Cardioprotective and vasomotor effects of HO activity during acute and chronic hypoxia

Prolonged hypoxia leads to the development of pulmonary hypertension. Recent reports have suggested enhancement of heme oxygenase (HO), the major source of intracellular carbon monoxide (CO), prevents hypoxia-induced pulmonary hypertension and vascular remodeling in rats. Therefore, we hypothesized that inhibition of HO activity by tin protoporphyrin (SnPP) would exacerbate the development of pulmonary hypertension. Rats were injected weekly with either saline or SnPP (50 micromol/kg) and exposed to hypobaric hypoxia or room air for 5 wk. Pulmonary and carotid arteries were catheterized, and animals were allowed to recover for 48 h. Pulmonary and systemic pressures, along with cardiac output, were recorded during room air and acute 10% O2 breathing in conscious rats. No difference was detected in pulmonary artery pressure between saline- and SnPP-treated animals in either normoxic or hypoxic groups. However, blockade of HO activity altered both systemic and pulmonary vasoreactivity to acute hypoxic challenge. Despite no change in baseline pulmonary artery pressure, all rats treated with SnPP had decreased ratio of right ventricular (RV) weight to left ventricular (LV) plus septal (S) weight (RV/LV + S) compared with saline-treated animals. Echocardiograms suggested dilatation of the RV and decreased RV function in hypoxic SnPP-treated rats. Together these data suggest that inhibition of HO activity and CO production does not exacerbate pulmonary hypertension, but rather that HO and CO may be involved in mediating pulmonary and systemic vasoreactivity to acute hypoxia and hypoxia-induced RV function.     

Developmental differences in pulmonary eNOS expression in response to chronic hypoxia in the rat.

Chronic hypoxia (CH) increases pulmonary endothelial nitric oxide synthase (eNOS) protein levels in adult rats but decreases eNOS protein levels in neonatal pigs. We hypothesized that this differing response to CH is due to developmental rather than species differences. Adult and neonatal rats were placed in either hypobaric hypoxia or normoxia for 2 wk. At that time, body weight, hematocrit, plasma nitrite/nitrate (NOx(-)), and right ventricular and total ventricular heart weights were measured. Percent pulmonary arterial wall area of 20-50 and 51-100 microm arteries were also determined. Total lung protein extracts were assayed for eNOS levels by using immunoblot analysis. Compared with their respective normoxic controls, both adult and neonatal hypoxic groups demonstrated significantly decreased body weight, elevated hematocrit, and elevated right ventricular-to-total ventricular weight ratios. Both adult and neonatal hypoxic groups also demonstrated significantly larger percent pulmonary arterial wall area compared with their respective normoxic controls. Hypoxic adult pulmonary eNOS protein and plasma NOx(-) were significantly greater than levels found in normoxic adults. In contrast, hypoxic neonatal pulmonary eNOS protein and plasma NOx(-) were significantly less compared with normoxic neonates. We conclude that there is a developmental difference in eNOS expression and nitric oxide production in response to CH.     

Attenuation of pulmonary arterial smooth muscle cell proliferation following hypoxic pulmonary hypertension by the Na+/H+ exchange inhibitor amiloride

Chronic hypoxia results in pulmonary hypertension. To investigate the role of Na+/H+ exchange in this process, we determined the effect of amiloride, a Na+/H+ exchange inhibitor, on hypoxic pulmonary hypertension and pulmonary arterial smooth muscle cell proliferation, both in vivo and in vitro. Sprague-Dawley rats were placed either in a hypobaric, hypoxic chamber (10.5% 02) or under normal 21% O2 atmosphere for 8 h each day for 3 weeks. Rats under hypoxic conditions received 1, 3, or 10 mg/kg/d amiloride or the vehicle alone. Hematologic indices, including red blood cells, hemoglobin, hematocrit and mean corpuscular hemoglobin increased in hypoxic rats, but these changes were prevented by treatment with amiloride. In the hypoxic rats, the right ventricular systolic pressure and right ventricular hypertension index (weight ratio of right ventricular to left and septum together) were increased by 88% and 129%, respectively. Arteriolar wall thickness and area in the hypoxia-treated animals increased 3- and 2-fold, respectively, over normoxic controls; the increase in each of these indices was attenuated by amiloride in a dose-dependent manner. In cultured pulmonary arterial smooth muscle cells, hypoxia greatly increased cellular proliferation, and this similarly showed a dose-dependent attenuation in the presence of amiloride. Amiloride did not affect blood pressure in vivo or cause cell damage in vitro. These data suggest that the Na+/H+ exchange inhibitor amiloride may represent an effective adjunctive therapy in pulmonary hypertension induced by chronic hypoxia.     

