Kenneth G. Morris

Rho Kinase–Mediated Vasoconstriction Is Important in Severe Occlusive Pulmonary Arterial Hypertension in Rats

Vascular remodeling, rather than vasoconstriction, is believed to account for high vascular resistance in severe pulmonary arterial hypertension (PAH). We have found previously that acute Rho kinase inhibition nearly normalizes PAH in chronically hypoxic rats that have no occlusive neointimal lesions. Here we examined whether Rho kinase-mediated vasoconstriction was also important in a rat model of severe occlusive PAH. Adult rats were exposed to chronic hypoxia (≈10% O2) after subcutaneous injection of the vascular endothelial growth factor receptor inhibitor SUGEN 5416. Hemodynamic measurements were made in anesthetized rats after 2 weeks of hypoxia (early group) and 3 weeks of hypoxia plus 2 weeks of normoxia (late group). Both groups developed PAH, with greater severity in the late group. In the early group, intravenous fasudil was more effective than intravenous bradykinin, inhaled NO, or intravenous iloprost in reducing right ventricular systolic pressure. Despite more occlusive vascular lesions, fasudil also markedly reduced right ventricular systolic pressure in late-stage rats. Blood-perfused lungs from late-stage rats showed spontaneous vasoconstriction, which was reversed partially by the endothelin A receptor blocker BQ123 and completely by fasudil or Y-27632. Phosphorylation of MYPT1, a downstream target of Rho kinase, was increased in lungs from both groups of rats, and fasudil (intravenous) reversed the increased phosphorylation in the late group. Thus, in addition to structural occlusion, Rho kinase-mediated vasoconstriction is an important component of severe PAH in SUGEN 5416/hypoxia-exposed rats, and PAH can be significantly reduced in the setting of a severely remodeled lung circulation if an unconventional vasodilator is used.

Angiotensin converting enzyme expression is increased in small pulmonary arteries of rats with hypoxia-induced pulmonary hypertension.

Previous studies suggest that while lung angiotensin converting enzyme (ACE) activity is reduced during chronic hypoxia, inhibitors of ACE attenuate hypoxic pulmonary hypertension. In an attempt to explain this paradox we investigated the possibility that whole lung ACE activity may not reflect local pulmonary vascular ACE expression. The experimental approach combined in vivo hemodynamic studies in control and chronically hypoxic rats, measurement of whole lung ACE activity, and evaluation of local pulmonary vascular ACE expression by in situ hybridization and immunohistochemistry. Total lung ACE activity was reduced to 50% of control activity by 5 d of hypoxia and remained low for the duration of the study. Immunohistochemistry showed a marked reduction of ACE staining in alveolar capillary endothelium. However, an increase in ACE staining was observed in the walls of small newly muscularized pulmonary arteries at the level of alveolar ducts and walls. In situ hybridization studies showed increased signal for ACE mRNA in the same vessels. Inhibition of ACE by captopril during chronic hypoxia attenuated pulmonary hypertension and markedly reduced distal muscularization of small pulmonary arteries. In addition, we demonstrated marked longitudinal variation in ACE expression along the normal pulmonary vasculature with the highest levels found in small muscular arteries associated with terminal and respiratory bronchioles. We conclude that local ACE expression is increased in the walls of small pulmonary arteries during the development of hypoxic pulmonary hypertension, despite a generalized reduction in alveolar capillary ACE expression, and we speculate that local arteriolar ACE may play a role in the vascular remodeling associated with pulmonary hypertension.

Relative contributions of endothelial, inducible, and neuronal NOS to tone in the murine pulmonary circulation

