An antifibrotic agent reduces blood pressure in established pulmonary hypertension in the rat

We have shown that administration of the antifibrotic agent cis-4-hydroxy-L-proline (cHyp) to rats at the onset of exposure to hypoxia prevents collagen accumulation in pulmonary arteries and the rise in pulmonary blood pressure. In this experiment, we tested whether cHyp is effective when administered after hypertension was already established. Rats were exposed to hypoxia (10% O2) for 21 days. Groups were hypoxic animals treated with cHyp (200 mg/kg sc twice daily) on days 10-21 (hypoxic cHyp) and saline-injected hypoxic animals (hypoxic). On day 21, we measured mean right ventricular pressure, hematocrit, collagen content of main and intrapulmonary arteries, and wall thickness of arterioles. Treatment reduced right ventricular pressure from 21 +/- 1 to 17 +/- 1 mmHg (P less than 0.05), hematocrit from 66 +/- 1 to 56 +/- 1% (P less than 0.05), hydroxyproline content of intrapulmonary arteries from 30 +/- 3 to 11 +/- 2 micrograms/vessel (P less than 0.05), and wall thickness from 27 +/- 3 to 16 +/- 2 microns (P less than 0.05). These results show that vascular collagen content is increased in established pulmonary hypertension and that cHyp treatment is effective in partially preventing the hemodynamic, structural, and biochemical changes if started after pulmonary hypertension is established. cHyp may also affect the rheological properties of blood.     

Hyperoxia and recovery from hypoxia alter collagen in peripheral pulmonary arteries similarly

Chronic hypoxia causes pulmonary hypertension, the mechanism of which includes altered collagen metabolism in the pulmonary vascular wall. This chronic hypoxic pulmonary hypertension is gradually reversible upon reoxygenation. The return to air after the adjustment to chronic hypoxia resembles in some aspects a hyperoxic stimulus and we hypothesize that the changes of extracellular matrix proteins in peripheral pulmonary arteries may be similar. Therefore, we studied the exposure to moderate chronic hyperoxia (FiO2 = 0.35, 3 weeks) in rats and compared its effects on the rat pulmonary vasculature to the effects of recovery (3 weeks) from chronic hypoxia (FiO2 = 0.1, 3 weeks). Chronically hypoxic rats had pulmonary hypertension (Pap = 26 +/- 3 mm Hg, controls 16 +/- 1 mm Hg) and right ventricular hypertrophy. Pulmonary arterial blood pressure and right ventricle weight normalized after 3 weeks of recovery in air (Pap = 19 +/- 1 mm Hg). The rats exposed to moderate chronic hyperoxia also did not have pulmonary hypertension (Pap = 18 +/- 1 mm Hg, controls 17 +/- 1 mm Hg). Collagenous proteins isolated from the peripheral pulmonary arteries (100-300 microm) were studied using polyacrylamide gel electrophoresis. A dominant low molecular weight peptide (approx. 76 kD) was found in hypoxic rats. The proportion of this peptide decreases significantly in the course of recovery in air. In addition, another larger peptide doublet was found in rats recovering from chronic hypoxia. It was localized in polyacrylamide gels close to the zone of alpha2 chain of collagen type I. It was bound to anticollagen type I antibodies. An identically localized peptide was found in rats exposed to moderate chronic hyperoxia. The apparent molecular weight of this collagen fraction suggests that it is a product of collagen type I cleavage by a rodent-type interstitial collagenase (MMP-13). We conclude that chronic moderate hyperoxia and recovery from chronic hypoxia have a similar effect on collagenous proteins of the peripheral pulmonary arterial wall.     