PAF antagonists inhibit pulmonary vascular remodeling induced by hypobaric hypoxia in rats.

Chronic hypoxia causes pulmonary hypertension and pulmonary vascular remodeling in rats. Because platelet-activating factor (PAF) levels increase in lung lavage fluid and in plasma from chronically hypoxic rats, we examined the effect of two specific, structurally unrelated PAF antagonists, WEB 2170 and BN 50739, on hypoxia-induced pulmonary vascular remodeling. Treatment with either agent reduced hypoxia-induced pulmonary hypertension and right ventricular hypertrophy at 3 wk of hypoxic exposure (simulated altitude 5,100 m) but did not affect cobalt (CoCl2)-induced pulmonary hypertension. The PAF antagonists had no effect on the hematocrit of normoxic or chronically hypoxic rats or CoCl2-treated rats. Hypoxia-induced pulmonary hypertension was associated with an increase in the vessel wall thickness of the muscular arteries and reduction in the number of peripheral arterioles. In WEB 2170-treated rats, these changes were significantly less severe than those observed in untreated chronically hypoxic rats. PAF receptor blockade had no acute hemodynamic effects; i.e., it did not affect pulmonary arterial pressure or cardiac output nor did it affect the magnitude of acute hypoxic pulmonary vasoconstriction in awake normoxic or chronically hypoxic rats. Isolated lungs from chronically hypoxic rats showed a pressor response to the chemotactic tripeptide N-formyl-Met-Leu-Phe (fMLP) and an increase in the number of leukocytes lavaged from the pulmonary circulation. In vivo treatment with WEB 2170 significantly reduced the fMLP-induced pressor response compared with that observed in isolated lungs from untreated chronically hypoxic rats. These results suggest that PAF contributes to the development of chronic pulmonary hypertension induced by chronic hypoxia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Exercise training improves lung gas exchange and attenuates acute hypoxic pulmonary hypertension but does not prevent pulmonary hypertension of prolonged hypoxia.

Our laboratory has previously shown an attenuation of hypoxic pulmonary hypertension by exercise training (ET) (Henderson KK, Clancy RL, and Gonzalez NC. J Appl Physiol 90: 2057-2062, 2001), although the mechanism was not determined. The present study examined the effect of ET on the pulmonary arterial pressure (Pap) response of rats to short- and long-term hypoxia. After 3 wk of treadmill training, male rats were divided into two groups: one (HT) was placed in hypobaric hypoxia (380 Torr); the second remained in normoxia (NT). Both groups continued to train in normoxia for 10 days, after which they were studied at rest and during hypoxic and normoxic exercise. Sedentary normoxic (NS) and hypoxic (HS) littermates were exposed to the same environments as their trained counterparts. Resting and exercise hypoxic arterial P(O2) were higher in NT and HT than in NS and HS, respectively, although alveolar ventilation of trained rats was not higher. Lower alveolar-arterial P(O2) difference and higher effective lung diffusing capacity for O2 in NT vs. NS and in HT vs. HS suggest ET improved efficacy of gas exchange. Pap and Pap/cardiac output were lower in NT than NS in hypoxia, indicating that ET attenuates the initial vasoconstriction of hypoxia. However, ET had no effect on chronic hypoxic pulmonary hypertension: Pap and Pap/cardiac output in hypoxia were similar in HS vs HT. However, right ventricular weight was lower in HT than in HS, although Pap was not different. Because ET attenuates the initial pulmonary vasoconstriction of hypoxia, development of pulmonary hypertension may be delayed in HT rats, and the time during which right ventricular afterload is elevated may be shorter in this group. ET effects may improve the response to acute hypoxia by increasing efficacy of gas exchange and lowering right ventricular work.     

