Hypoxic Responses of Vascular Cells

Mechanical Load Enhances the Stimulatory Effect of PDGF on Pulmonary Artery Fibroblast Procollagen Synthesis* Jill E.

Bishop , PhD; R. Butt; K. Dawes; and G. Lau rent

(CHEST 1998; 114:25S) he mechanical environ men t is a key regulator of pulmona ry artery wall remodeling in norm al growth and during the development of hypertension. Her e we det ermined th e direct effect of mechanical load and plat elet-derived growth factor (PD GF) on adventitial pulmonary art ery fibroblast replication and pro collagen metab olism-t\vo pro cesses stimulated during th e development of pulmonary hypertension. Cyclical mechanical load (20% elongation, 1.5 Hz ) was appli ed to cells grown on elastin-coate d flexible membranes using a strain unit (Flexcell; McKeesport, PA) in th e presen ce of fet al calf serum (FC S) or PDGF-BB. Procollagen production and degradation were measur ed by assessing hydroxyproline levels in th e culture wells. Cell replic ation was measured using a meth ylen e blu e dye binding assay and dir ect cell counting. Cell replication was stimulated by 85±1O% (mean± SD) with PDCF (80 nglmL, p
T

*From the Centre for Cardiopulmonary Biochemistry and Respiratory Medicine, University College London Medical School, The Rayne Institute, London. This work was funded by The Wellcome Trust and the British Heart Foundation.

Hypoxic Responses of Vascular Cells* Stella Kourembanas, MD ; Toshisuke Morita, MD , PhD; Helen Christon, MD ; ¥u xian g Liu , PhD ; Hid eo Koike, I',;1D; Dara Brod.sky, MD; Vic toria A rthur; and S. Alex MUsial, PhD

(CHEST 1998; 114:25S-28S)

I n recent years, it has been demonstrated that hypoxia-

induced vascular remodeling in th e lung is a result of

complex cell-cell and cell-matrix inter actions involving th e synthes is and release of growth factors , cytokines, basement membrane components, as well as biological messengers such as nitri c oxide and carbon monoxide (CO) . Recent investigations in our laboratory address th e cellular responses to hypoxia and role of CO . HYPOXIC REGULATION OF GENE EXPRESSION

Th e vascular respons es to hypoxia are mediated by th e endothelial cell lining of blood vessels as well as the underlying smooth muscl e cells, ind ep endent of the en doth elium. ' Endothelial cells respond to hypoxia by increasing th e expression of vasoconstrictors and growth promoters such as platelet-derived growth facto r-B (PDCF-B), endothe lin (ET-l ), and vascular endothe lial growth factor (VE GF )2-S while inhibiting th e production of th e vasodilator and smooth muscl e cell growth inhibitor, nitric oxide (NO).6-H Th e responses of smooth muscle cells to hypoxia include inhibition of potassium channels! and gene regulation. Hypoxia increases the transcriptional rate of VEGF4 .9.10 as well as hem e oxygenas e-l (HO-l), th e latt er leading to increased production of CO . 11 Th er efore , under hypoxia, when endothe lial-de rived NO production is suppressed , molecules such as CO produced by smooth muscle cells could be important regulators of vascular ton e. Like NO , CO is an endoge nously produced gas molecule th at activates guanylyl cyclase , resultin g in increased production of cyclic guanosine monophosphate (cGMP). A role for CO has been suggested in neuronal Signal transduction' < and accumulating evide nce implicate s CO to play a critical role in the mainten ance of vascular ton e. l 3 .l 4 CO is produced from th e breakdown of hem e to biliverdin via th e activity of th e enzyme HO. Two isoforms of HO have been identified : HO-l and HO-2. HO-l is inducible by multipl e agents while HO-2 is constitutively expressed.!" Th e CO gene rate d from th e activity of HO can elevate cC MP levels in th e same or neighboring cells, II thus regulating vascular ton e in a manner similar to NO. Recent studies by our group dem onstrated that on exposure of aorti c or pulmonary smooth muscle cells to hypoxia, the trans criptional rate of th e HO-l gene and ste ady-state messen ger RNA (mHNA) level wer e dr amatically increased . HO-l mR NA levels peaked sevenfold above control by 15 h of hypo xia and returned to basal levels by 48 h .1 1 A corresponding sixfold increase in CO production, as measured by carboxyhe moglobin gene ration following addition of hemo globin to th e ce ll culture s, was demonstrated with hypoxic exposur e of 12 hs. Th is increase was du e to th e activity of HO since inhibitors of HO such as tin protoporphyrin -9 (SnPP-9) decreased carboxyhemo globin levels in a dose-dep endent manner. Furthermore , thi s increase in CO levels resulte d in corre'From the Joint Program in Neonatology, Children's Hospital and Brigham and 'Nomen's Hospital, Harvard Medical School, Boston. This work was supported hy grants NIH (I P50 HL 46491, GM 44520, and ROJ HL 55454) and the American Heart Association. CHEST / 114 / 1 / JULY , 1998 SUPPLEMENT