Hypoxia-induced activation in small isolated pulmonary arteries from the cat

Effects of hypoxia on force development and membrane potential were studied in isolated small (less than 300 microns diam) and large (greater than 500 microns diam) pulmonary arteries from cats. There was a consistent and reproducible hypoxic constrictor response in small pulmonary arteries that began at PO2 values between 350 and 300 Torr and reached a maximum at PO2 between 50 and 30 Torr. In the small artery smooth muscle cell the membrane potential, which was -51 +/- 1.4 mV at a PO2 of 400 Torr, was depolarized to -37 +/- 2 mV at a PO2 of 50 Torr. In contrast, larger arteries did not exhibit significant hypoxic constriction or depolarization upon exposure to low PO2. Constriction in small arteries was not blocked by phentolamine. Treatment with a low dose of indomethacin (10(-9) M) augmented the response; however, a larger dose of indomethacin (10(-3) M) blocked the constriction to hypoxia but not to 30 mM KCl. Depolarization during hypoxia was not blocked by ouabain. Results of this study suggest that the hypoxic response of these isolated small pulmonary vessels may be like that seen in the intact lung. Furthermore, these data suggest that hypoxic vasoconstriction may be mediated by electrical events occurring at the pulmonary arterial muscle cell membrane either directly or via mediators released from the vessel wall.     

Polycythemia and vascular remodeling in chronic hypoxic pulmonary hypertension in guinea pigs.

Chronic hypoxia increases pulmonary arterial pressure (PAP) as a result of vasoconstriction, polycythemia, and vascular remodeling with medial thickening. To determine whether preventing the polycythemia with repeated bleeding would diminish the pulmonary hypertension and remodeling, we compared hemodynamic and histological profiles in hypoxic bled (HB, n = 6) and hypoxic polycythemic guinea pigs (H, n = 6). After 10 days in hypoxia (10% O2), PAP was increased from 10 +/- 1 (SE) mmHg in room air controls (RA, n = 5) to 20 +/- 1 mmHg in H (P less than 0.05) but was lower in HB (15 +/- 1 mmHg, P less than 0.05 vs. H). Cardiac output and pulmonary artery vasoreactivity did not differ among groups. Total pulmonary vascular resistance increased from 0.072 +/- 0.011 mmHg.ml-1.min in RA to 0.131 mmHg.ml-1.min in H but was significantly lower in HB (0.109 +/- 0.006 mmHg.ml-1.min). Hematocrit increased with hypoxia (57 +/- 3% in H vs. 42 +/- 1% in RA, P less than 0.05), and bleeding prevented the increase (46 +/- 4% in HB, P less than 0.05 vs. H only). The proportion of thick-walled peripheral pulmonary vessels (53.2 +/- 2.9% in HB and 50.6 +/- 4.8% in H vs. 31.6 +/- 2.6% in RA, P less than 0.05) and the percent medial thickness of pulmonary arteries adjacent to alveolar ducts (7.2 +/- 0.6% in HB and 7.0 +/- 0.4% in H vs. 5.2 +/- 0.4% in RA, P less than 0.05) increased to a similar degree in both hypoxic groups. A similar tendency was present in larger bronchiolar vessels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chronic in ovo hypoxia decreases pulmonary arterial contractile reactivity and induces biventricular cardiac enlargement in the chicken embryo

Although chronic prenatal hypoxia is considered a major cause of persistent pulmonary hypertension of the newborn, experimental studies have failed to consistently find pulmonary hypertensive changes after chronic intrauterine hypoxia. We hypothesized that chronic prenatal hypoxia induces changes in the pulmonary vasculature of the chicken embryo. We analyzed pulmonary arterial reactivity and structure and heart morphology of chicken embryos maintained from days 6 to 19 of the 21-day incubation period under normoxic (21% O(2)) or hypoxic (15% O(2)) conditions. Hypoxia increased mortality (0.46 vs. 0.14; P < 0.01) and reduced the body mass of the surviving 19-day embryos (22.4 +/- 0.5 vs. 26.6 +/- 0.7 g; P < 0.01). A decrease in the response of the pulmonary artery to KCl was observed in the 19-day hypoxic embryos. The contractile responses to endothelin-1, the thromboxane A(2) mimetic U-46619, norepinephrine, and electrical-field stimulation were also reduced in a proportion similar to that observed for KCl-induced contractions. In contrast, no hypoxia-induced decrease of response to vasoconstrictors was observed in externally pipped 21-day embryos (incubated under normoxia for the last 2 days). Relaxations induced by ACh, sodium nitroprusside, or forskolin were unaffected by chronic hypoxia in the pulmonary artery, but femoral artery segments of 19-day hypoxic embryos were significantly less sensitive to ACh than arteries of control embryos [pD(2) (= -log EC(50)): 6.51 +/- 0.1 vs. 7.05 +/- 0.1, P < 0.01]. Pulmonary vessel density, percent wall area, and periarterial sympathetic nerve density were not different between control and hypoxic embryos. In contrast, hypoxic hearts showed an increase in right and left ventricular wall area and thickness. We conclude that, in the chicken embryo, chronic moderate hypoxia during incubation transiently reduced pulmonary arterial contractile reactivity, impaired endothelium-dependent relaxation of femoral but not pulmonary arteries, and induced biventricular cardiac hypertrophy.     

