIL used in our experiments had no effect on nNOS.

Fig. 2. Selective prolonged inducible NOS (iNOS) inhibition with oral L-NIL. Data are means ⫾ SE; ns for both panels are in the parentheses in B. A: in control rats (not treated with NOS inhibitor), LPS injection elicited a marked, time-dependent rise in NO concentration in exhaled breath. This increase was substantially smaller in rats given 3 mg/l L-NIL in drinking water for 3 days before LPS. A higher L-NIL dose (8 mg/l) did not have any significant additional inhibitory effect, suggesting that a maximally effective dose had been reached. Exhaled NO concentration was reduced the most in rats treated with a nonselective NOS inhibitor, L-NAME (500 mg/l). Note that exhaled NO did not differ between rats treated with L-NAME and the higher L-NIL dose (8 mg/l). B: systolic systemic arterial blood pressure (SAP) measured in awake rats is elevated by the nonselective NOS inhibitor, L-NAME, but not by either of the tested dosed of L-NIL, suggesting that L-NIL at these doses does not inhibit endothelial NOS (eNOS). *P ⬍ 0.05 value differs from the corresponding value in the control group. AJP-Lung Cell Mol Physiol • VOL

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thick) were stained with hematoxylin and eosin, cresyl fast blue, aldehyde fuchsine, Gomori silver stain, and toluidine blue. After microwave oven antigen retrieval (52), endogenous alkaline phosphatase was inhibited by levamizole, and nonspecific binding was blocked by 10% BSA in Tris-buffered saline. The sections were then incubated for 24 h with monoclonal anti-iNOS antibody (1:25) at room temperature in a humid chamber. Rabbit anti-mouse polyclonal antibody labeled with alkaline phosphatase (1:50) was used as a secondary antibody for 30 min and then visualized with Fast Red TR/Naphthol AS-MX (Sigma FAST tablets). The slides were counterstained with hematoxylin and mounted in gelatin. The primary antibody was omitted in control measurements. Using an imageanalyzing software LUCIA General (Laboratory Imaging, Prague, Czech Republic), an observer (J. Uhlı´k) blinded to the group assignment of the specimens determined separately the percentage of vessels with positive immunostaining for small prealveolar arteries (20 –50 ␮m in diameter), small muscular arteries (50 –100 ␮m), and larger arteries (⬎100 ␮m). The slide areas (23– 81 mm2) and the total numbers of vessels per slide (141– 498) did not differ significantly among the groups. Exhaled NO in chronic hypoxia. Evidence is available for increased pulmonary NO production in chronic hypoxia (reviewed in Ref. 23). However, it is unknown whether this increase occurs soon enough to be able to contribute to the radical injury at the beginning of the pulmonary vascular remodeling. To find out, we measured exhaled NO in intact, conscious rats as described in Inhibition of NOS during 1-wk hypoxia (15-min collection in 2.1-l jar). The measurements were performed once every 1–2 days during a 26-day exposure to hypoxia (10% O2) and in subsequent 4 days recovery in room air. To distinguish the contribution of upper airways and lung tissue to the exhaled NO, a supplementary experiment was performed in which normoxic control rats and 4-day hypoxic rats were anesthetized (thiopental 40 mg/kg body wt ip) and intubated via a tracheotomy. NO in their exhaled breath was first measured after placing them for 10 min into the 2.1-l jar, as described above. Because NO production in the nose and paranasal sinuses is known to be very large (14, 39), we expected that NO would diffuse into the jar even in the absence of nasal ventilation. To measure NO production into the exhaled air from the lung tissue and lower airways, the exhalate for NO measurement was then collected directly from the tracheal tube as in the experiments with LPS (see Dose of L-NIL for long-term, peroral use in rats). Nitrotyrosine in lung tissue. To support our hypothesis that increased lung NO production at the beginning of hypoxic exposure contributes to oxidative tissue injury, we used competitive ELISA to

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Table 1. Body weight, cardiac index, right and left ventricle plus septum wet weight, and their ratio (RV/LV⫹S) in normoxic and 1-wk hypoxic rats Group

BW, g

CI, ml䡠min⫺1䡠kg⫺1

RV, mg

LV⫹S, mg

RV/LV⫹S

N H 1 wk H 1 wk ⫹ L-NAME H 1 wk ⫹ L-NIL

297⫾6*(8) 227⫾5 (7) 223⫾3 (8) 209⫾4† (9)

101⫾7 (6) 107⫾6 (5) 74⫾3*(7) 99⫾15 (3)

182⫾10 153⫾7‡ 149⫾9‡ 160⫾4

604⫾19* 440⫾8 460⫾7§ 420⫾14

0.30⫾0.02 (8) 0.35⫾0.02 (7) 0.32⫾0.02§ (8) 0.38⫾0.02‡ (9)

Data are means ⫾ SE, ns are in the parentheses. BW, body wt; CI, cardiac index; RV, right ventricle; LV ⫹ S, left ventricle plus septum; N, normoxia; H, hypoxia; H 1 wk ⫹ L-NAME, rats treated with NG-nitro-L-arginone methyl ester (500 mg/l) for 3 days before and during a 1-wk exposure to hypoxia; H 1 wk ⫹ L-NIL, rats treated with L-N6-(1-iminoethyl) lysine (8 mg/l) for 3 days before and during a 1-wk exposure to hypoxia. *P ⬍ 0.05 group differs from all other groups, †P ⬍ 0.05 group differs from other hypoxic groups, ‡P ⬍ 0.05 group differs from normoxic group, §P ⬍ 0.05 group differs from L-NIL-treated group.

than in the L-NIL-treated hypoxic rats, leading to higher RV/ LV⫹S in the latter group (Table 1). Reduction of pulmonary hypertension by NOS inhibition during the first week of a 3-wk hypoxia. Rats exposed to hypoxia for 3 wk had lower body weight than corresponding controls living in room air (Table 2). At the end of the exposure, NO concentration in the exhaled air was similarly elevated in all hypoxic groups (Fig. 4A). Chronic hypoxic pulmonary hypertension was evident from markedly elevated PAP at the end of 3-wk hypoxic exposure compared with normoxic controls (Fig. 4C). As expected, PAP was higher after 3 wk than after 1 wk of hypoxia. At the end of the 3-wk hypoxic exposure, PAP was higher in untreated, hypoxic controls than in rats treated during the first week of exposure with L-NIL, despite the 2-wk lag from the end of the L-NIL treatment (Fig. 4C). Although the PAP was numerically similar in the L-NAME group to that in the L-NIL group, the difference between the former and the untreated hypoxic controls did not reach statistical significance, perhaps due to the lower number of successful PAP measurements in the L-NAME group. If both NOS blockade groups were pooled, their PAP was significantly lower than that of the untreated hypoxic group (23.1 ⫾ 1.5 vs. 28.1 ⫾ 1.7 mmHg, P ⫽ 0.026). The groups did not differ in SAP and CI (Fig. 4B and Table 2). Compared with normoxic controls, all hypoxic groups had similarly increased RV/LV⫹S (Table 2).