255

sponding increas es of cGMP production by the smooth muscle cells. cGMP levels peaked around 12 to 15 h of hypoxia and returned to almost baseline values by 48 h. Thi s pattern of increase in intracellular cG MP paralleled the pattern of HO-1 mR NA expression under hypoxic conditions. In addition, inhibition of NO synth esis with inhibitors such as N"'-nitro-L-arginine (L-NNA), did not affect eithe r basal or hypoxic levels of cGMP in smooth muscle cells, thus eliminating endogenous NO as a regulator of cGMP in smooth muscle cells. cGMP is a wellcharacte rized medi ator of smooth muscle cell relaxation. Since CO is a regulator of cG MP in these cells, it can be a physiologic regulator of vascular tone. J 1. 1:3

CO

AND HYPOXIC G ENE EXPRESSION

Exogenous CO has been reported to inhibit the hypoxic induction of genes. Goldb erg et aPGfirst proposed ,that the oxygen sen sor is a heme-cont aining protein based on studi es using exogenous CO to inhibit the hypoxic indu ction of the erythropoietin gene . Thi s postulated heme sensor binds CO and att ains a relaxed configuration, thu s mimicking a "normoxic" state despite the absence of oxygen. In addition to th e erythropoietin gene, exogenous CO was shown to inhib it the hypoxic indu ction of ET-l , PDGF-B , and more recently, the VEGF gene .3J 7 To explore the molecular mechanisms by which CO might inhibit gene expression, we examined the regulation of the VEGF gene by this gaseous molecule. We have shown previously that endothe lial cells do not express VEGF under normoxic conditions but on exposure to hypoxia, VEGF mHNA levels were dramatically induced within a few hours .' We have furth er demon strated that this increase in trans cription was at least parti ally mediated by a 28-bp element in the 5' region of the gene that contains a hypoxia indu cible facto r-I (H IF-1) binding site .! vVang and Semen za!" first describ ed this motif in the hypoxia-respons e eleme nt of the erythropoietin gene and have subsequently characte rized HIF-1 and shown it to be a member of the helix-Ioop-helix-PAS family of tran scription factors.!? HIF-I regulates tran scription of a number of genes encoding glycolytic enzymes .w Th e HIF-1 site of the VEGF gene acts as an enhance r by stimulating expression of a reporter gene und er hypoxia. Disruption of the core H1F -1 motif inhibited hypoxia inducibil ity, but we found that additional sequences 3' to the HIF-1 binding site were also critical for enhance r fun ction .' In transfection experiments using smooth muscle cells, we test ed the effect of exogenous CO on the ability of this enhance r to regulate chloramphenicol acetyl transfer ase (CAT ) activity. Although this enhance r stimulated CAT expression 4 to lO-fold unde r hypoxic conditions, in the presenc e of 5% exogenous CO, hypoxia indu cibility was suppressed by 50%. In DNA binding expe riments, we demonstrated that bindin g of a hypoxia-inducible factor to the VEGF enhance r was Significantly reduce d in the presence of exogenous CO. Despite the suppressed bind ing activity, HIF-1 Q' and f3 proteins were still indu ced by hypoxia in the presenc e of excess CO, suggesting that CO inhibits the activity but not the production of HIF-l. 268

