INTRODUCTION

HYPOXIA IS A COMMON stress that affects anorganism’s homeostasis. Although much

is known of the mechanisms of cellular and bio-chemical responses to acute hypoxia, relativelylittle is known of the mechanisms underlyingresponses to prolonged or chronic hypoxia.During pregnancy, exposure to chronic hy-poxia is thought to be associated with an in-creased risk of preeclampsia and fetal in-trauterine growth restriction (IUGR) (Moore etal., 1982; Zamudio et al., 1995a). Studies in hu-mans have suggested that exposure to chronichypoxia during pregnancy may inhibit the nor-mal pregnancy-mediated vasodilation and re-

modeling that characterize the uteroplacentalcirculation. For instance, in pregnant womenresiding at high altitude (3100 m) throughoutpregnancy, uterine blood flow at 36 weeks isdecreased in comparison with those at low al-titude (the Denver altitude of 1600 m), primar-ily due to a decrease in vessel diameter (Za-mudio et al., 1995b). This reduction in vesseldiameter may result in part from hypoxic in-hibition of the normal remodeling that occursin the uterine artery during pregnancy, as sug-gested by studies in pregnant guinea pigs.Uterine arteries from animals exposed tochronic hypoxia (3962 m) for their entire ges-tation (63 days) had a diminished growth re-sponse as measured by DNA synthesis com-

HIGH ALTITUDE MEDICINE & BIOLOGYVolume 4, Number 2, 2003© Mary Ann Liebert, Inc.

Effects of Chronic Hypoxia on Maternal Vasodilation and Vascular Reactivity in Guinea Pig

and Ovine Pregnancy

MARGUERITTE M. WHITE1 and LUBO ZHANG2

ABSTRACT

White, Margueritte M., and Lubo Zhang. Effects of chronic hypoxia on maternal vasodilationand vascular reactivity in guinea pig and ovine pregnancy. High Alt Med Biol 4:157–169, 2003.—During pregnancy, exposure to chronic hypoxia is thought to be associated with an increasedrisk of preeclampsia and fetal intrauterine growth restriction (IUGR). While some studies sug-gest that this process may be mediated through effects of chronic hypoxia on uterine artery va-sodilation and growth, these observations are likely to be species specific and may represent ge-netic variability in maternal adaptation to hypoxia. This review is a comparative analysis of theeffects of chronic hypoxia on vascular reactivity in pregnant and nonpregnant guinea pig andsheep. Data suggest that exposure to chronic hypoxia is associated with enhanced uterine arteryblood flow in the sheep, whereas, in the guinea pig, blood flow is decreased.

Key Words: high altitude; vascular function; animal models; comparative analysis

1Division of Cardiology, University of Colorado Health Sciences Center, Denver, CO.2Center for Perinatal Biology, Loma Linda University School of Medicine, Loma Linda, CA.

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pared to their low altitude counterparts (Keyeset al., 1997).

The regulation of uterine blood flow is cru-cial for growth and survival of the fetus andfor maternal cardiovascular well-being. How-ever, the adaptive mechanisms of the uterinevasculature to chronic hypoxia during preg-nancy are likely to be species dependent andmay represent interspecies variability in ge-netic adaptation to high altitude. For example,while the cardiovascular adaptation to normalpregnancy is similar for the guinea pig andsheep, in the former, maternal exposure tochronic hypoxia is associated with growth re-tardation (Rockwell et al., 2000), which is notevident in the sheep after similar exposure(Kamitomo, 1992). The purpose of this reviewis to further explore interspecies differences inmaternal adaptation to chronic hypoxia by pre-senting a comparative analysis of the effects ofchronic hypoxia on vascular function in boththe uterine and nonuterine circulations in theguinea pig and sheep. Data are presented froma variety of study preparations, includingwhole animal, isolated vessels, and endothelialcell cultures.

EFFECTS OF PREGNANCY AND CHRONIC HYPOXIA ON

CARDIOVASCULAR PHYSIOLOGY IN THE GUINEA PIG AND SHEEP

Whole-animal studies

Both ovine and guinea pig pregnancies arecharacterized by changes in cardiovascularphysiology that mirror certain alterations inhuman pregnancy. In the guinea pig, preg-nancy increased cardiac output and uteropla-cental blood flow in conjunction with a markeddecrease in systemic vascular resistance (SVR)(Curran-Everett et al., 1991). The increase inuteroplacental blood flow was accounted for bysimilar corresponding increases in cardiac out-put of 43% and 35% in the guinea pig andsheep, respectively (Rosenfeld, 1977; Curran-Everett et al., 1991). In both guinea pig andsheep, the cardiac output increases progres-sively and reaches the maximum in late gesta-tion (Rosenfeld, 1977; Myers and Hsui-Yu,

1985). In evaluating regional contributions tothe pregnancy-associated decrease in SVR,Curran-Everett concluded that most (70%) ofthe decrease in SVR in the pregnant guineapig was attributed to decreased SVR of thenonuteroplacental circulation (Curran-Everettet al., 1991).