Fig. 3. NOS inhibition during a 1-wk hypoxia reduces pulmonary hypertension. Data are means ⫾ SE, ns are in the parentheses. A: elevation of NO concentration in the exhaled air, induced by 1-wk hypoxia, is blocked by both L-NAME and L-NIL treatment, proving their effectiveness. B: mean SAP is elevated by L-NAME, but not L-NIL, treatment, excluding eNOS inhibition by L-NIL. C: increase in mean pulmonary arterial pressure (PAP), induced by 1-wk hypoxia, is blunted by concomitant NOS inhibition. N, rats kept in normoxia; H 1 wk, rats exposed to hypoxia for 1 wk; H 1 wk⫹L-NAME, rats treated with L-NAME (500 mg/l) for 3 days before and during a 1-wk exposure to hypoxia; H 1 wk⫹L-NIL, rats treated with L-NIL (8 mg/l) for 3 days before and during a 1-wk exposure to hypoxia. *P ⬍ 0.05 vs. N, ⫹P ⬍ 0.05 vs. H 1 wk, #P ⬍ 0.05 vs. all other groups. AJP-Lung Cell Mol Physiol • VOL

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Inhibition of NOS during 1-wk hypoxia reduces pulmonary hypertension. As expected, all rats exposed to 1-wk hypoxia had lower body weight than normoxic controls. In addition, the rats treated in hypoxia with L-NIL were slightly but significantly lighter than the remaining hypoxic groups (Table 1). After a 15-min accumulation of exhaled NO, its concentration in a 2.1-l jar was significantly higher in 1-wk hypoxic rats compared with normoxic controls. This hypoxia-induced elevation of exhaled NO was prevented by each of the NOS inhibitors (Fig. 3A). Selectivity of L-NIL was indirectly confirmed by the fact that it did not elevate SAP, unlike the nonselective NOS inhibitor L-NAME (Fig. 3B). One week of hypoxia was sufficient to significantly elevate PAP compared with normoxic controls (Fig. 3C). In rats treated with the NOS inhibitors during hypoxia, PAP was slightly but significantly lower than in hypoxic controls, albeit still higher than in normoxic controls (Fig. 3C). PAP did not differ between the L-NAME- and L-NIL-treated hypoxic groups. Treatment with L-NAME caused a significant CI reduction (Table 1), as reported previously (20). This was not seen with L-NIL; however, the number of successful CI measurements in this group was low. One week of hypoxia was not sufficient to produce statistically significant right ventricular hypertrophy, and there were no differences among the hypoxic groups in the weight of the right ventricle (Table 1). Probably because of the L-NAME-induced systemic hypertension, the left ventricle plus septum weight was higher in the L-NAME-

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Table 2. BW, CI, RV, and LV⫹S wet weight and their ratio (RV/LV⫹S) in normoxic rats and rats treated with NOS inhibitors during the 1st week of a 3-week hypoxia Group

BW, g

CI ml䡠min⫺1䡠kg⫺1

RV, mg

LV⫹S, mg

RV/LV⫹S

N H 3 wk H 3 wk ⫹ L-NAME 1st wk H 3 wk ⫹ L-NIL 1st wk

385⫾15*(6) 253⫾8 (8) 265⫾5 (7) 249⫾3 (8)

127⫾15 (6) 104⫾5 (7) 127⫾12 (4) 109⫾13 (8)

183⫾7* 285⫾15 275⫾12 238⫾15†

703⫾21* 620⫾23 627⫾17 525⫾23†‡

0.26⫾0.01*(6) 0.46⫾0.02 (8) 0.44⫾0.03 (7) 0.45⫾0.03 (8)

Data are means ⫾ SE, ns are in the parentheses. NOS, nitric oxide synthase; H 3 wk ⫹ L-NAME 1st wk, rats treated with L-NAME (500 mg/l) for 3 days before and during the 1st week of a 3-wk hypoxic exposure; H 3 wk ⫹ L-NIL 1st wk, rats treated with L-NIL (8 mg/l) for 3 days before and during the 1st week of a 3-wk hypoxic exposure. *P ⬍ 0.01 normoxic group differs from all hypoxic groups, †P ⬍ 0.05 L-NIL-treated group differs from the untreated hypoxic group, ‡P ⬍ 0.05 L-NIL-treated group differs from the L-NAME-treated group.

and 7). The staining was localized predominantly to the smooth muscle layer, although, to a lesser degree, it was detected also in the intima and adventitia. In addition to the vessels, iNOS immunostaining was positive also in the pleura, airway walls, and focally in the interalveolar septa. The positive staining in the pleura and airways was also evident in the lungs of rats kept in hypoxia for 3 wk. However, the iNOS immunostaining in the walls of pulmonary vessels of all sizes was no longer apparent in these animals and did not differ from normoxic controls (Figs. 6C and 7). We observed an increase in the total number of large pulmonary vessels per mm2 in the 3-wk hypoxic group (0.70 ⫾ 0.05) compared with both the 4-day hypoxic (0.45 ⫾ 0.06) and control rats (0.42 ⫾ 0.07). There were no statistically significant differences in the numbers of other vessels among the groups. Exhaled NO increases early in chronic hypoxia. Concentration of NO in exhaled air was low when the animals lived in normoxia but increased markedly on the first day of hypoxic exposure. It continued to rise until day 5, then leveled off, and, despite daily and individual variations, remained essentially stable for the rest of the hypoxic exposure (Fig. 8A). The NO levels started to drop on the first day of room air recovery and approached the normally low values by day 4 of recovery (Fig. 8A). In the supplementary experiment, exhaled NO measured directly from the tracheal tube doubled after 4-day hypoxia compared with the normoxic group (Fig. 8B). The difference between the tracheal tube collection and jar accumulation,

Fig. 4. NOS inhibition during the 1st wk of a 3-wk hypoxia reduces pulmonary hypertension. Data are means ⫾ SE, ns are in the parentheses. A: 2 wk after cessation of NOS blockade there are no significant differences in exhaled NO levels among hypoxic animals. The dotted horizontal line indicates the level in normoxic controls from Fig. 3A. B: 2 wk after cessation of NOS blockade there are no significant differences in SAP. C: increase in PAP, induced by 3-wk hypoxia, is smaller in rats treated with L-NIL at the beginning of the exposure. H 3 wk, rats exposed to hypoxia for 3 wk; H 3 wk⫹L-NAME 1st wk, rats exposed to hypoxia for 3 wk and treated with L-NAME (500 mg/l) for 3 days before and during the 1st week of the exposure; H 3 wk⫹L-NIL 1st wk, rats exposed to hypoxia for 3 wk and treated with L-NIL (8 mg/l) for 3 days before and during the 1st week of the exposure. *P ⬍ 0.05 vs. N, ⫹P ⬍ 0.05 vs. H 3 wk. AJP-Lung Cell Mol Physiol • VOL

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NOS inhibition during the third week of hypoxia has little effect on pulmonary hypertension. Just before the initiation of the NOS blocker treatment, the exhaled NO levels were similarly elevated in all hypoxic groups. At the end of the NOS blockade during the third week of hypoxia, the exhaled NO was markedly reduced by both L-NAME and L-NIL (Fig. 5A). As expected, SAP was elevated by 1-wk L-NAME administered just before the measurement, whereas L-NIL had no effect on SAP (Fig. 5B). The groups treated with NOS blockers in the last week of hypoxia did not differ significantly in PAP from the untreated hypoxic controls, although there was a tendency for increase in the group given L-NAME and for reduction in the L-NIL group (Fig. 5C). There were no differences among the groups in CI (Table 3). Surprisingly, both the right ventricle weight and RV/L⫹S were lower in the group given L-NAME during the last week of hypoxia than in the remaining hypoxic groups (Table 3). We do not have any explanation for this perplexing observation, except perhaps some nonspecific effect of L-NAME (2, 3, 8, 46, 53). Left ventricular weights did not differ between these groups. L-NIL had no effect on the ventricular weights (Table 3). Hypoxia transiently elevates iNOS expression in lung vessels. Expression of iNOS was not detected in lung sections from control rats living in normoxia (Fig. 6A). By contrast, almost all pulmonary arteries of all sizes showed strong positive iNOS immunostaining after 4 days of hypoxia (Figs. 6B