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.     

High-altitude chronic hypoxia during gestation and after birth modifies cardiovascular responses in newborn sheep

Perinatal exposure to chronic hypoxia induces sustained pulmonary hypertension and structural and functional changes in both pulmonary and systemic vascular beds. The aim of this study was to analyze consequences of high-altitude chronic hypoxia during gestation and early after birth in pulmonary and femoral vascular responses in newborn sheep. Lowland (LLNB; 580 m) and highland (HLNB; 3,600 m) newborn lambs were cathetherized under general anesthesia and submitted to acute sustained or stepwise hypoxic episodes. Contractile and dilator responses of isolated pulmonary and femoral small arteries were analyzed in a wire myograph. Under basal conditions, HLNB had a higher pulmonary arterial pressure (PAP; 20.2 +/- 2.4 vs. 13.6 +/- 0.5 mmHg, P < 0.05) and cardiac output (342 +/- 23 vs. 279 +/- 13 ml x min(-1) x kg(-1), P < 0.05) compared with LLNB. In small pulmonary arteries, HLNB showed greater contractile capacity and higher sensitivity to nitric oxide. In small femoral arteries, HLNB had lower maximal contraction than LLNB with higher maximal response and sensitivity to noradrenaline and phenylephrine. In acute superimposed hypoxia, HLNB reached higher PAP and femoral vascular resistance than LLNB. Graded hypoxia showed that average PAP was always higher in HLNB compared with LLNB at any Po2. Newborn lambs from pregnancies at high altitude have stronger pulmonary vascular responses to acute hypoxia associated with higher arterial contractile status. In addition, systemic vascular response to acute hypoxia is increased in high-altitude newborns, associated with higher arterial adrenergic responses. These responses determined in intrauterine life and early after birth could be adaptive to chronic hypoxia in the Andean altiplano.     

Effect of increased alveolar surface tension on segmental pulmonary vascular resistance

The site of change in pulmonary vascular resistance (PVR) after surfactant displacement with the detergent diocytl sodium sulfosuccinate (OT) was studied in the isolated canine left lower lobe preparation. Changes in PVR were assessed using the arterial and venous occlusion technique and the vascular pressure-flow relationship. Changes in alveolar surface tension were confirmed from measurements of pulmonary compliance as well as from measurements of surface tension of extracts from lung homogenates. After surfactant depletion (the perfusion rate constant) the total pressure gradient (delta PT) across the lobe increased from 13.4 +/- 1 to 17.1 +/- 0.8 mmHg. This increase in delta PT was associated with a significant increase in the arterial and venous gradients (3.7 +/- 0.3 to 4.9 +/- 0.4 and 5.7 +/- 0.5 to 9.4 +/- 0.6 mmHg, respectively) and a decrease in middle pressure gradient (4.1 +/- 0.8 to 2.9 +/- 0.6 mmHg). The vascular pressure-flow relationship supported these findings and showed that the mean slope increased by 52% (P less than 0.05), whereas the pressure intercept decreased slightly but not significantly (3.7 +/- 0.7 to 3.2 +/- 0.8 mmHg). These results suggest that the resistance of arteries and veins increases, whereas the resistance of the middle segment decreases after surfactant depletion. These effects were apparently due to surface tension that acts directly on the capillary wall. Direct visualization of subpleural capillaries supported the notion that capillaries become distended and recruited as alveolar surface tension increases. In the normal lung (perfused at constant-flow rate) changes in alveolar pressure (Palv) were transmitted fully to the capillaries as suggested by equal changes in pulmonary arterial pressure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genistein rescues hypoxia-induced pulmonary arterial hypertension through estrogen receptor and β-adrenoceptor signaling