A hallmark of human pregnancy is the well-characterized decrease in vasoconstrictor re-sponse to vasopressors such as angiotensin II(AII) (Benjamin et al., 1991; Ramsay et al., 1992).Early studies in catheterized guinea pigs andsheep demonstrated that the nonuteroplacen-tal conductance response to AII (measured asblood flow) is increased in pregnant comparedto nonpregnant animals, supporting a refrac-toriness to the constrictor effects of AII (Anni-bale et al., 1989; Curran-Everett et al., 1991;Hariharan et al., 1987) . In the guinea pig, preg-nancy did not affect conductance response toAII in the uteroplacental circulation, whereasin sheep the uteroplacental conductance wasslightly decreased in response to AII, albeit lessthan the rest of the maternal circulation (Mag-ness and Rosenfeld, 1989). However, despitethe decrease noted in sheep, overall uteropla-cental blood flow was preserved in both sheepand guinea pig during AII infusion.

Studies in guinea pigs have evaluated the ef-fect of chronic hypoxia on several aspects ofthese normal systemic vascular changes duringpregnancy. In these studies, low altitude is de-fined as the Denver altitude of 1600 m. Preg-nant and nonpregnant guinea pigs maintainedat low (1600 m) (PaO2 70 mmHg) and high (3962m) (PaO2 35 mmHg) altitude for 6 weeks orthe entire length of gestation were catheter-ized and, after full recovery, studied in a quietawake state. Cardiac output, blood pressure,and heart rate were monitored at baseline andin response to a slow infusion of vasoactivesubstances. In the nonpregnant animals, therewas no difference in SVR or change in SVR re-sponse to PE or AII between the normoxic andhypoxic group (Harrison et al., 1986). Additionof the prostaglandin inhibitor meclofenamateincreased the SVR response to PE, but had noeffect on the response to AII, suggesting that inthe nonpregnant guinea pig chronic hypoxiaenhanced the production of dilator prostaglan-dins during a-adrenergic stimulation.

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In pregnant guinea pigs exposed to chronichypoxia, baseline SVR was increased comparedto normoxic pregnant animals (Harrison andMoore, 1990). However, chronic hypoxia didnot alter the SVR response to AII. Addition ofmeclofenamate did not equalize the differencein SVR between low and high altitude pregnantanimals, suggesting that during pregnancychronic hypoxia does not inhibit dilating pros-taglandin production. Results from these studypreparations suggest that in the guinea pig,while chronic hypoxia increases SVR duringpregnancy, this effect is mediated throughmechanisms other than increased sensitivity toPE or AII or through an inhibition of vasodi-lating prostaglandins.

Maternal responses to long-term hypoxemiawere studied in chronically catheterized sheepat 110 to 115 days gestation (term 140 days) andnormoxic controls (Kitanaka et al., 1989). Arte-rial PO2 was maintained at about 60 mmHg forup to 28 days. In previous studies in pregnantsheep, exposure to high altitude (3820 m) from30 to 120 days gestation was shown to decreasematernal PO2 from 102 to 64 mmHg (Kamit-omo, 1992). In hypoxic pregnant ewes, therewas a significant decrease in cardiac output by14% throughout the experimental period.Within the first 24 h of the onset of hypoxemia,uterine blood flow decreased about 15% from1180 to 990 mL/min. Thereafter it increasedsignificantly to 1360 mL/min by day 21 of hy-poxemia. Thus, during pregnancy, chronic hy-poxemia is associated with increased SVR inthe guinea pig. In the sheep, hypoxia was as-sociated with a gradual increase in uterineartery blood flow.

Isolated vessel rings studies

To further characterize the effects of preg-nancy and hypoxic exposure on vascular func-tion and to determine regional variability, numerous studies have been carried out in iso-lated rings. Through measurement of isomet-ric tension, it has been consistently demon-strated in the guinea pig uterine artery thatpregnancy decreases the contractile response toa number of agonists, including PE, norepi-nephrine (NE), epinephrine (EPI), serotoninand the thromboxane analog U46619 (Weiner