肺血管EDNO及其合酶在大鼠低氧性肺动脉高压形成中的作用

本研究观察了低氧对大鼠肺组织和血管内皮一氧化氮合酶（ＮＯＳ）活性及内皮衍生一氧化氮（ＥＤＮＯ）依赖性舒张反应的影响,以及ＮＯＳ抑制剂（Ｌ－ＮＡＭＥ）对常氧和低氧大鼠肺组织和血管内皮ＮＯＳ活性及颈、肺动脉血压（ＣＡＰｓ、ｍＰＡＰ）的作用。结果表明常氧大鼠肺泡内无肌性血管内皮未见ＮＯＳ活性,其肺血管床对ＥＤＮＯ依赖性舒血管物质ＢＫ没有反应,注射Ｌ－ＮＡＭＥ后大鼠ｍＰＡＰ略有降低,ＣＡＰｓ有所升高。低氧大鼠肺泡内无肌性血管内皮显示ＮＯＳ活性,对ＢＫ的ＥＤＮＯ依赖性舒张反应呈剂量依赖性增大,注射Ｌ－ＮＡＭＥ使低氧大鼠ｍＰＡＰ显著降低（Ｐ＜０．０１）,ＣＡＰｓ显著升高（Ｐ＜０．０５）。提示肺血管ＥＤＮＯ及其合酶在维持正常成年大鼠肺循环低压低阻中的生理作用值得进一步探讨;低氧引起肺血管内皮ｅｃＮＯＳ活性增加和ＥＤＮＯ生成增多可能起到限制肺动脉压过度升高的调制作用,也可能对肺血管内皮产生毒性作用,反而促进肺动脉高压的发生和发展。     

N-acetylcysteine inhibits hypoxic pulmonary hypertension most effectively in the initial phase of chronic hypoxia

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 (F(iO2) = 0.1) and treated with the antioxidant, N-acetylcysteine (NAC, 20 g/l in drinking water). NAC was given "early" (7 days before and the first 7 days of hypoxia) or "late" (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 +/- 0.9 mmHg) than late NAC treatment (30.5 +/- 1.0 mmHg, P < 0.05; hypoxic without NAC 32.6 +/- 1.2 mmHg, normoxic controls 14.9 +/- 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.     

Neonatal dexamethasone treatment increases the risk for pulmonary hypertension in adult rats

Dexamethasone (Dex) treatment during a critical period of lung development causes lung hypoplasia in infant rats. However, the effects of Dex on the pulmonary circulation are unknown. To determine whether Dex increases the risk for development of pulmonary hypertension, we treated newborn Sprague-Dawley rats with Dex (0.25 microg/day, days 3-13). Litters were divided equally between Dex-treated and vehicle control (ethanol) rats. Rats were raised in either room air until 10 wk of age (normoxic groups) or room air until 7 wk of age and then in a hypoxia chamber (inspired O(2) fraction = 0.10; hypoxic groups) for 3 wk to induce pulmonary hypertension. Compared with vehicle control rats, Dex treatment of neonatal rats reduced alveolarization (by 42%; P < 0.05) and barium-filled pulmonary artery counts (by 37%; P < 0.05) in 10-wk-old adults. Pulmonary arterial pressure and the ratio of right ventricle to left ventricle plus septum weights (RV/LV+S) were higher in 10-wk-old Dex-treated normoxic rats compared with those in normoxic control rats (by 16 and 16% respectively; P < 0.05). Small pulmonary arteries of adult normoxic Dex-treated rats showed increased vessel wall thickness compared with that in control rats (by 15%; P < 0.05). After 3 wk of hypoxia, RV/LV+S values were 36% higher in rats treated with Dex in the neonatal period compared with those in hypoxic control rats (P < 0.05). RV/LV+S was 42% higher in hypoxic control rats compared with those in normoxic control rats (P < 0.05). We conclude that Dex treatment of neonatal rats caused sustained lung hypoplasia and increased pulmonary arterial pressures and augmented the severity of hypoxia-induced pulmonary hypertension in adult rats.     