Pulmonary vascular remodeling is an important pathological feature of pulmonary arterial hypertension (PAH), which is characterized by thickening of the medial smooth muscle layer. Hypertrophy of pulmonary artery smooth muscle cells (PASMCs) participates in the development of medial thickening. Genistein can attenuate PAH and inhibit medial thickening of pulmonary arteries. Since hypoxia is one of the main causes of pulmonary hypertension, this study aims to investigate the mechanism of genistein in inhibiting hypertrophic responses in PASMCs induced by hypoxia. Cells isolated from the chick embryo were cultured with or without genistein and subjected to hypoxia or not. The increase of cell surface area and α-smooth muscle actin (α-SMA) of PASMCs was significantly suppressed by genistein during hypoxia. This result was confirmed by the incorporation of puromycin into peptide chains and flow cytometry analysis. Constrained mRNA and protein hypoxia-inducible factor (HIF)-1α expression was improved by genistein under hypoxia condition. Genistein restored redox homeostasis by fluorescent probe determination. The effect of genistein on hypertrophic response was blocked by estrogen receptor inhibitor, β1-adrenoceptor agonist and β2-adrenoceptor antagonist. In conclusion, genistein potently attenuates hypoxia-induced hypertrophy of PASMCs, which may enable a novel therapy for PAH.     

Development of the pulmonary vasculature in newborn lambs: structure-function relationships

Our objectives were 1) to describe the quantitative light microscopy and ultrastructure of newborn lamb lungs and 2) to correlate hemodynamic changes during normoxia and hypoxia with the morphology. By light microscopy, we measured the percent muscle thickness (%MT) and peripheral muscularization of pulmonary arteries and veins from 25 lambs aged less than 24 h, 2-4 days, 2 wk, and 1 mo. At the same ages, lungs were isolated and perfused in situ and, after cyclooxygenase blockade with indomethacin, total, arterial (delta Pa), middle (delta Pm), and venous pressure gradients at inspired O2 fractions of 0.28 (mild hyperoxia) and 0.04 (hypoxia) were determined with inflow-outflow occlusion. During mild hyperoxia, delta Pa and delta Pm fell significantly between 2-4 days and 2 wk, whereas during hypoxia, only delta Pm fell. The %MT of all arteries (less than 50 to greater than 1,000 microns diam) decreased, and peripheral muscularization of less than 100-microns-diam arteries fell between less than 4 days and greater than 2 wk. Our data suggest that 1) the %MT of arteries determines normoxic pulmonary vascular resistance, because only arterial and middle segment resistance fell, 2) peripheral muscularization is a major determinant of hypoxic pulmonary vasoconstriction, because we observed a fall with age in peripheral muscularization of less than 100-micron-diam arteries and in delta Pm with hypoxia, and 3) the arterial limit of the middle segment defined by inflow-outflow occlusion lies in 100- to 1,000-microns-diam arteries.     

Anti-platelet agents reduce morphological changes of chronic hypoxic pulmonary hypertension