et al., 1989; Weiner et al., 1992a; Weiner et al.,1992b; Kim et al., 1994; White et al., 1998). Ef-fects of pregnancy have also been noted innonuterine vessels, though results vary de-pending on the pharmacologic agent em-ployed, as well as vessel type and size. Whilethe carotid and mesenteric artery segmentsfrom pregnant animals showed a decrease inthe contractile response to serotonin and thethromboxane mimetic U46619, respectively,(Weiner et al., 1992b), no effect of pregnancywas noted in thoracic and mesenteric artery re-sponse to PE (White et al., 1998). Pregnancy hasalso been shown to enhance relaxation toacetylcholine (ACH), A23187, and sodium ni-troprusside (SNP) in the guinea pig uterineartery, suggesting an effect on receptor- andnonreceptor-mediated, endothelium-dependentrelaxation, as well as downstream signals in thesmooth muscle (Weiner et al., 1991; White etal., 2000). In an attempt to create a more phys-iologic preparation, a dual pipette system wasused in which both ends of a vessel are can-nulated and flow through the vessel can becarefully controlled (Sillau et al., 2002). Underthese conditions, uterine artery segments frompregnant guinea pigs showed diminished con-tractile sensitivity to PE and enhanced va-sodilator response to ACH and to flow whenpreconstricted with PE (Mateev et al., 2002).

In sheep, uterine artery relaxation to A23187was enhanced during pregnancy (Xiao et al.,2001b). In these studies, however, unlike in theguinea pig, uterine artery relaxation to SNPwas unaltered by pregnancy. Further evalua-tion of pregnancy-related alterations in smoothmuscle cell function in sheep have revealedthat, although pregnancy increases endothe-lial-mediated relaxation and decreases proteinkinase C-mediated contraction in the uterineartery, it paradoxically increases acute contrac-tile response of the uterine artery to adrenergicstimulation. During ovine pregnancy, the uter-ine artery when denuded of endothelium wasless responsive to the circulating vasoconstric-tor angiotensin II, but increased its contractilesensitivity by threefold to a-agonists (Annibaleet al., 1989; Xiao and Zhang, 2002). Similarly,uterine arteries from late pregnant rats increasedthe contractile response to a-adrenergic stimu-lation (D’Angelo and Osol, 1993, 1994). These

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studies suggest that in sheep and other speciesdecreased vascular tone in the uterine artery inpregnancy is accompanied by an increase invasoconstriction reserve and contractile capa-bility to a-agonists in the smooth muscle cell.

Chronic hypoxia has been shown to alter re-sponses to agonist stimulation in uterine andnonuterine isolated vessel preparations, but theeffects are quite different in the guinea pig andsheep and vary among vascular beds even within

the same species. In evaluating changes in iso-metric tension, chronic hypoxic exposure did notalter the pregnancy-associated decrease in con-tractile response to PE in term guinea pig uterineartery segments (White et al., 1998). Furthermore,though the pregnancy-associated enhanced re-laxation to ACH was less in hypoxic comparedto normoxic pregnant animals, this difference didnot reach statistical significance (White et al.,2000) (Fig. 1). In more recent studies using the

FIG. 1. Pregnancy-enhanced relaxation to Ach in the guinea pig uterine artery at both altitudes: (A) low altitude;(B) high altitude. p-Values indicate comparisons of relaxation dose–response curves between nonpregnant and preg-nant groups by nonlinear regression analyses. Solid symbols, nonpregnant vessels; open symbols, pregnant vessels.Circles, low altitude vessels; triangles, high altitude vessels. Addition of 200-mM L-NNA to vessel bath diminishedrelaxation response to Ach in the uterine artery from (C) low altitude and (D) high altitude pregnant animals, but theeffect of L-NNA was diminished at high altitude. p-Values indicate comparisons of relaxation dose–response curvesbetween vehicle (without L-NNA) and L-NNA-treated (with L-NNA) groups by nonlinear regression analyses. Dashedlines connect values from vessels treated with L-NNA; solid lines connect values from vessels without L-NNA. s , lowaltitude vessels; D, high altitude vessels (n 5 10 low altitude nonpregnant; n 5 16 low altitude pregnant; n 5 13 highaltitude nonpregnant; n 5 9 high altitude pregnant). (Reprinted with permission from White et al., 2000.)

CHRONIC HYPOXIA ON MATERNAL VASODILATION 161

cannulated vessel preparation described above,chronic hypoxia similarly did not alter contrac-tile sensitivity to PE or inhibit relaxation to ACH(Mateev et al., 2002) in uterine artery segmentsfrom mid-gestation animals (day 30). However,uterine artery segments from hypoxic pregnantanimals exhibited a decreased vasodilator re-sponse (Fig. 2) and increased wall stress to flowcompared to their normoxic counterparts. Un-known is whether, in the latter preparation, theeffects of hypoxia on responses to PE and ACHwould have been similar in uterine arteries fromterm as opposed to mid-gestation animals; how-ever, these studies suggest that local mechanismscontrolling blood flow are potentially importanttargets through which chronic hypoxia inhibitsuterine artery blood flow in pregnant women athigh altitude (Zamudio et al., 1995b). Responsesin the nonuterine circulation are differentially al-tered by chronic hypoxia such that the middlecerebral artery exhibits increased contractile re-sponse to the thromboxane mimetic U-46619 (Sil-lau et al., 2002), while the mesenteric artery dem-onstrates decreased contractile response to PE(White et al., 1998).