Pulmonary pressure reduction attenuates expression of proteins identified by lung proteomic profiling in pulmonary hypertensive rats

The present study was designed to analyze protein expression in lungs from pulmonary hypertensive rats in order to identify novel signaling pathways. This was achieved by proteomic studies in which proteins from lung homogenates from hypoxic were compared to normoxic rats. The expression of these proteins was then investigated in lungs from hypoxic rats treated with either an activator of soluble guanylyl cyclase, BAY 412272, or an inhibitor of phosphodiesterase type 5, sildenafil. The proteomic study revealed an up-regulation of guanine nucleotide-binding protein β, GST-ω-1, cathepsin D, chloride intracellular channel subunit 5, annexin A4, F-actin capping protein CapZ (CapZα), and the translation factor elongation factor 1 δ in lungs from chronic hypoxic rats with pulmonary hypertension. Immunohistochemistry revealed that CapZα, cathepsin D, and annexin A4 were expressed in the pulmonary vascular wall and immunoblotting showed these proteins correlated to alterations in muscularization. Both drugs inhibited hypoxia-induced increase in right ventricular systolic pressure and pulmonary arterial muscularization, and prevented most of the protein regulations observed after hypoxia. These findings suggest that pulmonary pressure is an important factor for initiating signaling pathways leading to protein expression and muscularization in the pulmonary vasculature.     

Generation of oxidative stress contributes to the development of pulmonary hypertension induced by hypoxia.

Chronic hypoxia causes pulmonary hypertension and right ventricular hypertrophy associated with pulmonary vascular remodeling. Because hypoxia might promote generation of oxidative stress in vivo, we hypothesized that oxidative stress may play a role in the hypoxia-induced cardiopulmonary changes and examined the effect of treatment with the antioxidant N-acetylcysteine (NAC) in rats. NAC reduced hypoxia-induced cardiopulmonary alterations at 3 wk of hypoxia. Lung phosphatidylcholine hydroperoxide (PCOOH) increased at days 1 and 7 of the hypoxic exposure, and NAC attenuated the increase in lung PCOOH. Lung xanthine oxidase (XO) activity was elevated from day 1 through day 21, especially during the initial 3 days of the hypoxic exposure. The XO inhibitor allopurinol significantly inhibited the hypoxia-induced increase in lung PCOOH and pulmonary hypertension, and allopurinol treatment only for the initial 3 days also reduced the hypoxia-induced right ventricular hypertrophy and pulmonary vascular thickening. These results suggest that oxidative stress produced by activated XO in the induction phase of hypoxic exposure contributes to the development of chronic hypoxic pulmonary hypertension.     

Impaired iNOS-sGC-cGMP signalling contributes to chronic hypoxic and hypercapnic pulmonary hypertension in rat

Nitric oxide (NO) is an important vascular modulator in the development of pulmonary hypertension. NO exerts its regulatory effect mainly by activating soluble guanylate cyclase (sGC) to synthesize cyclic guanosine monophosphate (cGMP). Exposure to hypoxia causes pulmonary hypertension. But in lung disease, hypoxia is commonly accompanied by hypercapnia. The aim of this study was to examine the changes of sGC enzyme activity and cGMP content in lung tissue, as well as the expression of inducible nitric oxide synthase (iNOS) and sGC in rat pulmonary artery after exposure to hypoxia and hypercapnia, and assess the role of iNOS-sGC-cGMP signal pathway in the development of hypoxic and hypercapnic pulmonary hypertension. Male Sprague-Dawley rats were exposed to hypoxia and hypercapnia for 4 weeks to establish model of chronic pulmonary hypertension. Weight-matched rats exposed to normoxia served as control. After exposure to hypoxia and hypercapnia, mean pulmonary artery pressure, the ratio of right ventricle/left ventricle+septum, and the ratio of right ventricle/body weight were significantly increased. iNOS mRNA and protein levels were significantly increased, but sGC α(1) mRNA and protein levels were significantly decreased in small pulmonary arteries of hypoxic and hypercapnic exposed rat. In addition, basal and stimulated sGC enzyme activity and cGMP content in lung tissue were significantly lower after exposure to hypoxia and hypercapnia. These results demonstrate that hypoxia and hypercapnia lead to the upregulation of iNOS expression, downregulation of sGC expression and activity, which then contribute to the development of pulmonary hypertension.     