CO was shown to inhibit VEGF protein secret ed by smooth muscle cells in parallel with changes in gen e tran scription. VEGF protein was quantifi ed by enzymelinked immunosorbent assay while controlling for CO levels in the culture s. CO in the media was redu ced by eithe r inhibiting HO activity with SnPP -9 or scavenging CO with hemo globin. No significant VEGF protein could be measured in the conditioned med ia of norm oxic smooth muscle cells and significant but small amo unts of VEGF could be det ected und er hypoxia. Int erestin gly, in the presence of CO inhibitors, we demon strated a significant sever al-fold hyperinduction in the amount of VEGF protein secrete d by hypoxic smooth muscle cells. CO, therefore, serves to modulate the hypoxic induction of the VEGF gene leading to decreased amounts of VEGF secret ed by smooth muscle cells. Combined, the above studies indicate that CO is an auto crine regulator of smooth muscle cell behavior leadin g to increased cG MP cont ent and decreased production of VEGF . In addition to its autocrine effects, CO can easily diffuse across cell membranes and can have paracrin e effects on adjace nt cells. We investigated whethe r CO, at the physiologic concentra tions released by vascular smooth muscle cells (VSMC ), could regulate endothe lial cell gene expression. We first examined whethe r the presence of smooth muscle cells could regulate ET-l and PDGF-B gene expression in coculture d endothelial cells. North ern blot analysis of endothe lial RNA showed that, in the absence of smooth muscle cells, ET-l mRNA levels increased 5.8-fold by 12 h of hypoxia and 7.2-fold by 48 h above control levels. In contrast, ET-l mRNA in endothelial cells cocultu rcd with smooth muscle cells was not induce d until 24 h of hypoxia « 2-fold), and the maximal indu ction was 5.7-fold above control levels at 48 h. Th ese findings suggest that the factor responsible for suppressing the hypoxic increases in ET-l and PDGF-B mRNA is produced by smooth muscle cells in the early hypoxic period and its production decreased by 48 h of hypoxia. Further more, its effects are specific for the ET-l and PDGF-B genes, that is, genes regulated by hypoxia in these cells. To examine if smooth muscle cell-de rived CO is the factor respon sible for the changes in gene expression by endothe lial cells, we inhibited CO release by eithe r inhibiting HO-1 activity with SnPP-9 or by scavenging CO with hemoglobin. Given that NO is produced by vascular cells and can regulate gene expression, we inhibit ed its production by addin g L-NNA, a known inhibitor of NO synthesis, to the cultures. Und er normo xia, none of these agents affecte d the expression of ET-l . However , under hypoxia (although L-NNA had no effect), both SnPP and hemo globin reversed the inhibitory effects of coculture d VSMC on endothe lial cell gene expression, thus allowing for high expression of ET-l und er hypoxia, similar to that observed in endothelial cells culture d alone. Collectively, th ese findings suggest that CO, the product of HO , released by hypoxic smooth muscle cells into the media , is responsible for the suppression of hypoxia-indu ced increases in ET-l expression by endothe lial cells. Identi cal respon ses were noted when the expression of the PDGF-B gene was studied.

Thomas L. Petty 40th Annu al Aspen Lung Conf erence : Biology & Pathobiology of the Lung Circulation