In pregnant ewes exposed to chronic hypoxia(3820 m) from day 30 to approximately day 140

of gestation (maternal PaO2 values decreasedfrom 102 6 2 to 64 6 2), uterine artery seg-ments exhibited decreased contractile responseto a number of agonists, such as 5-HT and NE(Fig. 3), and enhanced relaxation to A23187 inprecontracted vessels (Hu et al., 1996b; Hu andZhang, 1997; Xiao et al., 2001c). The decreasedcontractile response was attributed to decreaseda1-adrenergic and 5-HT receptor density andagonist binding affinity in uterine arteries fromhypoxic versus normoxic animals (Hu et al.,1996a). In pregnant sheep, in nonuterine circu-lations such as cerebral vessels, chronic hypoxiaenhanced endothelium-dependent relaxation toA23187 similar to effects in the uterine artery(Longo et al., 1993). Unlike the uterine artery,however, in these studies cerebral arteries fromhypoxic sheep demonstrated decreased relax-ation to SNAP, whereas hypoxia had no effecton SNAP or 8-Br-cGMP-mediated relaxation inthe uterine artery (Xiao et al., 2001a). BecauseSNAP directly releases NO, which in turn stim-ulates vascular guanylate cyclase and cGMPsynthesis, these results suggest that chronic hy-poxia may affect some component of the cGMPrelaxation pathway in cerebral but not uterinearteries (Xiao et al., 2001a).

FIG. 2. The percent maximal vasodilation at each level of flow was measured in uterine arteries (UtA) 50% maxi-mally preconstricted with phenylephrine from pregnant (closed circles) and nonpregnant (open circles) guinea pigsthat had been housed under (A) normoxic and (B) chronically hypoxic conditions. Pregnancy increased UtA flow va-sodilation in the normoxic group animals, but the vasodilator response to flow was not sustained in the vessels fromchronically hypoxic animals, resulting in net vasoconstriction at high flow. p-values refer to comparisons betweengroups using a linear mixed effects modeling approach, which accounts for variability between animals as well asmultiple measurements on the same animal. * 5 p , 0.05 for specified comparisons. n 5 7 low altitude nonpregnant,5 low altitude pregnant; n 5 6 high altitude nonpregnant, 6 high altitude pregnant). (Reprinted with permission fromMateev et al., 2002.)

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In summary, data from guinea pig isolatedvessels suggest that the effects of chronic hy-poxia are primarily aimed at inhibiting flow-mediated relaxation, with no significant effectson agonist-induced contractile or relaxation re-sponses. In sheep, however, the net effect ofchronic hypoxia appears to be enhanced uter-ine artery relaxation as evidenced by a decreasein contractile and an increase in relaxation re-sponses to a number of agonists.

MECHANISM UNDERLYING EFFECTS OFPREGNANCY AND CHRONIC HYPOXIA

ON MATERNAL VASCULARADAPTATION

Role of the endothelium

Nitric oxide (NO) is a potent endothelium-derived vasodilator whose role in the controlof vascular tone and reactivity has been exten-

sively studied in a number of vascular beds.The contributions of NO to the enhanced re-laxation and depressed contractile responsesduring pregnancy vary among vascular bedsand with specific agonists employed. In theguinea pig uterine artery, addition of the NOSinhibitor NLA abolished the difference in con-tractile response to PE between term pregnantand nonpregnant vessels, suggesting that basalNO accounted for the pregnancy-associateddecrease in contractile response to PE (White etal., 1998). However, NLA did not completelyreverse the relaxation response to ACH in thepregnant vessels, implicating a role for addi-tional vasodilators to the enhanced endothe-lium-dependent relaxation during pregnancy(White et al., 2000). In recent flow studies, whileNLA decreased the dilator response to flow,considerable residual vasodilation remainedafter both NO and cyclooxygenase inhibition,implicating another dilator, such as EDHF, ascontributing to flow-mediated vasodilation(Mateev et al., 2002). Even within the same ves-sel, results vary depending on the agonist em-ployed. While NO partially contributes to thedecreased contractile response of guinea piguterine arteries to thromboxane (Weiner et al.,1992a), it is not responsible for the decreasedsensitivity to serotonin observed in uterine andcarotid arteries (Weiner et al., 1992b).