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reflecting the contribution of the upper airways, did not differ between the hypoxic and normoxic groups (Fig. 8B), indicating that the source of the elevated NO exhalation in hypoxia is the lower respiratory tract. Although the contribution of the upper airways remains remarkable, it does not rise in hypoxia. Nitrotyrosine concentration in the lungs is elevated after 4 days of hypoxia. The concentration of nitrotyrosine was higher (P ⫽ 0.023) in extracts from lung tissue of rats exposed to hypoxia for 4 days (1,065 ⫾ 96 nmol/g of extracted protein; n ⫽ 6) than in extracts from control lungs (791 ⫾ 57 nmol/g; n ⫽ 8). This implies greater exposure to peroxynitrite, the product of reaction of NO with superoxide (5). Increased nitrotyrosine levels in lung tissue after 4 days of hypoxia was also confirmed by immunoblot analysis (data not shown).

end of a 3-wk hypoxia. On the other hand, 10 days’ treatment with NOS inhibitors, started after the hypoxic pulmonary hypertension had already developed, did not have significant effect on PAP. The role of iNOS in the first days of hypoxia is further supported by the finding of markedly and transiently elevated expression of iNOS in lung vessels on the fourth day of hypoxia. Our exhaled NO data document the elevated lung NO production at the very onset of hypoxia. NO is best known for its marked vasodilator activity. As such, exogenous NO is useful in clinical management of acute pulmonary hypertensive crises (for review, see e.g., Ref. 19). However, NO is also a reactive radical capable of tissue injury either directly or after reacting with other radicals. For example, an extremely fast reaction of NO with superoxide yields a highly cytotoxic peroxynitrite (5). There is evidence that oxidative injury of the pulmonary vascular wall at the beginning of hypoxia initiates the process of vascular remodeling that eventually results in pulmonary hypertension (28, 35). Our present data show that endogenous NO affects the initial development of chronic hypoxic pulmonary hypertension. The vasodilator and antiproliferative properties of NO do not prevail at this initial phase because otherwise the NOS inhibitors would have to aggravate, rather than reduce, pulmonary hypertension. The conclusion that increased NO production by iNOS at the initial stage of hypoxia promotes the development of pulmonary hypertension by contributing to oxidative vascular wall injury is supported by our observation of increased

DISCUSSION

This study shows that increased NO production in (or near) the pulmonary vascular wall during the first days of hypoxia, mostly by the iNOS isoform, contributes to the initiation of a process that eventually results in pulmonary hypertension. Of our results, especially the following support this conclusion: the PAP rise in the initial days of hypoxic exposure was partly inhibited by concomitant NOS inhibition. In this regard, selective iNOS inhibitor was as effective as the nonselective blocker of all NOS isoforms. If iNOS blockade was then discontinued and the hypoxic exposure continued, the pulmonary hypertension did not completely “catch up”; it was still reduced at the

Table 3. BW, CI, RV and LV⫹S wet weight and their ratio (RV/LV⫹S) in rats treated with NOS inhibitors during the last week of a 3-wk hypoxia Group

BW, g

CI, ml䡠min⫺1䡠kg⫺1

RV, mg

LV⫹S, mg

RV/LV⫹S

H 3 wk H 3 wk ⫹ L-NAME 3rd wk H 3 wk ⫹ L-NIL 3rd wk

253⫾5 (8) 265⫾4*(10) 247⫾6 (8)

81⫾6 (7) 97⫾9 (5) 97⫾15 (6)

269⫾18 214⫾9*† 275⫾14

531⫾18 551⫾22 509⫾8

0.51⫾0.02 (8) 0.39⫾0.02*† (10) 0.54⫾0.02 (8)

Data are means ⫾ SE, ns are in the parentheses. H 3 wk ⫹ L-NAME 3rd wk, rats treated with L-NAME (500 mg/l) during the last week of a 3-wk hypoxic exposure; H 3 wk ⫹ L-NIL 3rd wk, rats treated with L-NIL (8 mg/l) during the last week of a 3-wk hypoxic exposure. *P ⬍ 0.05 group treated with L-NAME differs form L-NIL-treated group, †P ⬍ 0.05 group treated with L-NAME differs from untreated hypoxic group. AJP-Lung Cell Mol Physiol • VOL

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Fig. 5. NOS inhibition during the last 10 days of a 3-wk hypoxia. Data are means ⫾ SE, ns are in the parentheses. A: in 3 groups exposed to hypoxia, NO concentration in exhaled air was similar before the initiation of NOS blockade after 2 wk of hypoxia and decreased significantly after 10 days of L-NIL or L-NAME treatment. The dotted horizontal line indicates the level in normoxic controls from Fig. 3A. B: SAP in rats kept in hypoxia for 3 wk was increased by L-NAME treatment during the last 10 days before the measurement. L-NIL had no effect. C: PAP, elevated by 3 wk of hypoxia, was not significantly reduced by NOS inhibition during the last 10 days of the exposure. H 3 wk⫹L-NAME 3rd wk, rats exposed to 3-wk hypoxia and treated with L-NAME (500 mg/l) during the last 10 days; H 3 wk⫹L-NIL 3rd wk, rats exposed to 3-wk hypoxia and treated with L-NIL (8 mg/l) during the last 10 days. *P ⬍ 0.0005 vs. pre NOS block, **P ⬍ 0.05 vs. pre NOS block, ⫹P ⬍ 0.05 vs. all other groups, #P ⬍ 0.05 vs. H 3 wk⫹L-NIL 3rd wk.

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lung tissue levels of peroxynitrite marker, nitrotyrosine, after 4 days of hypoxia. Further support is provided by our previous finding that serum levels of nitrotyrosine are elevated after 4 days of hypoxia but normalized at the end of a 3-wk exposure (27). Consistently elevated lung nitrotyrosine was reported in human patients with pulmonary hypertension (7). Because our conclusions rely to a large extent on the L-NIL selectivity for iNOS, it was necessary first to determine an effective and selective L-NIL dose for chronic peroral administration. The effectiveness of various L-NIL doses in inhibiting NO production was assessed by measurements of exhaled NO levels after a challenge with a well-known iNOS inducer, LPS (41), whereas selectivity toward iNOS rather than eNOS was inferred from the absence of an effect on SAP. L-NIL at 8 mg/l in drinking water markedly reduced exhaled NO levels after LPS, albeit not as much as the nonselective NOS blocker, L-NAME, at 500 mg/l. This is consistent with reports that, in addition to iNOS induction, LPS treatment can increase expression of the remaining NOS isoforms (12, 24, 30, 32). Somewhat surprisingly, even the high L-NAME dose used did

Fig. 7. Transient elevation of iNOS immunostaining in pulmonary vessels at the beginning of CH. Percentage (means ⫾ SE) of prealveolar, small muscular, and large pulmonary vessels showing iNOS immunostaining in normoxia, after 4 days of hypoxia, and after 3 wk of hypoxia. d, Days. *P ⬍ 0.0001 vs. the other groups. AJP-Lung Cell Mol Physiol • VOL

not prevent the rise in exhaled NO after LPS completely. It is possible that some of the NO is formed in compartments not well accessible from the circulation. For example, macrophages could encounter LPS in the bloodstream, begin the process of iNOS induction, and then leave the vessels toward alveolar spaces, where the penetration of L-NAME may be limited. The reduction in PAP by NOS inhibition during the first week of hypoxia was not accompanied by a corresponding mitigation of right ventricular hypertrophy. Enlargement of the right ventricle in chronic hypoxia is a recognized consequence of PAP elevation. However, several studies suggest that right ventricular weight and PAP must not always be closely tied. For example, various treatments of experimental pulmonary hypertension reduced right ventricular hypertrophy less (and at higher doses) than PAP (56, 57). Chronic pulmonary hypertension can be elicited experimentally without an accompanying right ventricular hypertrophy (10). Right ventricular weight was reported to rise in proportion to PAP elevation in rats exposed to chronic intermittent hypoxia from fourth day of age, but no significant correlation could be detected in older animals (34). Thus it is quite likely that chronic hypoxia has other effects on the ventricular size in addition to those mediated by increased PAP. This possibility is supported by observations that left ventricle can also be somewhat enlarged (relative to body weight) in chronic hypoxia (34, 43). Because NO has antiproliferative effects, it is also possible that NOS inhibition causes some degree of heart enlargement that may offset the reducing effect of the lessened afterload. This possibility is strongly supported by our earlier observation that chronic L-NAME treatment in normoxia enlarges both ventricles (20). The effects of NOS inhibition on chronic hypoxic pulmonary hypertension have been studied previously, but not specifically at the initial stage of the exposure. Numerous studies demonstrated that acutely applied, nonselective NOS inhibitors cause considerably greater vasoconstriction in lungs of chron290 • JANUARY 2006 •