Pressure load: the main factor for altered gene expression in right ventricular hypertrophy in chronic hypoxic rats

Background

The present study investigated whether changes in gene expression in the right ventricle following pulmonary hypertension can be attributed to hypoxia or pressure loading.

Methodology/Principal Findings

To distinguish hypoxia from pressure-induced alterations, a group of rats underwent banding of the pulmonary trunk (PTB), sham operation, or the rats were exposed to normoxia or chronic, hypobaric hypoxia. Pressure measurements were performed and the right ventricle was analyzed by Affymetrix GeneChip, and selected genes were confirmed by quantitative PCR and immunoblotting. Right ventricular systolic blood pressure and right ventricle to body weight ratio were elevated in the PTB and the hypoxic rats. Expression of the same 172 genes was altered in the chronic hypoxic and PTB rats. Thus, gene expression of enzymes participating in fatty acid oxidation and the glycerol channel were downregulated. mRNA expression of aquaporin 7 was downregulated, but this was not the case for the protein expression. In contrast, monoamine oxidase A and tissue transglutaminase were upregulated both at gene and protein levels. 11 genes (e.g. insulin-like growth factor binding protein) were upregulated in the PTB experiment and downregulated in the hypoxic experiment, and 3 genes (e.g. c-kit tyrosine kinase) were downregulated in the PTB and upregulated in the hypoxic experiment.

Conclusion/Significance

Pressure load of the right ventricle induces a marked shift in the gene expression, which in case of the metabolic genes appears compensated at the protein level, while both expression of genes and proteins of importance for myocardial function and remodelling are altered by the increased pressure load of the right ventricle. These findings imply that treatment of pulmonary hypertension should also aim at reducing right ventricular pressure.     

Three-week neonatal hypoxia reduces blood CGRP and causes persistent pulmonary hypertension in rats

To increase understanding of persistent pulmonary hypertension, we examined chronic pulmonary effects of hypoxia at birth and their relationships with immunoreactive levels of the potent vasodilator, calcitonin gene-related peptide (CGRP). Rats were born in 10% hypobaric hypoxia, where they remained for 1-2 days, or in 15% hypoxia, where they remained for 21 days. All were then reared in normoxia for 3 mo followed by reexposure to 10% hypoxia for 7 days (H-->H) or continued normoxia (H-->N); age-matched normoxic rats were hypoxic for the last 7 days (N-->H) or normoxic throughout (N-->N). Results are as follows. Pulmonary arterial pressure (P(PA)) in 10% H-->N rats was normal at the end of the experiment (13 wk), but in rats reexposed to hypoxia (H-->H), pressure rose to 19% above N-->H controls. In 15% H-->N rats, P(PA) remained high, similar to that of N-->H rats, and increased further by 40% on reexposure (H-->H). Medial thickness of small pulmonary arteries in 10% H-->H rats also increased by 40% over N-->H controls and was equally high in 15% H-->N and H-->H rats. In N-->H rats from both experiments, right ventricular hypertrophy index (RVH) was increased after hypoxia at 15-16 wk. Also, in the 15% study, RVH remained elevated in H-->N rats and increased in H-->H rats by 19% above N-->H controls. Blood CGRP was reduced by neonate and adult hypoxia, and hypoxic reexposure (H-->H) further lowered blood CGRP in the 15% but not 10% study. Declining left ventricular blood CGRP correlated highly with logarithmically increasing P(PA) in the 15% study (r = -0.81, P = 0.000). In conclusion, 1) short perinatal exposure to 10% O(2) exacerbated pulmonary hypertension with hypoxia later in life, 2) 15% O(2) at birth and for 21 days caused persistent pulmonary hypertension and exacerbation with reexposure, and 3) P(PA) correlated highly with declining blood CGRP levels in the 15% study.     