The pathophysiologic mechanism by which chronic hypoxia causes pulmonary hypertension is unknown. If anti-platelet agents, or other pharmacologic interventions, altered the pulmonary vascular changes induced by hypoxia, information concerning the pathogenesis of the pulmonary hypertension or the potential therapeutic usefulness of the drugs might be obtained. In Study 1, rats exposed to chronic hypobaric hypoxia (PB = 520 mmHg) had a pulmonary arterial medial thickness of 6.7 +/- 0.6 mu compared to 4.1 +/- 0.2 mu* for control, normoxic rats (*p less than 0.05). Administration of dipyridamole (2mg/kg/day), or sulfinpyrazone (11 mg/kg/day) in the drinking water reduced the medial thickness to 5.0 +/- 0.3 mu* and 5.4 +/- 0.5 mu* respectively, thus suggesting the possible involvement of platelets in the response of the media to chronic hypoxia. In Study 2, hypoxic rats treated with the calcium blocker, flunarizine, were found to have less medial hypertrophy than a control group of hypoxic rats. This observation suggests that a decrease in transmembrane calcium flux may also reduce medial hypertrophy.     

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.     

Evidence that hypoxic pulmonary vascular remodeling in rats is polyamine dependent

This study tested the hypothesis that the polyamines, a family of low-molecular-weight organic cations with documented regulatory roles in cell growth and differentiation, are mediators of chronic hypoxia-induced pulmonary vascular remodeling. Relative to room air controls, chronically hypoxic animals (inspired O2 fraction = 0.1; 21 days) exhibited higher pulmonary arterial pressures (measured in room air), thicker medial layers in pulmonary arteries of 50-100 microns diam, increased hematocrits, and right ventricular hypertrophy. In addition, lung contents of the polyamines, putrescine, spermidine, and spermine were greater in hypoxic animals than in controls. alpha-Difluoromethylornithine (DFMO), an inhibitor of polyamine synthesis, attenuated the hypoxia-induced elevations in lung putrescine and spermidine content and blunted the increases in pulmonary arterial pressure and medial thickness. Neither the increased hematocrit nor right ventricular hypertrophy associated with chronic hypoxia were abrogated by DFMO. In addition, DFMO failed to influence vasoconstrictor responses provoked by acute hypoxic ventilation in isolated, buffer-perfused rat lungs. These observations suggest that depression of polyamine biosynthesis with DFMO blunts the sustained increase in pulmonary arterial pressure by attenuating hypoxia-induced medial thickening.     

Cellular disposition of transported polyamines in hypoxic rat lung and pulmonary arteries

The polyamines putrescine, spermidine (SPD), and spermine are a family of low-molecular-weight organic cations essential for cell growth and differentiation and other aspects of signal transduction. Hypoxic pulmonary vascular remodeling is accompanied by depressed lung polyamine synthesis and markedly augmented polyamine uptake. Cell types in which hypoxia induces polyamine transport in intact lung have not been delineated. Accordingly, rat lung and rat main pulmonary arterial explants were incubated with [(14)C]SPD in either normoxic (21% O(2)) or hypoxic (2% O(2)) environments for 24 h. Autoradiographic evaluation confirmed previous studies showing that, in normoxia, alveolar epithelial cells are dominant sites of polyamine uptake. In contrast, hypoxia was accompanied by prominent localization of [(14)C]SPD in conduit, muscularized, and partially muscularized pulmonary arteries, which was not evident in normoxic lung tissue. Hypoxic main pulmonary arterial explants also exhibited substantial increases in [(14)C]SPD uptake relative to control explants, and autoradiography revealed that enhanced uptake was most evident in the medial layer. Main pulmonary arterial explants denuded of endothelium failed to increase polyamine transport in hypoxia. Conversely, medium conditioned by endothelial cells cultured in hypoxic, but not in normoxic, environments enabled hypoxic transport induction in denuded arterial explants. These findings in arterial explants were recapitulated in rat cultured main pulmonary artery cells, including the enhancing effect of a soluble endothelium-derived factor(s) on hypoxic induction of [(14)C]SPD uptake in smooth muscle cells. Viewed collectively, these results show in intact lung tissue that hypoxia enhances polyamine transport in pulmonary artery smooth muscle by a mechanism requiring elaboration of an unknown factor(s) from endothelial cells.     