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Fig. 6. Transient elevation of iNOS immunostaining in pulmonary vessels at the beginning of CH. A: small prealveolar arteriole from a normoxic rat showing relatively thin wall, single elastic lamina and negative iNOS immunostaining. B: small prealveolar arteriole from a rat exposed to hypoxia for 4 days demonstrating distinct wall thickening and strongly positive iNOS immunostaining. C: small prealveolar arteriole from a rat exposed to hypoxia for 20 days showing wall thickening, double elastic laminae, and negative iNOS immunostaining. D: small prealveolar arteriole from a rat exposed to hypoxia for 4 days with primary antibody omitted (negative control). Original magnification was ⫻40 in all panels, and the scale bars represent 50 ␮m.

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Fig. 8. Concentration of NO in exhaled breath increases at the very beginning of CH exposure. A: accumulation of breath NO during a 15-min stay of awake rats in a closed jar (n ⫽ 8 in the 1st wk, 5 thereafter) is negligible before exposure to CH but is significantly increased on the 1st day of exposure, remains elevated throughout a 3-wk hypoxia, and drops toward minimal, control level within days of normoxic recovery. All data points in hypoxia differ significantly from day 0. B: total exhaled NO, measured as 10-min accumulation in a closed jar, is higher in 4-day hypoxic rats than in normoxic controls, as is the contribution from lower airways, estimated from breath collection into a small bag through tracheal tube. The difference between these 2 measurements, corresponding to the contribution from the upper airways, is large but unchanged by hypoxia. Data are means ⫾ SE. *P ⬍ 0.05 vs. normoxia.

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prolonged hypoxia (2– 4 wk) in whole lung homogenates (16, 29, 36, 44, 48). We are aware of two publications that focus on lung iNOS expression during the first week of hypoxia (51, 59); using rats, both show increased iNOS protein in whole lung homogenates. Our present data further supplement this information by showing that the iNOS induction in the first days of hypoxia is localized to a major extent in the pulmonary vessels, especially their media (Fig. 6). Furthermore, our data indicate that with prolonged hypoxia, iNOS remains expressed in airway epithelia, but its expression in the pulmonary vascular wall returns to the barely detectable baseline level. Thus it appears that the chronic hypoxia-induced elevation of whole lung iNOS protein in some (29, 36) but not all (16) studies can be attributed to the extravascular iNOS expression. Together, our data are consistent with the reports of others and with the hypothesis that the pulmonary vascular wall iNOS expression rises during the first few days of hypoxia and returns towards baseline thereafter. Enhanced iNOS expression not accompanied by NO overproduction has been occasionally reported (9, 13, 37). Thus to support our hypothesis of a causative role of elevated NO in the initiation of hypoxic pulmonary hypertension, it was necessary not only to show early iNOS induction in lung vessels (Figs. 6 and 7), but also to prove that NO synthesis actually rises rapidly after the onset of hypoxia. Elevated pulmonary NO production in prolonged hypoxia has been reported previously (for review, see Ref. 23). High-altitude residents have higher exhaled NO compared with lowlanders (4). However, changes in NO production during the first days of hypoxia have not been studied. We show rapidly rising NO production into the exhaled air in the first days of hypoxia (Fig. 8A). Although this measurement is relatively easy to perform, its main limitation is ambiguity in respect to the source of the detected NO. It has been demonstrated that a major source of exhaled NO are the upper airways, especially the nasal and paranasal cavities (14, 39). Therefore, we performed a supplementary experiment to show that while the majority of NO in exhaled air comes from upper airways both in normoxia and hypoxia, the respiratory tract below the level of the trachea is responsible for the increase in exhaled NO seen in the first days of hypoxia (Fig. 8B). Furthermore, our data show that most of the exhaled NO increase in hypoxia is attributable to iNOS, as it was similarly and almost completely inhibited by L-NAME and L-NIL, both at the beginning of hypoxia (Fig. 3A) and after a more pro290 • JANUARY 2006 •

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ically hypoxic compared with normoxic rats, implying elevated lung NO activity in chronic hypoxia (for review, see Ref. 23). In an apparent contrast, rats given L-NAME during the entire 3-wk hypoxic exposure did not have more severe pulmonary hypertension than rats exposed to the same hypoxia, but with no inhibitor treatment (20). Our current results offer one possible interpretation of that paradox. It could be a combination of a sustained reduction of pulmonary hypertension by iNOS blockade in the first week (Fig. 3D) counteracted by augmentation of pulmonary hypertension by inhibition of elevated eNOS activity in the more advanced phase of the hypoxic exposure. There are also studies of chronic administration of selective iNOS inhibitors (L-NIL and aminoguanidine) during the entire 3- to 4-wk hypoxic exposure, showing no effect on pulmonary hypertension (48, 55). In our study, iNOS blockade during the first week of hypoxia reduced pulmonary hypertension. A possible explanation of this discrepancy could be the fact that we started the administration of NOS blockers 3 days before hypoxic exposure to make sure that their blood and tissue levels were at effective levels at the moment of the onset of hypoxia. It is thus possible that the role of iNOS in vascular wall injury promoting pulmonary hypertension is confined to the very beginning of hypoxia. In addition, ours is the only study demonstrating the selective effectiveness of the iNOS blocker at the dose and route of administration used. As far as we know, this is the first description of a chronic, peroral use of L-NIL for selective iNOS inhibition in vivo. Continuous infusion or repeated injections of L-NIL were reported previously (48, 49); however, our method of adding L-NIL to drinking water is much more practical. It has a possible disadvantage of variations in water consumption. This is especially important at the beginning of hypoxia, which is typically associated with reduced drinking. However, in our study the L-NIL intake in 1-wk hypoxia was sufficient, as evidenced by the suppression of exhaled NO to the low level similar to that in normoxic controls (Fig. 3A). Furthermore, the exhaled NO was similar after L-NIL treatment in 1-wk hypoxia and at the end of a 3-wk hypoxia, when water intake is known to be normalized (Fig. 5A). Many authors have reported elevated NOS mRNA and protein in lungs and pulmonary vessels in chronic hypoxia (for review, see Ref. 23). Most of those studies either focused on eNOS or did not discriminate between NOS isoforms. Several studies show elevated iNOS mRNA or protein at the end of a

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10.

11.

12.

13.

14. 15.

16.

17.

18. ACKNOWLEDGMENTS The authors are grateful to Dr. Ludek Cervenka for invaluable help with systolic arterial pressure measurements in awake rats, to Dr. Ludek Vajner for involvement in the histochemical evaluation, to Dr. Richard Vytasek for performing peroxynitrite ELISA, to Michal Snorek and Petr Zdaril for assistance with exhaled NO measurements in awake rats, and to Kveta Venclı´kova´ for general technical and logistical support.