CO MODULATES THE HYPOXIC R ESPONSES OF VASC ULAR C ELLS

Th e above data indi cate that smooth mu scle cellderived CO suppressed the production of both ET-1 and PDGF-B by hypoxic endothelial cells in a paracrin e way. We spec ulated that smooth muscle cell-de rived CO could regulate smooth muscle cell proliferation rate indirectly by supp ressing the expression of these two potent smooth muscle cell mitogen s produced by endothe lial cells. To test this hypothesis, we studi ed the proliferation of smooth muscle cells in the presence of endothelial cells und er normoxia and hypoxia. We found that in the presenc e of endothelial cells, a modest increase of smooth muscle cell number was see n by 24 h und er either normoxia or hypoxia. When CO production was inhibited with SnPP-9, ther e was no change in smooth muscle cell numbe r under normoxia, however , ther e was a th reefold rise in smooth muscle cell number under hypoxia that was revers ed by anti-PDGF and anti -ET-1 antibodies add ed to the cultur es. Th ese findings confirm that CO indeed can regulate smooth muscle cell growth via the production ofET-1 and PDGF in adjacent endothelial cells. We explored pot enti al auto crin e effects of smooth muscle cell-de rived CO on smooth muscle cell proliferation , und er normoxia and hypoxia, and mor e specifically its effect on the respon se of smooth muscle cells to exogenous mitogens . Smooth muscle cells were exposed to hypoxia or norm oxia for various periods of time and their growth as well as their respons e to seru m (10%) or ET-1 were studi ed . Hypoxic smooth muscle cells were shown to have a more pronounced proliferativ e respon se to ET -1 or to seru m than their normoxic counterparts. Unde r hypoxic conditions, smooth muscle cell number increased about 2.3-fold in 48 h, compared with a 1.7-fold increase und er normoxia for the same time period. Similar findin gs have been reported for fibrobl asts that were shown to proliferate at higher rates in respons e to serum and growth factors und er hypoxic compared with normoxic condltions .>' In the absen ce of serum or exogenous growth factors , smooth muscle cell number did not increase significantly und er hypoxia and increased only mod estly und er norm oxia. To examine if CO modulates the proliferative respon se of smooth muscle cells to exogenous mito gens , we altered CO production and studi ed this response. Treatment of cells with hemin, a pot ent indu cer of HO activity and CO production, did not affect smooth muscle cell prolifer ation und er eithe r basal or ET-1- stimulat ed conditi ons. Thi s is not unexpected because hypoxic VSMC already produces high levels of CO. In contrast, inhibition of HO by SnPP -9 or removal of CO with hemo globin resulted in increase d proliferation of hypoxic smooth muscle cells even in the absence of growth factors, while these agents had no effect und er normoxia. Int er estingly, althou gh maximal proliferation was observed in hypoxic cells treated with ET-1 , inhibitors of CO had an equal or more striking relative proliferative effect on hypoxic smooth muscle cells und er basal conditi ons. These findings argue for an impo rtant role of CO as an inhibitor of smooth muscle cell growth und er hypoxia indep endent of exogenous mito gens . To further characte rize the effec ts of CO in cell cycle

control, we examined the expression of the cell cyclespec ific tran scription factor E2F-1. E2F-1 mR NA levels wer e increased by 12 h of hypoxia whe n cells wer e exposed to mitogen s such as ET -1 or seru m. This increase was furth er augmente d to 4.5-fold in the presenc e of hemoglobin or the inhibitor of CO production, SnPP. The presenc e of hemin, an ind uce r of HO-1 activity, result ed in low E2F-1 mR NA levels. CO, ther efore, regulates E2F-1 mRNA levels in mitogen-stimulated smooth mu scle cells in a fashion that parallels smooth muscle cell proliferation rate. Since cG MP is th e second messen ger molecule that might med iate CO action , we examined the effect of exogenous cGMP on E2F-1 mRN A expression and smooth muscle cell proliferation. Exogenous 8-BromocGMP dram atically reduced E2F-1 mRNA levels und er hypoxic conditions whe reas the presen ce of ODQ, a spe cific inhibitor of guanylyl cyclase, hyperindu ced E2F-1 mR NA levels. Thi s indicates that cGMP is the second messenger molecule mediatin g the effects of CO on E2F-1, and, in agreem ent with these findings, exogenous cGMP inhibited smooth mu scle cell proliferation in a dose-d ependent mann er. Th e above in vitro expe rime nts suggest th at hypoxia has both tran sient and prolonged effects on vascular cells (Fig 1). Th e prol onged effects are mediated by the endothe lial cells, wher eby the transcriptional rates of ET-l and PDGF-B genes are dram atically increased resulting in incr eased production of these two vasoconstrictors and smooth muscle cell mitogens. This leads to enhanced smooth muscle cell growth and contractility. Th e suppressed production of endothe lial-de rived NO further contributes to the unopposed smooth muscle cell prolifer ation. Th e transient effects of hypoxia are mediated by the smooth muscle cells via the increased expression of HO-1 resulting in incr eased CO production . Increased CO levels inhibit smooth muscle cell growth in an autocrine mann er via E2F-1 and modulate the endothe lial expression of ET-l and PDGF-B , thereby inhibiting smooth muscle cell contractility and prolifer ation in a paracrin e mann er. However , und er prolonged per iods of hypoxia, CO production is not sustain ed and thus the endothelial cell pathway predominates, leading to active