In sheep, several additional lines of evidencesupport the importance of NO in pregnancy-me-diated vascular alterations. Plasma nitrate con-centrations as determined by a chemilumines-cence assay in pregnant ewes were significantlyelevated compared with those of nonpregnantewes (Zhang et al., 1998) (Fig. 4A). In perfusedovine uterine arteries (Xiao et al., 1999), basalNO release was significantly higher in pregnantthan in nonpregnant uterine arteries. The ago-nist (A23187 and ATP)-induced NO release wasalso significantly enhanced in the pregnant uter-ine artery. Subsequent studies point to a mech-anism through which pregnancy increases NOthat may involve alterations in [Ca21] homeo-stasis (Xiao et al., 2001c). Simultaneous mea-surement of tension and [Ca21]i in smoothmuscle demonstrated a linear correlation withthe slope of unity between A23187-induced re-laxation and the reduction of [Ca21]

i in bothnonpregnant and pregnant uterine arteries.

FIG. 3. Pregnant (day 30) sheep were divided betweennormoxic control and chronic hypoxic (maintained at highaltitude, 3820 m, PaO2: ,60 mmHg for 110 days) groups(n5 7 per group). The fourth branch of main uterine arter-ies was obtained from near-term (140 day) pregnant sheep,and isometric contractions by norepinephrine were mea-sured. The fraction of a1-adrenoceptors occupied at eachNE concentration ([RA]/[RT]) was calculated by Furchgott’smethod. The relative intrinsic efficacy of NE was obtainedfrom the antilog of the distance between the two curvesalong the abscissa at 30% of the maximal control response.The relative intrinsic efficacy of NE in chronic hypoxic, ascompared with control arteries, was 14%.

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The A23187-induced reduction of [Ca21]i wassignificantly enhanced in pregnant uterine ar-teries. The results indicated that pregnancy in-creased NO release, which, through decreasing[Ca21]i in smooth muscle, accounted for the in-creased endothelium-dependent relaxation ofthe uterine artery.

In both guinea pig and sheep, the effect ofpregnancy on NO is associated with increasedeNOS protein expression and activity (Weiner

et al., 1994; Magness, 1997; Xiao et al., 2001a).eNOS protein expression in pregnant sheeputerine artery was 197% of that in nonpregnantartery (Xiao et al., 1999), while pregnancy in-creased eNOS protein expression fivefold inguinea pig uterine but not thoracic arteries(White et al., 2001).

Mechanisms underlying increases in NOduring pregnancy may also involve modifica-tions of downstream signaling pathways suchas in the ERK-1/2. While ERK-2 is increased inpregnant ovine uterine artery (Xiao and Zhang,2002), studies in cultured ovine uterine arteryendothelial cells confirmed that ERK-1/2 andCa21 were both required for NO and PGI2 pro-duction in pregnant uterine artery endothelialcells, as well as for PGI2 production in non-pregnant cells (Bird et al., 2000; Di et al., 2001).They also revealed that cPLA2 was much moreCa21 sensitive than eNOS in nonpregnant uter-ine artery endothelial cells and that both eNOSand cPLA2 activation showed further increasesin Ca21 sensitivity in association with ERK-phosphorylation in pregnant uterine artery en-dothelial cells. These studies suggest that Ca21

sensitivity of eNOS may be regulated in anERK-1/2 sensitive manner and that changes inCa21 and ERK-1/2 signaling are potentially amajor underlying factor in the observed preg-nancy-specific changes in NO and PGI2 pro-duction in uterine artery endothelial cells.

In addition to the selective increase in plasmanitrate levels in pregnant sheep, chronic hy-poxia has been associated with increased (1)eNOS protein and mRNA in uterine artery en-dothelium (Fig. 4B), (2) basal and the calciumionophore A23187-induced NO release from theuterine artery, and (3) endothelium-dependentrelaxation of phenylephrine-precontracted uter-ine artery rings (Xiao et al., 2001c). Further-more, the effects of chronic hypoxia appearedto be tissue specific, since eNOS was unaffectedin femoral and renal arteries from either preg-nant or nonpregnant sheep. In pregnant uter-ine artery endothelium, chronic hypoxia wasassociated with a ninefold increase in steady-state eNOS mRNA levels, with a onefold in-crease in eNOS protein. The finding that chronichypoxia did not change the apparent transla-tional efficiency of eNOS mRNA, but increasedeNOS mRNA levels, suggests that increased

FIG. 4. (A) Nonpregnant (n 5 7) and pregnant (day 30)(n 5 17) sheep were divided between normoxic controland chronic hypoxic (maintained at high altitude, 3820 m,PaO2: ,60 mmHg for 110 days) groups. Blood sampleswere taken from the jugular veins in nonpregnant andnear-term (140 day) pregnant sheep, and plasma nitrateconcentration was measured by the chemiluminescencemethod. (B) Western analysis of eNOS was performed infreshly isolated endothelial cells of uterine arteries ob-tained from the four groups of animals: control nonpreg-nant (n 5 6), control pregnant (n 5 12), hypoxic non-pregnant (n 5 6), and hypoxic pregnant (n 5 12) sheep. a,p , 0.05, pregnant versus nonpregnant; b, p , 0.05, hy-poxia versus control.