Impaired iNOS–sGC–cGMP signalling contributes to chronic hypoxic and hypercapnic pulmonary hypertension in rat

Nitric oxide (NO) is an important vascular modulator in the development of pulmonary hypertension. NO exerts its regulatory effect mainly by activating soluble guanylate cyclase (sGC) to synthesize cyclic guanosine monophosphate (cGMP). Exposure to hypoxia causes pulmonary hypertension. But in lung disease, hypoxia is commonly accompanied by hypercapnia. The aim of this study was to examine the changes of sGC enzyme activity and cGMP content in lung tissue, as well as the expression of inducible nitric oxide synthase (iNOS) and sGC in rat pulmonary artery after exposure to hypoxia and hypercapnia, and assess the role of iNOS–sGC–cGMP signal pathway in the development of hypoxic and hypercapnic pulmonary hypertension. Male Sprague–Dawley rats were exposed to hypoxia and hypercapnia for 4 weeks to establish model of chronic pulmonary hypertension. Weight‐matched rats exposed to normoxia served as control. After exposure to hypoxia and hypercapnia, mean pulmonary artery pressure, the ratio of right ventricle/left ventricle + septum, and the ratio of right ventricle/body weight were significantly increased. iNOS mRNA and protein levels were significantly increased, but sGC α₁ mRNA and protein levels were significantly decreased in small pulmonary arteries of hypoxic and hypercapnic exposed rat. In addition, basal and stimulated sGC enzyme activity and cGMP content in lung tissue were significantly lower after exposure to hypoxia and hypercapnia. These results demonstrate that hypoxia and hypercapnia lead to the upregulation of iNOS expression, downregulation of sGC expression and activity, which then contribute to the development of pulmonary hypertension. Copyright © 2012 John Wiley & Sons, Ltd.     

Alterations in atrial and plasma atrial natriuretic factor (ANF) content during development of hypoxia-induced pulmonary hypertension in the rat

Distension of the atrial wall has been proposed as a signal for the increased release of atrial natriuretic factor (ANF) from atrial myocytes in response to perceived volume overload. To determine whether pressure changes resulting from hypertension in the pulmonary circulation may stimulate release of ANF, rats were exposed to chronic hypobaric hypoxia for 3 or 21 days and the ANF concentration in the atria and plasma were determined by specific radioimmunoassay. Exposure to chronic hypoxia resulted in significant increases in hematocrit at both 3 (p less than 0.025) and 21 days (p less than 0.005) and in the development of right ventricular hypertrophy (RVH) expressed as the ratio of the weight of the right ventricle to the weight of the left ventricle and septum (RV/LV+S) at both 3 (RV/LV+S = 0.278 +/- 0.005) and 21 days (RV/LV+S = 0.536 +/- 0.021). After 21 days, left atrial (LA) ANF content was significantly increased in hypoxic rats compared to controls (508 +/- 70 ng/mg tissue vs 302 +/- 37 ng/mg), while right atrial (RA) ANF content was significantly reduced (440 +/- 45 vs 601 +/- 58 ng/mg). At this time, plasma ANF concentration was significantly elevated compared to controls (238 +/- 107 pg/ml vs 101 +/- 10 pg/ml). These results suggest that the development of pulmonary hypertension following chronic hypobaric exposure induces altered atrial ANF content and increased plasma ANF concentration as a result of altered distension of the atrial wall.     