Proposed Model EC Prolonged .....--:::-_ _;::;;..;'-effect .-+----", 0. NOS

-,

L- r9_ NO + Citrulline

Ie

IHypoxial

ET-' PDGF-B

o

°

SMC growth

contraction

0 Transient effect Heme ~ biliverdin + CO

active vasoconstriction

~a nd

'-------V'structural remodeling

SMC FIG UR E 1. Proposed mod el shows th at hypo xia has both tra nsient and prolonged effec ts on vascula r cells.

CHEST / 114/ 1 / JUL Y, 1998 SUPPLEMENT

278

and sustained vas oc onstrict ion with structural remodeling of th e pulmonary vasculature . Bas ed on this model , a sustained exp re ssion of HO-l leadin g to in cre ased C O le vel s could prevent or reduce the d evelopment of hypoxi c pulmonary hypertension in vivo . Usi ng th e adult rat model of hypoxia-induced pulmonary hypertension, sustain ed C O production was acco m plished with se rial inj ections of h emin or NiCI 2 . Right ventricular pressures (RVPs) were m e asured to as sess th e d e gre e of pulmonary hypertension and vasoreact ivity . As expecte d , RVP s were significantly hi gh er unde r hypoxic co m pared with norm oxic co nd itions. Mo st in te res tingly, this rise in RVP under hypoxia was co m p le te ly inhibited when ani m als were tre ated with N iC l2 o r hemin. Sustain ed in creases in HO-l production with th e se age n ts also prevented th e sm ooth m uscle ce ll hyperpl asia characte ristic of pulmonary hyp ertension, and led to d e creased ET-l mRl'\A levels in the lungs, in ag ree ment wi th our in vitro data. C ONCLUSION

U nder normal conditions, th e basal production of C O in th e vascula ture is low and re gulation of vascula r tone is under th e co n tro l of NO. H owe ver, on ce th e production of NO in e ndo thelial ce lls is impaired by co nd ition s suc h as hypoxia, th e smooth muscl e cell-derive d C O may b e a cru cial regulato r of vasc ula r tone b y modulating vas oc onstrict o r exp re ssion an d cGM P le vel s. In this m odel, C O produced by sm ooth muscle ce lls pl ays an import ant physiologic rol e in modulating th e tran sient hypoxic resp o nse of vascu lar cell s to hypoxi a. Therapi e s aimed at e n h anci ng and sus taini ng C O production for lon ger periods may p reven t th e proliferative ce llular re sponses and m ini mize inju ry seco nd a ry to hypoxia. ACKNOWLED GM E NTS: We tha nk Amy Elias for her expe rt assistance in preparation of this manuscript. R EFERE NCES

'Weir EK, Archer SL. The mechanism of acute hypoxic pulm onary vasoconstriction: the tale of two chaunels. FASE B J 199.5; 9: \83-89 2 Kourembanas S, Hannan RL, Faller DV. Oxygen tension regulates the expression of the platelet-derived grO\vl:h factor-B chain gene in hum an endothelial cells. J Clin Invest 1990; 86:670-74 3 Kourembanas S, Marsden PA, McQuillan LP, et al. Hypoxia indu ces endothelin gene expression and secretion in cultured human endothe lium. J Clin Invest 1991; 88:10.54-.57 4 Lin Y, Cox SR, Morita T, et a!' Hypoxia regulates VECF gene expression in endothelial cells: identification of a .5' enhance r. Circ Res 199.5; 77:638-43 .5 Namiki A, Brogi E, Kearney M, et al. Hypoxia induces vascular en dothelial growth factor in culture d hum an endothelial cells. J BioI Che m 1995; 270:31189-95 6 McQuillan LP, Leun g GK, Marsden PA, et a!' Hypoxia inhibit s expression of e NOS via transcript ional and po sttran scriptional mechanisms. Am J Physiol 1994; 267:H I 921-27 7 Shaul P\V, Fa rrar MA, Magness RR. Pulmonary endothelial nitric oxide production is developmentally regulated in the fetus and newborn. Am J Physiol 1993; 26.5:H IO.56-63 8 Liao JK, Zulucta JJ, Yu F-S, et a!' Regulation of bovine endothelial constitutive nitric oxide synthase by oxygeu. J Clin Invest 199.5; 96:2661-66 285