Acute and chronic hypoxic pulmonary hypertension in guinea pigs

To determine whether the strength of acute hypoxic vasoconstriction predicts the magnitude of chronic hypoxic pulmonary hypertension, we performed serial studies on guinea pigs. Unanesthetized, chronically catheterized guinea pigs increased mean pulmonary arterial pressure (PAP) from 11 +/- 0.5 to 13 +/- 0.7 Torr in acute hypoxia (10% O2 for 65 min). The response was maximal at 5 min, remained stable for 1 h, and was reversible on return to room air. Cardiac index did not change with acute hypoxia or recovery. Guinea pigs exposed to chronic hypoxia increased PAP, measured in room air 1 h after removal from the hypoxic chamber, to 18 +/- 1 Torr by 5 days with little further increase in PAP to 20 +/- 1 Torr after 21 days. Cardiac index fell from 273 +/- 12 to 206 +/- 7 ml.kg-1.min-1 (P less than 0.05) after 21 days of hypoxia. Medial thickness of pulmonary arteries adjacent to terminal bronchioles and alveolar ducts increased significantly by 10 days. The magnitude of the pulmonary vasoconstriction to acute hypoxia persisted and was unabated during the development and apparent stabilization of chronic hypoxic pulmonary hypertension, suggesting that if vasoconstriction is the stimulus for remodeling, then the importance of the stimulus lessens with duration of hypoxia. In individual animals followed serially, we found no correlation between the magnitude of the acute vasoconstrictor response before chronic hypoxia and the severity of chronic pulmonary hypertension that subsequently developed either because the initial response was small and variable or because vasoconstriction may not be the sole stimulus for vascular remodeling in the guinea pig.     

Pulmonary vascular remodelling in a high-altitude Aymara Indian

A histological study of the pulmonary vasculature in a young male high-altitude Aymara Indian revealed four aspects of interest. There was muscularization of the terminal portion of the pulmonary arterial tree to involve pulmonary arterioles as small as 15 m in diameter, thus forming a basis for the slightly increased pulmonary vascular resistance of native highlanders. Intimal longitudinal muscle was found in pulmonary arteries and arterioles and thought to be due to chronic alveolar hypoxia. Inner muscular tubes similar to those found in chronic obstructive lung disease were present. Pulmonary veins and venules also showed intimal muscularization suggesting that alveolar hypoxia affects vascular smooth muscle cells per se irrespective of their situation. The nature of the remodelling in a pulmonary blood vessel depends on a combination of hypoxia and haemodynamics.     

Role of platelet-activating factor in pulmonary vascular remodeling associated with chronic high altitude hypoxia in ovine fetal lambs

Platelet-activating factor (PAF) is implicated in pathogenesis of chronic hypoxia-induced pulmonary hypertension in some animal models and in neonates. Effects of chronic hypoxia on PAF receptor (PAF-R) system in fetal pulmonary vasculature are unknown. We investigated the effect of chronic high altitude hypoxia (HAH) in fetal lambs [pregnant ewes were kept at 3,801 m (12,470 ft) altitude from approximately 35 to 145 days gestation] on PAF-R-mediated effects in the pulmonary vasculature. Age-matched controls were kept at sea level. Intrapulmonary arteries were isolated, and smooth muscle cells (SMC-PA) were cultured from HAH and control fetuses. To determine presence of pulmonary vascular remodeling, lung tissue sections were subjected to morphometric analysis. Percentage medial wall thickness was significantly increased (P < 0.05) in arteries at all levels in the HAH lambs. PAF-R protein expression studied by immunocytochemistry and Western blot analysis on lung tissue SMC-PA demonstrated greater PAF-R expression in HAH lambs. PAF-R binding (femtomoles per 10(6) cells) in HAH SMC-PA was 90.3 +/- 4.08 and 66% greater than 54.3 +/- 4.9 in control SMC-PA. Pulmonary arteries from HAH fetuses synthesized >3-fold PAF than vessels from controls. Compared with controls SMC-PA of HAH lambs demonstrated 139% and 40% greater proliferation in 10% FBS alone and with 10 nM PAF, respectively. Our data demonstrate that exposure of ovine fetuses to HAH will result in significant upregulation of PAF synthesis, PAF-R expression, and PAF-R-mediated effects in pulmonary arteries. These findings suggest that increased PAF-R protein expression and increased PAF binding contribute to pulmonary vascular remodeling in these animals and may predispose them to persistent pulmonary hypertension after birth.     