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eNOS protein expression in the pregnant uterineartery endothelium may not be regulated at thetranslational level. In addition, it is unlikely thatincreased steady-state eNOS protein levelsfound in hypoxic, compared with control, preg-nant ovine uterine artery endothelium are dueto increased protein stability, because that eNOSprotein versus message is constant among dif-ferent groups. It is not clear, however, from thesestudies to what extent increased steady-statemRNA levels resulted from increased transcrip-tion or enhanced message stability. Furthermore,whether the disproportional reduction in eNOSprotein relative to mRNA may be due to de-creased eNOS protein stability in hypoxic tissuesremains unclear.

In the sheep uterine artery, chronic hypoxiaproduced a close correlation of onefold in-creases in both basal NO release and eNOS pro-tein levels, suggesting that the increased basalNO release is predominantly due to the in-creased eNOS protein in the hypoxic arteries.On the other hand, the fold-increase of NO re-lease induced by the calcium ionophore A23187was higher than that of eNOS protein expres-sion in hypoxic uterine arteries, suggesting ahypoxic-mediated increase in the sensitivity ofthe calcium signaling pathway of eNOS in ad-dition to the enhanced protein expression. Al-though chronic hypoxia increased eNOS mRNAin the endothelium of nonpregnant ovine uter-ine arteries, it had no effect on their NOS pro-tein levels, NO production, or endothelium-de-pendent relaxation, suggesting that the effect ofmoderate chronic hypoxia is selective to preg-nant uterine arteries in sheep. The reason thatchronic hypoxia increased eNOS mRNA bythreefold, but did not significantly change eNOSprotein levels in nonpregnant uterine artery en-dothelium, may have resulted at least in partfrom the low apparent translational efficiency(0.12) of eNOS mRNA determined in ovine uter-ine artery. In fact, it has been shown that manymammalian genes translate relatively poorly(Kozak, 1991).

In the guinea pig uterine artery, the overall ef-fect of chronic hypoxia during pregnancy ap-pears to be consistent with a decrease in thecontribution of NO, depending on the studypreparation and vessel bed examined. Whilethere was no significant difference in ACH-me-

diated relaxation, the effect of NO inhibitionwas significantly less in chronically hypoxicversus normoxic uterine artery segments, sug-gesting an inhibitory effect of hypoxia on NO(White et al., 2000). This is consistent with afinding of decreased eNOS protein expressionin uterine arteries from hypoxic versus nor-moxic pregnant animals (White et al., 2001). Inaddition, the increase in eNOS protein expres-sion associated with pregnancy in normoxic an-imals was not evident in uterine arteries fromhypoxia animals. In the guinea pig middle cere-bral artery, increased contractile response to U-46619 in vessels from hypoxic animals wascompletely eliminated by the addition of NLA,suggesting that in this vessel decrease in basalNO may be responsible for the increase in con-tractile sensitivity (Sillau et al., 2002). In theevaluation of flow-mediated vasodilation inthe uterine artery, the effect of chronic hypoxiadoes not appear to be mediated through NO.Addition of the NO inhibitor NLA increasedrather than decreased the vasodilator responseto flow in uterine arteries of the chronically hy-poxic pregnant guinea pigs (Mateev et al.,2002). The addition of meclofenamate furtherraised, not lowered, flow vasodilation. Both ofthese observations indicate that decreased NOand prostaglandin vasodilator productionwere not responsible for the reduced vasodila-tor response to flow in chronic hypoxia andsuggest that a possible contribution of anothervasodilator such as EDHF to flow-mediated va-sodilation. The rise in uterine artery flow va-sodilation following NOS inhibition may havebeen due to restoration of EDHF production,given the ability of NOS inhibition to stimulatea compensatory increase in EDHF.