9 Ikeda E, Achen MG, Breier G, et al. Hypoxia-indu ced transcriptional activation and increased mRNA stability of vascular endothelial growth factor in C6 glioma cells. J Bioi Che rn 1995; 270:19761-66 10 Brogi E, Wu T, Namiki A, et al. Indirect angiogenic cytokines up regulate VEC F and hFC F gene expression in vascular smooth muscle cells, whe reas hypoxia upregulates VEC F expression only. Circulation 1994; 90:649-.52 II Morita T, Perr ella MA, Lee M-E , et al. Smooth muscle cell-der ived carbon monoxide is a regulator of vascular cG MP. Proc Natl Acad Sci USA 199.5; 92:147.5-79 12 Verm a A, Hirsch OJ, Glatt CE, et al. Carbon monoxide: a put ative neu ral messenger. Science 1993; 2.59:381-84 13 Cook NlN , Nakatsu K, Marks GS, et al. Heme oxygenase activity in the adult rat aorta and liver ;L~ measured by carbon monoxide formation. Can J Physiol Pharmacol 199.5; 73:51.5-\ 8 14 Morita T, Kourembanas S. End othelial cell expression of vasoconstrictors and growth factors is regulated by smooth muscle cell-derived carbon monoxide. J Clin Invest 1995; 96:2676-82 \.5 Maines MD, Trakshel GM, Kutty RK. Characte rization of two constitut ive forms of rat liver microsomal heme oxygenase: only one molecular species of the enzyme is inducible. J Bioi Chem 1986; 261:4II- 19 16 Goldbe rg MA, Dun ning SP, Bunn HF . Regulation of th e erythropoietin gene: evidence that the oxygen sensor is a heme protein. Science 1988; 242:1412-1.5 17 Goldbe rg MA, Schneide r TJ. Similarities between the oxygen-se nsing mechanisms regulating the expression of vascular endothelial growth factor and erythropoietin . J Bioi Che rn 1994; 269:43.5.5-.59 18 Wang GL, Sem en za GL. Purifi cation and cha racte rization of hypoxia-inducibl e factor 1. J Biol Che rn 199.5; 270: 1230-37 19 Wang GL , Jiang B-H , Rue EA, et al. Hypoxia-inducibl e factor \ is a basic-helix-loop -helix-PAS het e rodi mer regulated by cellul ar O 2 ten sion . Proc Natl Acad Sci USA 199.5 ; 92:.5.5 10-14 20 Seme nza GL, Roth PH , Fang H-M, et a!' Transcriptional regulation of genes encoding glycolytiC enzymes by hypoxiainducible factor 1. J Bioi Che m 1994; 269:237.57-63 21 Storch T G, Talley GO. Oxygen concentration regulates the proliferati ve respon se of human fibroblasts to serum and growth factor s. Exp Cell Res 1988; 17.5:3\7-2.5

Hypoxia Stimulates Proliferation of a Unique Cell Population Isolated From the Bovine Vascular Media* Maria G. Frid, PhD ; Al ma z A Aldas hec, PhD ; Ca ry F. Cabirac, PhD ; Edward C. Dempsey, MD , FCC P; and Kurt R. Stenmark, MD

(CHEST 1998; 114:28S-29S)

A bno rmal replication o f vascu lar sm oo th muscle cell

(SMC) plays a pi votal role in th e p athogene sis of many c h ro nic pulmonary vascu la r d ise ases . Increased

*From the Department of Development al Lung Biology, University of Colorado Health Sciences Ce nte r, Denver.

Tho ma s L. Pett y 40th Annual Aspen Lung Conference: Biolog y & Pathobiolog y of the Lung Circulation