Targeted blocking of gene expression for CGRP receptors elevates pulmonary artery pressure in hypoxic rats

We previously described the protection by calcitonin gene-related peptide (CGRP) against hypoxic pulmonary hypertension. Here, we examine the roles of its putative receptor RDC-1 and receptor activity-modifying protein (RAMP) 1 in mediating this protection by selectively inhibiting their synthesis. RAMP1 is an accessory protein for another putative CGRP receptor, calcitonin receptor-like receptor. Antisense oligodeoxyribonucleotides (ASODNs, 5 mg.kg-1.day-1 or 5 and 10 mg.kg-1.day-1 for RDC-1) targeting RAMP1 and RDC-1 mRNAs were chronically infused to the pulmonary circulation of male Sprague-Dawley rats during 7 days of normoxia or hypobaric hypoxia (380 mmHg), and alpha-CGRP ASODN was used as a technical control. CGRP, RAMP1, and RDC-1 ASODNs significantly elevated pulmonary artery pressure (PPA) in chronic hypoxic rats compared with hypoxic mismatched ASODN (MMODN) and saline vehicle controls. CGRP and RAMP1 ASODNs raised PPA in normoxic rats briefly exposed to 10% O2 above MMODN and saline controls. Moreover, normoxic rats treated with CGRP ASODN had higher basal pulmonary vascular tone compared with controls. These data confirm the protective role of CGRP in the pulmonary circulation and suggest that endogenous RAMP1 and RDC-1 are essential in regulation of PPA in hypoxia. This is the first in vivo evidence supporting RDC-1 and RAMP1 as functional CGRP receptor and receptor component.     

Effect of prenatal hypoxia on heat stress-mediated cardioprotection in adult rat heart

Fetal programming has profound effects on cardiovascular function in later adult life. We tested the hypothesis that chronic hypoxic exposure during fetal development downregulates endogenous cardioprotective mechanisms in adult rats. Time-dated pregnant rats were divided between normoxic and hypoxic (10.5% O2 from days 15 to 21 of gestation) groups. The male progeny were studied at 2 mo of age. Rats were subjected to heat stress (42 degrees C for 15 min). After 24 h, hearts were excised and subjected to 30 min of global ischemia and 1 h of reperfusion. Prenatal hypoxia did not change adult rat body weight and heart weight, but significantly increased the cross-sectional area of a left ventricular (LV) myocyte. Heat stress significantly improved postischemic recovery of LV function in normoxic control rats, but not in prenatally hypoxic rats. The infarct size in the LV resulting from ischemia-reperfusion was reduced by the heat stress pretreatment in control rats, but not in prenatally hypoxic rats. In accordance, heat stress significantly increased LV myocardial content of heat shock protein 70 only in normoxic control rats. In addition, there was a significant decrease in the LV myocardial content of the PKC-epsilon isoform in prenatally hypoxic rats compared with control rats. We conclude that prenatal hypoxia causes in utero programming of hsp70 gene in the LV, leading to an inhibition of its response to heat stress and a loss of cardioprotection in later adult life.     

Reduction of chronic hypoxic pulmonary hypertension in the rat by beta-aminopropionitrile

We administered antifibrotic agent beta-aminopropionitrile (BAPN) to rats exposed to 10% O2-90% N2 for 3 wk to prevent excess vascular collagen accumulation. Groups of Sprague-Dawley rats studied were air breathing, hypoxic, and hypoxic treated with BAPN, 150 mg/kg twice daily intraperitoneally. After the 3-wk period, we measured mean right ventricular pressure (RVP), the ratio of weight of right ventricle to left ventricle plus septum (RV/LV + S), and hydroxyproline content of the main pulmonary artery (PA) trunk. Hypoxia increased RVP from 14 to 29 mmHg; RVP was 21 mmHg in hypoxic BAPN-treated animals. Hypoxia increased the RV/LV + S ratio from 0.28 to 0.41; the ratio was 0.32 in hypoxic BAPN-treated animals. Hypoxia increased PA hydroxyproline from 20 to 239 micrograms/artery; hydroxyproline was 179 micrograms/artery in hypoxic BAPN-treated animals. Thus BAPN prevented pulmonary hypertension, right ventricular hypertrophy, and excess vascular collagen produced by hypoxia. We conclude that vascular collagen contributes to the maintenance of chronic hypoxic pulmonary hypertension.     