Mechanism of endothelium-dependent effect

It is not clear why chronic hypoxia has vari-able effects on eNOS protein expression and activity and NO production among differentspecies and vascular beds. In whole-lung ho-mogenates from chronically hypoxic piglets,eNOS protein levels were decreased in con-junction with decreased plasma and lung per-fusate NOx measurements (Fike et al., 1998). Inrat pulmonary vasculature, chronic hypoxia in-creased endothelial NO release and upregu-

CHRONIC HYPOXIA ON MATERNAL VASODILATION 165

lated endothelial (eNOS) and inducible (iNOS)gene and protein expression (Isaacson et al.,1994; Le Cras et al., 1996; Resta and Walker,1996). In contrast, in rat aorta it has been shownthat chronic hypoxia results in a decrease ineNOS protein and mRNA and impaired endo-thelium-dependent relaxation (Toporsian et al.,2000), while in the rat pulmonary circulationchronic hypoxia is associated with upregula-tion of eNOS protein (Resta et al., 1999). Fur-thermore, numerous studies in cultured endo-thelial cells have yielded conflicting results onthe effects of hypoxia (24 h) on eNOS proteinand mRNA (McQuillan et al., 1994; Liao, 1995;Arnet, 1996; Le Cras et al., 1996, Phelan andFaller, 1996; Toporsian et al., 2000). Whetherchronic hypoxia has been associated with up-or downregulation of eNOS activity, similarunderlying mechanisms have been proposed.In a recent study, chronic hypoxia increasedeNOS production of NO by increasing hsp90association and eNOS serine phosphorylation(Shi et al., 2002), while in pulmonary artery en-dothelial cells hypoxia decreased NOS activityby reducing hsp90 levels (Su and Block, 2000).In addition to hypoxic modulation of regula-tors of eNOS activity such as hsp90, substrateavailability represents another level of control.Studies in lung suggest that chronic hypoxiaimpairs L-arginine uptake (Fike et al., 2000). Inthe guinea pig MCA, addition of L-arginine tothe drinking water had no effect on contractilesensitivity of MCA to U-46619 in vessel seg-ments from chronically hypoxic animals, sug-gesting that this was not a factor to the de-creased production of NO.

Previous studies in the sheep have deter-mined the importance of Ca21 and ERK-1/2signaling in the pregnancy-specific changes inNO and PGI2. Recent findings that chronic hy-poxia upregulated ERK-2 levels and increasedCa21 sensitivity of endothelium-dependent re-laxation of pregnant ovine uterine artery indi-cate a potential role for the ERK-1/2 signalingpathway in hypoxic-mediated regulation ofsheep uterine artery endothelial function.

While hypoxia per se may have different reg-ulatory effects on eNOS protein and mRNA ex-pression, other factors that result from systemicin vivo hypoxia are also likely to be importantcontributors to the selective responses of vas-

cular beds to hypoxia. Human studies haveclearly demonstrated that uterine artery bloodflow velocity (hence the shear stress) is signif-icantly increased by chronic moderate high al-titude hypoxia in pregnant women (Zamudioet al., 1995b). This increase in blood flow isthought to be due to decreased vessel diame-ter due to hypoxic inhibition of vessel growth(Rockwell et al., 2000). Consistent with this ob-servation is the finding in recent studies thatuterine artery vasodilation response to flow isreduced in vessels from pregnant animals ex-posed to chronic hypoxia. The reduction indilator response to flow in the hypoxic guineapig uterine artery is due to factors other thanhypoxic inhibition of NO and may be associ-ated with structural changes in vessel growth,inhibition of EDHF, or possible hypoxic stim-ulation of vasoconstrictors (Mateev et al., 2002).In sheep, uterine blood flow is increased in re-sponse to chronic hypoxia in pregnant sheep(Kitanaka et al., 1989). Shear stress has beenshown to increase eNOS mRNA and proteinlevels in cultured endothelial cells (Ranjan etal., 1995), and vessels exposed to chronic ele-vations in shear stress exhibit augmented en-dothelium-dependent relaxation (Miller andVanhoutte, 1988). Thus, in the sheep uterineartery, this may represent one mechanism con-tributing to hypoxia-mediated enhancement ofvasodilation.

Effects of pregnancy on vascular smooth musclefunction in sheep

The vascular smooth muscle cell plays an im-portant role in mediating response to chronichypoxia. A number of elegant studies in sheephave suggested that chronic hypoxia alters receptor-mediated excitation–contraction cou-pling and/or signal transduction in the vascu-lar smooth muscle. A decrease in agonist bind-ing affinity to the receptor may be a generalizedeffect of chronic hypoxia on vascular smoothmuscle. Decreased binding affinities of nor-adrenaline and serotonin to a1-adrenoceptorsand serotonin2 receptors, respectively, havebeen observed in uterine and umbilical arter-ies in response to chronic hypoxia (Hu et al.,1996a; Hu et al., 1996b; Hu and Zhang, 1997).In addition to the effects of chronic hypoxia on

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agonist–receptor interaction, for a given num-ber of a1-adrenoceptors occupied, less IP3 wasproduced in the hypoxic tissues, suggestingthat chronic hypoxia attenuated coupling effi-ciency of a1-adrenoceptors to IP3 synthesis inthe uterine artery. The mechanisms underlyingthe decreased coupling efficiency of a1-adreno-ceptors to IP3 synthesis are not clear, but mayinvolve decreases in signal amplification for Gprotein coupled receptors at various levels. Gproteins coupling a1-adrenoceptors to phos-pholipase C may be decreased in response tohypoxia. This is supported by the finding thatchronic exposure to catecholamines causesdownregulation of a1-adrenoceptor coupled Gproteins (Hadcock and Malbon, 1993; Zhou etal., 1995).