Effect of acute hypoxia on microcirculatory and tissue oxygen levels in rat cremaster muscle.

Repeated exposure to brief periods of hypoxia leads to pathophysiological changes in experimental animals similar to those seen in sleep apnea. To determine the effects of such exposure on oxygen levels in vivo, we used an optical method to measure PO2 in microcirculatory vessels and tissue of the rat cremaster muscle during a 1-min step reduction of inspired oxygen fraction from 0.21 to 0.07. Under control conditions, PO2 was 98.1 +/- 1.9 Torr in arterial blood, 52.2 +/- 2.8 Torr in 29.0 +/- 2.7-microm arterioles, 26.8 +/- 1.7 Torr in the tissue interstitium near venous capillaries, and 35.1 +/- 2.6 Torr in 29.7 +/- 1.9-microm venules. The initial fall in PO2 during hypoxia was significantly greater in arterial blood, being 93% complete in the first 10 s, whereas it was 68% complete in arterioles, 47% at the tissue sites, and 38% in venules. In the 10- to 30-s period, the fall in normalized tissue and venular PO2 was significantly greater than in arterial PO2. At the end of hypoxic exposure, PO2 at all measurement sites had fallen very nearly in proportion to that in the inspired gas, but tissue oxygen levels did not reach critical PO2. Significant differences in oxyhemoglobin desaturation rate were also observed between arterial and microcirculatory vessels during hypoxia. In conclusion, the fall in microcirculatory and tissue oxygen levels in resting skeletal muscle is significantly slower than in arterial blood during a step reduction to an inspired oxygen fraction of 0.07, and tissue PO2 does not reach anaerobic levels.     

Acetazolamide prevents hypoxic pulmonary vasoconstriction in conscious dogs.

Acute hypoxia increases pulmonary arterial pressure and vascular resistance. Previous studies in isolated smooth muscle and perfused lungs have shown that carbonic anhydrase (CA) inhibition reduces the speed and magnitude of hypoxic pulmonary vasoconstriction (HPV). We studied whether CA inhibition by acetazolamide (Acz) is able to prevent HPV in the unanesthetized animal. Ten chronically tracheotomized, conscious dogs were investigated in three protocols. In all protocols, the dogs breathed 21% O(2) for the first hour and then 8 or 10% O(2) for the next 4 h spontaneously via a ventilator circuit. The protocols were as follows: protocol 1: controls given no Acz, inspired O(2) fraction (Fi(O(2))) = 0.10; protocol 2: Acz infused intravenously (250-mg bolus, followed by 167 microg.kg(-1).min(-1) continuously), Fi(O(2)) = 0.10; protocol 3: Acz given as above, but with Fi(O(2)) reduced to 0.08 to match the arterial Po(2) (Pa(O(2))) observed during hypoxia in controls. Pa(O(2)) was 37 Torr during hypoxia in controls, mean pulmonary arterial pressure increased from 17 +/- 1 to 23 +/- 1 mmHg, and pulmonary vascular resistance increased from 464 +/- 26 to 679 +/- 40 dyn.s(-1).cm(-5) (P < 0.05). In both Acz groups, mean pulmonary arterial pressure was 15 +/- 1 mmHg, and pulmonary vascular resistance ranged between 420 and 440 dyn.s(-1).cm(-5). These values did not change during hypoxia. In dogs given Acz at 10% O(2), the arterial Pa(O(2)) was 50 Torr owing to hyperventilation, whereas in those breathing 8% O(2) the Pa(O(2)) was 37 Torr, equivalent to controls. In conclusion, Acz prevents HPV in conscious spontaneously breathing dogs. The effect is not due to Acz-induced hyperventilation and higher alveolar Po(2), nor to changes in plasma endothelin-1, angiotensin-II, or potassium, and HPV suppression occurs despite the systemic acidosis with CA inhibition.