19.

20.

21.

GRANTS This study was supported by the Grant Agency of the Czech Republic Grant 305/05/0672.

22.

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longed exposure (Fig. 5A). In normoxia, almost all exhaled NO is derived from eNOS (58). The measurement of NO accumulation in an airtight container is a simple method for checking NO production in an intact, awake rat. Although we use it as an indicator of lung NO production, other possible NO sources, such as colonic denitrification bacteria or acidic nitrate reduction in the stomach, should be considered. However, the hypoxia-induced elevation of exhaled NO was completely prevented by each of the NOS inhibitors, implying that the collected NO was mostly exhaled since the alternative NO sources are NOS independent (38). Although our data prove iNOS involvement in the initial phase of the development of hypoxic pulmonary hypertension, they do not show whether iNOS induction is a cause or a consequence of injury to the pulmonary vessels that underlies their remodeling. It is possible that the aggravating influence of NO on the initial phase of pulmonary hypertension can contribute to the findings that inhaled NO does not improve long-term outcome in acute respiratory distress syndrome (31) and in persistent pulmonary hypertension of the newborn (11, 33). The most clinically relevant correlate to our experimental situation are probably acute exacerbations of chronic hypoxic conditions. Our data suggest that in these situations, adding even more NO (by inhalational therapy) may have drawbacks. This issue should also be considered in attempts to treat high-altitude pulmonary edema with inhaled NO (1).

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31. Kaisers U, Busch T, Deja M, Donaubauer B, and Falke K. Selective pulmonary vasodilation in acute respiratory distress syndrome. Crit Care Med 31: S337–S342, 2003. 32. Kang WS, Tamarkin FJ, Wheeler MA, and Weiss RM. Rapid upregulation of endothelial nitric-oxide synthase in a mouse model of Escherichia coli lipopolysaccharide-induced bladder inflammation. J Pharmacol Exp Ther 310: 452– 458, 2004. 33. Kinsella JP, Walsh WF, Bose CL, Gerstmann DR, Labella JJ, Sardesai S, Walsh-Sukys MC, McCaffrey MJ, Cornfield DN, Bhutani VK, Cutter GR, Baier M, and Abman SH. Inhaled nitric oxide in premature neonates with severe hypoxaemic respiratory failure: a randomised controlled trial. Lancet 354: 1061–1065, 1999. 34. Kola´rˇ F, Osˇt’a´dal B, Procha´zka J, Pelouch V, and Widimsky´ J. Comparison of cardiopulmonary response to intermittent high-altitude hypoxia in young and adult rats. Respiration 56: 57– 62, 1989. 35. Lachmanova´ V, Hnilicˇkova´ O, Povy´sˇilova´ V, Hampl V, and Herget J. N-acetylcysteine inhibits hypoxic pulmonary hypertension most effectively in the initial phase of chronic hypoxia. Life Sci 77: 175–182, 2005. 36. Le Cras TD, Xue C, Rengasamy A, and Johns RA. Chronic hypoxia upregulates endothelial and inducible NO synthase gene and protein expression in rat lung. Am J Physiol Lung Cell Mol Physiol 270: L164 – L170, 1996. 37. Lovchik J, Lipscomb M, and Lyons C. Expression of lung inducible nitric oxide synthase protein does not correlate with nitric oxide production in vivo in a pulmonary immune response against Cryptococcus neoformans. J Immunol 158: 1772–1778, 1997. 38. Lundberg J, Weitzberg E, Lundberg J, and Alving K. Intragastric nitric oxide production in humans: measurements in expelled air. Gut 35: 1543–1546, 1994. 39. Lundberg JON, Farkas-Szallasi T, Weitzberg E, Rinder J, Lindholm ¨ nggard A, Ho¨kfelt T, Lundberg JM, and Alving K. High nitric J, A oxide production in human paranasal sinuses. Nat Med 1: 370 –373, 1995. 40. Moore WM, Webber RK, Jerome GM, Tjoeng FS, Misko TP, and Currie MG. L-N6-(1-iminoethyl)lysine: a selective inhibitor of inducible nitric oxide synthase. J Med Chem 37: 3886 –3888, 1994. 41. Nathan C and Xie QW. Regulation of biosynthesis of nitric oxide. J Biol Chem 269: 13725–13728, 1994. 42. Novotna´ J, Bı´bova´ J, Hampl V, Deyl Z, and Herget J. Hyperoxia and recovery from hypoxia alter collagen in peripheral pulmonary arteries similarly. Physiol Res 50: 153–163, 2001. 43. Osˇt’a´dal B, Mirˇejovska´ E, Hurych J, Pelouch V, and Procha´zka J. Effect of intermittent high altitude hypoxia on the synthesis of collagenous and noncollagenous proteins of the right and left ventricular myocardium. Cardiovasc Res 12: 303–308, 1978. 44. Palmer LA, Semenza GL, Stoler MH, and Johns RA. Hypoxia induces type II NOS gene expression in pulmonary artery endothelial cells via HIF-1. Am J Physiol Lung Cell Mol Physiol 274: L212–L219, 1998. 45. Pecha´nˇova´ O and Berna´tova´ I. Effect of long-term NO synthase inhibition on cyclic nucleotide content in rat tissues. Physiol Res 45: 305–309, 1996.

Int. J. Exp. Path. (2006), 87, 383–391 doi: 10.1111/j.1365-2613.2006.00493.x

ORIGINAL ARTICLE

Acute and chronic hypoxia as well as 7-day recovery from chronic hypoxia affects the distribution of pulmonary mast cells and their MMP-13 expression in rats Ludeˇk Vajner*, Richard Vyta´sˇek §, Veˇra Lachmanova´à§, Jirˇı´ Uhlı´k*, Va´clava Konra´dova´*, Jana Novotna´ §, Va´clav Hamplৠand Jan Hergetৠ*Department of Histology and Embryology,  Department of Medical Chemistry and Biochemistry, 2nd Faculty of Medicine, Charles §

à

University, Prague, Centre for Cardiovascular Research, Prague, and Department of Physiology, 2nd Faculty of Medicine, Charles University, Prague, Czech Republic

INTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY

Received for publication: 30 March 2006 Accepted for publication: 30 May 2006 Correspondence: Ludeˇk Vajner, MVDr, CSc Department of Histology and Embryology 2nd Faculty of Medicine, Charles University Plzenˇska´ 130/221 Prague 5 – Motol CZ 150 00 Czech Republic Tel.: +420 257 296 250 Fax: +420 224 435 820 E-mail: [email protected]

Summary Chronic hypoxia results in pulmonary hypertension due to vasoconstriction and structural remodelling of peripheral lung blood vessels. We hypothesize that vascular remodelling is initiated in the walls of prealveolar pulmonary arteries by collagenolytic metalloproteinases (MMP) released from activated mast cells. Distribution of mast cells and their expression of interstitial collagenase, MMP-13, in lung conduit, small muscular, and prealveolar arteries was determined quantitatively in rats exposed for 4 and 20 days to hypoxia as well as after 7-day recovery from 20-day hypoxia (10% O2). Mast cells were identified using Toluidine Blue staining, and MMP-13 expression was detected using monoclonal antibody. After 4, but not after 20 days of hypoxia, a significant increase in the number of mast cells and their MMP-13 expression was found within walls of prealveolar arteries. In rats exposed for 20 days, MMP-13 positive mast cells accumulated within the walls of conduit arteries and subpleurally. In recovered rats, MMP-13 positive mast cells gathered at the prealveolar arterial level as well as in the walls of small muscular arteries; these mast cells stayed also in the conduit part of the pulmonary vasculature. These data support the hypothesis that perivascular pulmonary mast cells contribute to the vascular remodelling in hypoxic pulmonary hypertension in rats by releasing interstitial collagenase. Keywords mast cells, matrix-metalloproteinase 13, normobaric hypoxia, pulmonary hypertension, rat