Regulatory effect of AMP-activated protein kinase on pulmonary hypertension induced by chronic hypoxia in rats: in vivo and in vitro studies

Activation of AMP-activated protein kinase (AMPK) plays an important role in cardiovascular protection. It can inhibit arterial smooth muscle cell proliferation and cardiac fibroblast collagen synthesis induced by anoxia. However, the role of AMPK-dependent signalling cascades in the pulmonary vascular system is currently unknown. This study aims to determine the effects of AMPK on pulmonary hypertension and pulmonary vessel remodelling induced by hypoxia in rats using in vivo and in vitro studies. In vivo study: pulmonary hypertension, right ventricular hypertrophy and pulmonary vascular remodelling were found in hypoxic rats. Meanwhile, AMPKα₁ and phosphorylated AMPKα₁ were increased markedly in pulmonary arterioles and lung tissues. Mean pulmonary arterial pressure, index of right ventricular hypertrophy and parameters of pulmonary vascular remodelling, including vessel wall area/total area, density of nuclei in medial smooth muscle cells, and thickness of the medial smooth muscle cell layer were markedly suppressed by AICAR, an AMPK agonist. In vitro study: the expression of AMPKα₁ and phosphorylated AMPKα₁ was increased in pulmonary artery smooth muscle cells (PASMCs) under hypoxic conditions. The effects of PASMC proliferation stimulated by hypoxia were reinforced by treatment with Compound C, an AMPK inhibitor. AICAR inhibited the proliferation of PASMCs stimulated by hypoxia. These findings suggest that AMPK is involved in the formation of hypoxia-induced pulmonary hypertension and pulmonary vessel remodelling. Up-regulating AMPK can contribute to decreasing pulmonary vessel remodelling and pulmonary hypertension induced by hypoxia.     

Enhanced alpha1-adrenergic trophic activity in pulmonary artery of hypoxic pulmonary hypertensive rats

Mechanisms that induce the excessive proliferation of vascular wall cells in hypoxic pulmonary hypertension (PH) are not fully understood. Alveolar hypoxia causes sympathoexcitation, and norepinephrine can stimulate alpha(1)-adrenoceptor (alpha(1)-AR)-dependent hypertrophy/hyperplasia of smooth muscle cells and adventitial fibroblasts. Adrenergic trophic activity is augmented in systemic arteries by injury and altered shear stress, which are key pathogenic stimuli in hypoxic PH, and contributes to neointimal formation and flow-mediated hypertrophic remodeling. Here we examined whether norepinephrine stimulates growth of the pulmonary artery (PA) and whether this is augmented in PH. PA from normoxic and hypoxic rats [9 days of 0.1 fraction of inspired O(2) (Fi(O(2)))] was studied in organ culture, where wall tension, Po(2), and Pco(2) were maintained at values present in normal and hypoxic PH rats. Norepinephrine treatment for 72 h increased DNA and protein content modestly in normoxic PA (+10%, P < 0.05). In hypoxic PA, these effects were augmented threefold (P < 0.05), and protein synthesis was increased 34-fold (P < 0.05). Inferior thoracic vena cava from normoxic or hypoxic rats was unaffected. Norepinephrine-induced growth in hypoxic PA was dose dependent, had efficacy greater than or equal to endothelin-1, required the presence of wall tension, and was inhibited by alpha(1A)-AR antagonist. In hypoxic pulmonary vasculature, alpha(1A)-AR was downregulated the least among alpha(1)-AR subtypes. These data demonstrate that norepinephrine has trophic activity in the PA that is augmented by PH. If evident in vivo in the pulmonary vasculature, adrenergic-induced growth may contribute to the vascular hyperplasia that participates in hypoxic PH.