Release of intracellular Ca21 from the sar-coplasmic reticulum by IP3 is a major mech-anism of pharmacomechanical coupling insmooth muscle (Somlyo and Somlyo, 1994;Zhang et al., 1995). Through the developmentof a method to estimate coupling efficiency ofthe receptor to its second messenger, it wasdemonstrated that hypoxia decreased the in-trinsic ability of a1-adrenoceptors to couple toIP3 synthesis. Chronic hypoxia reduced boththe potency and the maximal response to nor-

epinephrine-induced IP3 synthesis. Further-more, a reduction in tension development fora given amount of IP3 formation in response tochronic hypoxia was noted. In pregnant sheeputerine artery, while chronic hypoxia did notchange the density of IP3 receptors, it signifi-cantly decreased IP3 binding affinity to its re-ceptors (Hu et al., 1999).

Chronic hypoxia also affects Ca21 mobiliza-tion and Ca21 sensitivity of myofilaments inpregnant ovine uterine arteries (Zhang andXiao, 1998). Chronic hypoxia suppressed bothCa21 mobilization and agonist-mediated Ca21

sensitization induced by 5-HT in pregnant uter-ine arteries. In contrast, chronic hypoxia dif-ferentially regulated NE-induced Ca21 mobi-lization and Ca21 sensitization by increasingNE-mediated Ca21 release, but decreasing NE-induced Ca21 sensitization of myofilaments inpregnant ovine uterine arteries.

SUMMARY

In sheep, moderate high altitude exposureduring pregnancy alters endothelial and smoothmuscle cell function in such a way as to enhancevasodilation and inhibit contractile responses to

TABLE 1. COMPARISON OF THE EFFECTS OF CHRONIC HYPOXIA AND PREGNANCY ON THE CONSTRICTOR AND RELAXATION

RESPONSES IN THE GUINEA PIG AND SHEEP UTERINE AND MIDDLE CEREBRAL ARTERIES

Guinea pig Sheep

Middle MiddleUterine cerebral Uterine cerebralartery artery artery artery Author

Vasoconstrictor responsePE or NE No change Decrease Hu et al., 1996;

Hu and Zhang, 1997White et al., 1998

5HT Decrease Hu et al., 1996;Hu and Zhang, 1997

U46619 Increase Sillau et al., 2002

Vasodilator responseAch No change Increase Increase Longo, 1993

White et al., 2000;Xiao et al., 2001

A23187 Increase Increase Long et al., 1993Xiao, 2001

SNP No change Decrease Longo et al., 1993;Xiao et al., 2001

Flow-mediated dilationU46619 Decrease Mateev et al., 2002

CHRONIC HYPOXIA ON MATERNAL VASODILATION 167

a number of pharmacologic agents (Table 1).These effects are in part mediated through fac-tors that increase (1) eNOS message, protein lev-els, and enzyme activity, (2) alter receptor num-ber and affinity, and (3) modulate downstreamsignaling pathways. In sheep, these factors mayserve to maintain uterine artery blood flow,thereby sparing the development of intrauter-ine growth retardation. Guinea pig adaptationto high altitude exposure suggests that chronichypoxia opposes uterine artery flow-mediatedvasodilation seen with normal pregnancy andinhibits the contribution of NO to agonist-me-diated dilation (Table 1). While hypoxic inhi-bition of additional vasodilators such as EDHFare also implicated, inhibitory effects occur inconjunction with decreased vessel growth.These alterations are likely contributors to thedevelopment of intrauterine growth retarda-tion in the guinea pig fetus. We speculate thatthese differing adaptive responses may repre-sent interspecies variability in the ability of thematernal vascular system to adjust to chronichypoxia. This adaptability may be geneticallydetermined, as demonstrated by studies in hu-mans in which long-term inhabitants of highaltitude regions do not demonstrate the reduc-tion in uterine artery blood flow or growth re-tardation that is evident in more recent inhab-itants (Zamudio et al., 1993; Moore et al., 2001).

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Address reprint requests to:Margueritte White, MD

University of Colorado Health Sciences CenterDivision of Cardiology, Campus Box B133

4200 E. Ninth Ave.Denver, CO 80262-0001Telephone: 303-315-3926

Fax: 303 315-4871

E-mail: Margueritte.White@UCHSC.edu

Received December 18, 2002; accepted infinal form March 11, 2003