Introduction Exposure to chronic hypoxia induces hypoxic pulmonary hypertension (HPH), which results from structural remodel-

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ling of peripheral pulmonary blood vessels and lung vasoconstriction (Reeves & Herget 1984; Reid 1986). The vascular remodelling starts as early as in the first week of exposure (Rabinovitch et al. 1979) and is characterized by 383

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medial hypertrophy and proliferation of smooth muscle in peripheral lung arterioles and by fibrotization of vascular walls (Hislop & Reid 1976; Herget et al. 1978; Rabinovitch et al. 1979). The turnover of vascular collagen is increased (Bishop et al. 1990; Poiani et al. 1990). As a result of increased collagenolytic activity (Novotna´ & Herget 1998), low molecular weight fragments of collagen type I molecules accumulate in the walls of peripheral pulmonary arteries (Novotna´ & Herget 1998). Collagen breakdown is highest in the first days of exposure to hypoxia (Novotna´ & Herget 2001). Experimental inhibition of collagen metabolism reduced vascular remodelling in hypoxic rats and inhibited the development of HPH (Kerr et al. 1984, 1987; Poiani et al. 1991; Herget et al. 2003). Elastolytic activity is also elevated during the development of chronic hypoxic pulmonary hypertension, and blockade of this activity inhibits pulmonary hypertension (Rabinovitch 1999). We hypothesize that breakdown of matrix protein molecules induced by hypoxic tissue injury may be one of the triggering mechanisms of vascular remodelling in chronic hypoxia (Hampl & Herget 2000; Novotna´ & Herget 2002). Important sources of collagenolytic enzymes are mast cells. Tozzi et al. (1998) have shown that an increase in collagenolytic activity in the lung mast cells plays an important role in the normalization of lung vascular structure during recovery from exposure to chronic hypoxia. The number of perivascular lung mast cells increases in hypoxic animals (Kay et al. 1974; Mungall 1976; Tucker et al. 1977; Williams et al. 1981; Migally et al. 1983). It is increased in human high altitude residents as well (Heath 1992). In chronically hypoxic rats, inhibition of mast cell degranulation reduced the development of hypoxic pulmonary hypertension (Mungall 1976; Kay et al. 1981). We tested the hypothesis that in rats exposed to chronic hypoxia, collagenolytic metalloproteinases are released from perivascular mast cells that accumulate in the walls of peripheral pulmonary arteries.

Methods Experimental animals Fifty male Wistar rats (initial body weight 210 ± 8 g; Anlab, Prague, Czech Republic) started the experiment. Thirty animals were exposed to hypoxia (FiO2 ¼ 0.1) in a normobaric hypoxic chamber (Hampl & Herget 1990). Experimental rats were examined after 4 (n ¼ 10) and 20 (n ¼ 8) days of hypoxic exposure (body weight 198 ± 5 g and 241 ± 8 g, respectively) as well as after 7-day recovery from 20-day hypoxia (n ¼ 8; body weight 323 ± 23 g). Two groups of

control animals (n ¼ 10 and 9, body weight 245 ± 19 g and 374 ± 35 g, respectively) were housed at atmospheric air in the same room for 4 and 28 days, respectively. All groups of rats received standard rat diet and tap water ad libitum. During the night on the day 11 of hypoxic exposure, four animals died. All protocols and procedures employed in this study were reviewed and approved by the Animals Protection Expert Commission of the Faculty.

Pathologic anatomy At the end of the experiment, all animals were anaesthetized with chloral hydrate (300 mg/kg b.w., i.p.; Tamda, Olomouc, Czech Republic) and killed by cutting the cervical vertebral column. The left lungs were fixed with Baker’s fluid, longitudinally cut and embedded in paraffin. Parallel sections 4–6 lm thick were cut and a series of stainings was carried out on each specimen: haematoxylin & eosin (HE), cresyl fast blue (FB), aldehyde fuchsine (AF), and gomori silver stain. To identify the mast cells, Toluidine blue (TB) staining was used. The immunohistochemical detection of the rodent-type interstitial collagenase, matrix metalloproteinase-13 (MMP-13), on paraffin sections was performed after microwave-oven antigen retrieval (Thomas et al. 2000). Having blocked the endogenous alkaline phosphatase (AP) with levamizole and the non-specific binding with 10% bovine serum albumin (BSA) in tris-buffered saline (TBS), the sections were incubated with a monoclonal anti MMP-13 antibody (24 h at the room temperature in a humid chamber). The antibody was obtained as the ascitic fluid from mice at the Department of Medical Chemistry and Biochemistry (R. Vyta´sˇek and J. Novotna´, personal communication) and diluted 1 : 50 with TBS. The secondary antibody, rabbit anti-mouse polyclonal AP-labelled (Sigma-Aldrich), was used in the second step, in the dilution 1 : 50 with TBS for 30 min. The binding reaction was visualized using Fast Red TR/Naphthol AS-MX Sigma FAST tablets (Sigma-Aldrich), slides were counterstained with haematoxylin and mounted in gelatin. For the control reaction, the primary antibody was omitted. The right medial lung lobes were submerged in 4% cacodylate-buffered paraformaldehyde, immediately cut and tiny pieces of the central and peripheral portions of the lobes were fixed, dehydrated and embedded in medium-grade LR White Resin (London Resin Comp., Reading, UK) using a hot cure. Semi-thin sections were cut, mounted on slides using acetone and stained with TB to identify mast cells in perivascular locations. Then, selected serial semi-thin sections were incubated with 10% BSA in TBS, followed by incubation with the same monoclonal anti MMP-13

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Hypoxia affects the activity of pulmonary mast cells antibody as in the paraffin technique for 24 h at room temperature in a humid chamber. A goat anti-mouse polyclonal gold conjugate (Sigma-Aldrich) was used in the second step in the dilution 1 : 50 with TBS for 60 min, followed by intensification by reaction with silver using Silver Enhancer Kit (Sigma-Aldrich). Semi-thin sections were counterstained with TB to colocalize the metachromatic reaction of mastcell granules and the metallic silver precipitate detecting MMP-13 sites. Paraffin slides were quantitatively evaluated using Lucia G Image Analysis software (Laboratory Imaging, Prague, Czech Republic). The area of each section of the lung was measured (mm2) and mast cells in the subpleural, peribronchial and perivascular locations were counted. Mast cells found within walls of prealveolar arterioles, small muscular arterioles or arteries, and conduit arteries (Meyrick et al. 1978) were counted separately in both TB and anti-MMP13 stainings. The same evaluation was performed in the large pulmonary veins, i.e. veins with myocardium within their walls (Paes de Almeida et al. 1975). Absolute numbers of mast cells in each six locations in both stainings were expressed as number per 50 mm2. The hearts were separated in parts and right ventricle to left ventricle plus septum weight index (RV/LV + S) was used as an indicator of presence of chronic pulmonary hypertension (Fulton et al. 1952).

Statistical analysis Separately in TB- and anti-MMP-13-staining, data were compared between control and hypoxic groups using a one-way analysis of variance (anova) in conjunction with Tukey post-hoc test and Kruskal–Wallis one-way anova

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in conjunction with Dunn’s method of the multiple comparison procedure, respectively, when appropriate (SigmaStat 2.0; SPSS Inc., Chicago, IL, USA). The results are presented as means ± SEM. Differences were considered significant at P < 0.05.

Results Control animals No pathological findings were observed in lungs of 4-day control animals. Mast cells were found scattered in subpleural, peribronchial and perivascular locations. Rare individual, MMP-13 positive neutrophilic granulocytes were found in alveolar septa or in subpleural, but never in perivascular locations. Similar findings were observed in 28-day control animals except one male in which we noticed increased cellularity of the lung interstitium and especially of the walls of bronchi and vessels, consisting of plasma cells, lymphocytes and neutrophilic granulocytes. Significant differences were encountered in numbers of his mast cells as well; that is why mast cell counts of this animal were discarded.

Four-day hypoxia In rats exposed to 4-day hypoxia, mast cells significantly accumulated in the prealveolar portion of pulmonary vasculature (Figure 1). Similarly, in contrast to the two control groups, a significantly higher number of mast cells in prealveolar vessels expressed MMP-13 (Figure 2). Newly muscularized peripheral pulmonary arteries were observed at the prealveolar level, usually accompanied with MMP-13

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Figure 1 Distribution of pulmonary Toluidine Blue-detected mast cells in control and hypoxic rats. Means (± SEM) of absolute number of mast cells per 50 mm2 of the left lung section.

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Figure 3 Newly muscularized double-laminated (small arrows) prealveolar arteriole with MMP-13-positive mast cells (long arrows) in its adventitia. 4-day hypoxic rat. Anti-MMP-13/Fast Red and Haematoxylin. Bar ¼ 50 lm.

positive mast cells (Figure 3). Mast cells relatively often surrounded the initial portion of a small muscular artery branched from a conduit one (supernumerary arteries, Elliott & Reid 1965) (Figure 4). MMP-13 positive neutrophilic granulocytes were found not numerous near mast cells in adventitia of some prealveolar and small muscular arteries and bronchioles. Similar granulocytes were found in several bronchioles. The number of mast cells detected peribronchially was low (Figure 1), and they rarely expressed MMP-13 (Figure 2). Occasionally, we observed MMP-13 positive alveolar macrophages. Four days of hypoxia were too short to produce haemodynamically significant pulmonary hypertension, as RV/LV + S weight ratio did not differ between 4 days hypoxic rats and normoxic controls.

Figure 2 Distribution of pulmonary MMP-13-expressing mast cells in control and hypoxic rats. Means (± SEM) of absolute number of mast cells expressing MMP-13 per 50 mm2 of the left lung section.

Figure 4 A group of MMP-13-positive mast cells (long arrow) in adventitia of a newly muscularized supernumerary arteriole (small arrow) outbranched from a conduit artery (asterisk). 4-day hypoxic rat. Anti-MMP-13/Fast Red and Haematoxylin. Bar ¼ 50 lm.

Twenty-day hypoxia The rats exposed to hypoxia for 20 days developed hypoxic pulmonary hypertension, their RV/LV + S weight index was significantly higher than in normoxic controls. It was 0.28 ± 0.02 in controls and 0.39 ± 0.23 in experimental animals (P ¼ 0.0006). The major difference from 4-day hypoxia group, however, was that the mast cells accumulated in the conduit part of the pulmonary vasculature and subpleurally (Figure 1). Many mast cells surrounding conduit pulmonary arteries expressed MMP-13 (Figures 2 and 5). The numbers of mast cells at the prealveolar level and around the small pulmonary arteries in rats exposed to hypoxia for 20 days, however, did not differ from normoxic controls in

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MMP-13 negative; tiny intra-alveolar haemorrhage was sometimes found.

Common features

Figure 5 Small muscular artery with well developed elastic laminae and elastic network within its tunica media (small arrows) and a MMP-13-positive mast cell with released granules (long arrow). 20-day hypoxic rat. Anti-MMP-13/gold conjugate/silver enhanced and Toluidine Blue, semi-thin section. Bar ¼ 20 lm.

spite of their slight increase (Figure 2). MMP-13 was expressed also in smooth muscle cells in tunica media of some conduit arteries. The number of the MMP-13 positive mast cells in the walls of the large veins significantly increased in comparison with other groups (Figure 2). Multiple atelectatic foci were occasionally encountered within lung parenchyma in all rats of the 20-day hypoxia group. In these foci, some alveoli were filled with clotted plasma and MMP-13 positive macrophages with abundant foamy cytoplasm, sometimes accompanied with red blood cells. A few conduit and small muscular arteries presented inflammatory infiltration composed of neutrophilic granulocytes or lymphocytes in their adventitia. Focally, pleural thickening occurred together with subpleural accumulation of mast cells; these mast cells were mostly MMP-13-negative.

Twenty-day hypoxia and 7-day recovery After 20-day period of hypoxia, a group of rats were given 7-day recovery period in normoxic conditions. In this group, the moderate increase in number of the mast cells at the prealveolar arterial level was encountered, again, even MMP-13 positive. Nevertheless, the mast cells, even MMP-13 positive, again accumulated in the conduit part of the pulmonary vasculature. Increased number of MMP-13 positive mast cells occurred also in the walls of small muscular arteries (Figures 1 and 2). Minor isolated atelectatic foci were occasionally encountered within lung parenchyma in three rats of the recovery group. In these foci, some alveoli were filled with clotted plasma, alveolar macrophages were rare and mostly

The total number of mast cells in the left lung was not significantly different among all groups, although it was slightly higher in the rats exposed to hypoxia for 20 days. This difference was due to the increase in number of the subpleurally located mast cells. No differences were encountered among all groups in numbers of the total mast cells found in walls of the large veins, either. Silver-enhanced visualization of MMP-13 on semi-thin sections stained with toluidine blue verified the presence of MMP-13 in mast cell granules (Figure 5).

Discussion The main finding of the present study is that in 4 days of exposure to hypoxia, the interstitial collagenase (MMP-13) producing mast cells conspicuously accumulated in the small prealveolar pulmonary arteries. Later, after 20 days of exposure, when hypoxic pulmonary hypertension is fully developed, the majority of MMP-13 producing mast cells reside in the walls of conduit portion of pulmonary vasculature. Seven-day recovery after 20-day hypoxia causes again moderate increase in number of MMP-13 producing mast cells at the levels of prealveolar and small muscular arteries; accumulation of these mast cells is still encountered in the walls of conduit arteries as well. We exposed rats to the hypoxic environment (10% O2) in a normobaric hypoxic chamber. According to our repeated experience, this procedure in <3 weeks consistently induces stable pulmonary hypertension, characterized by an increase in pulmonary arterial blood pressure, increase in the weight of the right heart ventricle, and hypertrophy of media and fibrotization in the walls of peripheral pulmonary arteries (Herget et al. 1978; Hampl & Herget 1990; Herget et al. 2003). Thus, the increase in the right to left ventricles weight ratio in the current experiment confirms the presence of pulmonary hypertension in the group exposed to hypoxia for 20 days. Previous findings of our and other groups demonstrated that significant morphological, biochemical and haemodynamic changes can be detected even during the first week of hypoxic exposure (Rabinovitch et al. 1979; Herget et al. 2000; Lachmanova´ et al. 2005). Increase in pulmonary arterial blood pressure and right heart weight, however, is usually moderate or not present at this time. The accumulation of mast cells in the pulmonary vasculature in chronic hypoxia was described as early as three

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decades ago (Kay et al. 1974; Tucker et al. 1977) in studies associated with a search for a mediator of pulmonary vasoconstriction induced by hypoxia. In the present study, we did not find significant increase of the total number of mast cell (toluidine blue detected); on the other hand in the three experimental groups, the numbers of MMP-13 expressing mast cells increased. Tucker et al. (1977) in their original study found that the increase in the total number of perivascular mast cells was present only in species with the highest chronic hypoxia-induced pulmonary hypertension (calves and pigs). In rats and other species with moderate hypoxic pulmonary hypertension, the lung accumulation of mast cells was small. They concluded that the increase in mast cell density is related to the increase in pulmonary blood pressure rather than to the lung hypoxia. We hypothesize that the accumulation of mast cells is the result of hypoxic lung injury, which is a main cause of structural remodelling of peripheral pulmonary arteries and hypoxic pulmonary hypertension (Herget et al. 2000; Novotna´ & Herget 2002). One would assume that greater hypoxic lung injury in early phases of exposure in hypoxia would induce more prominent remodelling of vascular walls and more severe hypoxic pulmonary hypertension. Therefore, the positive correlation between the mast cells density and the severity of pulmonary hypertension is not surprising even if the mechanism is not directly linked to the increase in intravascular pressure. In fact, both mechanisms – hypoxic lung injury and increase in intravascular blood pressure – may participate. They may have, however, different importance in different stages of hypoxic pulmonary hypertension. In the first days of exposure, injury to the walls of prealveolar blood vessels attracts mast cells into this region, and they participate in the mechanism of remodelling of peripheral portion of pulmonary vascular bed. In the developed hypoxic pulmonary hypertension (after 20 days of exposure in our study), mast cells accumulate in the walls of conduit arteries, which are exposed to the increased intravascular pressure due to developed pulmonary hypertension. An increase in wall tension of conduit pulmonary arteries is known to stimulate matrix protein turnover (Riley & Gullo 1988). A similar mechanism may explain the increase in the number of mast cells localized subpleurally. Exposure to chronic hypoxia results in sustained increase in functional lung capacity (Barer et al. 1978), which may cause mechanical stress of the lung pleura. Why the vascular and subpleural mast cells differ in the expression of interstitial collagenase is not clear. Finally, the return to normoxia causes again gradual vascular wall remodelling in both prealveolar and conduit arteries. However, the splitting behaviour of the interstitial collagenase is now different as evidenced by Novotna´ et al. (2001).

Mast cells are detected by various methods. The reliable and most common technique is metachromatic staining with toluidine blue, metachromatic blue or thionin (Williams et al. 1977; Churukian & Schenk 1981; Henwood 2002; Masuda et al. 2003; Mene´trey et al. 2003). Other methods, e.g. polychromatic (Unna’s methylene blue or Giemsa) or mucopolysaccharide (alcian blue, aldehyde fuchsin and acridine orange) methods (Henwood 2002; Chong et al. 2003) are used in special needs as well as immunohistochemical evidence of mast cell tryptase (Cai et al. 2003), especially in immunofluorescent co-localization. Chloroacetate esterase activity is detected in both mast cells and neutrophilic granulocytes (Gomori 1953). Cytoplasmic granules of mast cells contain several biologically active substances, which may be involved in hypoxia-induced remodelling of peripheral pulmonary vasculature (e.g. biogenic amines, proteolytic enzymes, neutral proteases, metalloproteinases and cytokines). As MMPs are potentially dangerous for extracellular matrix, they are strictly regulated. They are usually not stored but transcribed under the influence of cytokines immediately before their secretion. Moreover, they are secreted as precursors activated by cleaving. Mast cells express interstitial collagenase (Di Girolamo & Wakefield 2000), which can be activated by mast cell neutral proteases, tryptase and chymase (Gruber et al. 1988, 1989; Saarinen et al. 1994). In active MMPs, the ratio between MMP and the tissue inhibitors of metalloproteinases (TIMPs) decides of the proper activity. Finally, sites of MMPs’ activity are usually compartmentalized (Elkington & Friedland 2006). The interstitial collagenase, in the rat species identified as MMP-13 (rodent-like interstitial collagenase), is the principal enzyme responsible for initiation of collagen breakdown. In previous studies, we observed that extracts from the walls of peripheral pulmonary arteries of rats exposed to hypoxia had increased activity of MMP-13 (Novotna´ & Herget 1998; Herget et al. 2003) and a consequent presence of collagen type I cleavages (Novotna´ & Herget 1998). Pharmacological inhibition of collagenolytic activity partly inhibited the development of hypoxic pulmonary hypertension (Herget et al. 2003). Atkinson et al. (2001) observed collagen type I degradation also by the membrane type 1 matrix metalloproteinase (MT-1–MMP) in a transfected human fibrosarcoma cell line in co-operation with MMP-2. As MT-1–MMP is membrane-bound, this breakdown can be highly localized. In addition, MT-1– MMP forms a complex with TIMP-2 on the cell surface, which activates MMP-2. Than, MMP-2 can serve as an activator of other MMPs, namely MMP-13 (Li et al. 2000). MT-1–MMP expression in mast cells is thus another candidate to be traced because it could be a part of the

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Hypoxia affects the activity of pulmonary mast cells collagenolytic cascade in the walls of pulmonary vessels during their remodelling. According to our hypothesis (Hampl & Herget 2000; Herget et al. 2000), collagen degradation products may represent one of the mechanisms, which stimulate growth of the vascular smooth muscle (Bacˇa´kova´ et al. 1997) and fibroproduction (Gardi et al. 1994) in peripheral pulmonary blood vessels in hypoxia. In addition, it was shown that release of tryptase from activated mast cells can also directly stimulate production of collagen type I (Cairns & Walls 1997). Mast cells play a key role in the early inflammatory response of systemic microvasculature to hypoxia (Dix et al. 2003; Steiner et al. 2003). The mechanism of activation of mast cells in systemic hypoxia is explained by alteration of balance between oxygen radicals (ROS) and nitric oxide (NO) production (Steiner et al. 2002). Similarly, in the pulmonary blood vessels, an increase in ROS and NO production seems to be one of the triggering mechanisms of pulmonary hypertension induced by chronic hypoxia (Hampl & Herget 2000). Administration of an antioxidant (N-acetylcysteine) inhibits the development of hypoxic pulmonary hypertension most effectively if it is applied in a short period at the beginning of exposure to hypoxia (Lachmanova´ et al. 2005). Antioxidant therapy is therefore most effective at the time when mast cells accumulate in the prealveolar pulmonary vessels. Frantz et al. (1988) concluded that mast cell products are not important mediators of the short-time hypoxia-induced pulmonary hypertension because 20 minintravenous infusion of cromolyn sodium did not inhibit 10 min hypoxia-induced pulmonary hypertension in newborn and young lambs. For the cellular response to hypoxia, hypoxia-inducible factor-1 (HIF-1) plays a master role. Under normoxic conditions, the turnover of its subunit HIF-1a is controlled by ubiquitination and degradation in proteasomes. In hypoxia, HIF-1a is stabilized by ROS (Griffiths et al. 2005) and binds the HIF-1b subunit in the nucleus. Thus, HIF-1 mediates transcription of genes encoding proteins engaged in reactions to hypoxia. At least two of these proteins, inducible NOsynthase and vascular endothelial growth factor certainly participate in mechanism of the chronic hypoxia – induced pulmonary vascular remodelling. Redistribution of mast cells to the peripheral parts of pulmonary vasculature and expression and release of interstitial collagenase is in concordance with our hypothesis that increased collagenolysis in peripheral pulmonary arteries is probably one of the important mechanisms that triggers pulmonary vascular remodelling in chronic hypoxia.

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Acknowledgements The work was supported by the grants from the Grant Agency of the Czech Republic (Nos 304/02/1348 and 305/ 05/0672) and Grant Agency of the Charles University, Prague (No 53/2002). We are also obliged to Erik B. Johnson, B.C. Pharm. (Department of Physiology, Charles University 2nd Medical Faculty) for his kind language review and correction.

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