Nitric oxide plays an important role in modulating pulmonary vascular tone. All three isoforms of nitric oxide synthase (NOS), neuronal (nNOS, NOS I), inducible (iNOS, NOS II), and endothelial (eNOS, NOS III), are expressed in the lung. Recent reports have suggested an important role for eNOS in the modulation of pulmonary vascular tone chronically; however, the relative contribution of the three isoforms to acute modulation of pulmonary vascular tone is uncertain. We therefore tested the effect of targeted disruption of each isoform on pulmonary vascular reactivity in transgenic mice. Isolated perfused mouse lungs were used to evaluate the effect of selective loss of pulmonary nNOS, iNOS, and eNOS with respect to hypoxic pulmonary vasoconstriction (HPV) and endothelium-dependent and -independent vasodilation. eNOS null mice had augmented HPV (225 +/- 65% control, P < 0.02, mean +/- SE) and absent endothelium-dependent vasodilation, whereas endothelium-independent vasodilation was preserved. HPV was minimally elevated in iNOS null mice and normal in nNOS null mice. Both nNOS and iNOS null mice had normal endothelium-dependent vasodilation. In wild-type lungs, nonselective NOS inhibition doubled HPV, whereas selective iNOS inhibition had no detectable effect. In intact, lightly sedated mice, right ventricular systolic pressure was elevated in eNOS-deficient (42.3 +/- 1.2 mmHg, P < 0.001) and, to a lesser extent, in iNOS-deficient (37.2 +/- 0.8 mmHg, P < 0.001) mice, whereas it was normal in nNOS-deficient mice (30.9 +/- 0.7 mmHg, P = not significant) compared with wild-type controls (31.3 +/- 0.7 mmHg). We conclude that in the normal murine pulmonary circulation 1) nNOS does not modulate tone, 2) eNOS-derived nitric oxide is the principle mediator of endothelium-dependent vasodilation in the pulmonary circulation, and 3) both eNOS and iNOS play a role in modulating basal tone chronically.Nitric oxide plays an important role in modulating pulmonary vascular tone. All three isoforms of nitric oxide synthase (NOS), neuronal (nNOS, NOS I), inducible (iNOS, NOS II), and endothelial (eNOS, NOS III), are expressed in the lung. Recent reports have suggested an important role for eNOS in the modulation of pulmonary vascular tone chronically; however, the relative contribution of the three isoforms to acute modulation of pulmonary vascular tone is uncertain. We therefore tested the effect of targeted disruption of each isoform on pulmonary vascular reactivity in transgenic mice. Isolated perfused mouse lungs were used to evaluate the effect of selective loss of pulmonary nNOS, iNOS, and eNOS with respect to hypoxic pulmonary vasoconstriction (HPV) and endothelium-dependent and -independent vasodilation. eNOS null mice had augmented HPV (225 ± 65% control, P < 0.02, mean ± SE) and absent endothelium-dependent vasodilation, whereas endothelium-independent vasodilation was preserved. HPV was minimally elevated in iNOS null mice and normal in nNOS null mice. Both nNOS and iNOS null mice had normal endothelium-dependent vasodilation. In wild-type lungs, nonselective NOS inhibition doubled HPV, whereas selective iNOS inhibition had no detectable effect. In intact, lightly sedated mice, right ventricular systolic pressure was elevated in eNOS-deficient (42.3 ± 1.2 mmHg, P< 0.001) and, to a lesser extent, in iNOS-deficient (37.2 ± 0.8 mmHg, P < 0.001) mice, whereas it was normal in nNOS-deficient mice (30.9 ± 0.7 mmHg, P = not significant) compared with wild-type controls (31.3 ± 0.7 mmHg). We conclude that in the normal murine pulmonary circulation 1) nNOS does not modulate tone, 2) eNOS-derived nitric oxide is the principle mediator of endothelium-dependent vasodilation in the pulmonary circulation, and 3) both eNOS and iNOS play a role in modulating basal tone chronically.

Effects of chronic hypoxia and altered hemodynamics on endothelial nitric oxide synthase expression in the adult rat lung.

Mechanisms that regulate endothelial nitric oxide synthase (eNOS) expression in normal and hypoxic pulmonary circulation are poorly understood. Lung eNOS expression is increased after chronic hypoxic pulmonary hypertension in rats, but whether this increase is due to altered hemodynamics or to hypoxia is unknown. Therefore, to determine the effect of blood flow changes on eNOS expression in the normal pulmonary circulation, and to determine whether the increase in eNOS expression after chronic hypoxia is caused by hemodynamic changes or low oxygen tension, we compared eNOS expression in the left and right lungs of normoxic and chronically hypoxic rats with surgical stenosis of the left pulmonary artery (LPA). LPA stenosis in normoxic rats reduced blood flow to the left lung from 9.8+/-0.9 to 0.8+/-0.4 ml/100 mg/min (sham surgery controls vs. LPA stenosis, P < 0.05), but there was not a significant increase in right lung blood flow. When compared with the right lung, eNOS protein and mRNA content in the left lung was decreased by 32+/-7 and 54+/-13%, respectively (P < 0.05), and right lung eNOS protein content was unchanged. After 3 wk of hypoxia, LPA stenosis reduced blood flow to the left lung from 5.8+/-0.6 to 1.5+/-0.4 ml/100 mg/min, and increased blood flow to the right lung from 5.8+/-0.5 to 10.0+/-1.4 ml/ 100 mg/min (sham surgery controls vs. LPA stenosis, P < 0.05). Despite reduced flow and pressure to the left lung and increased flow and pressure to the right lung, left and right lung eNOS protein and mRNA contents were not different. There were also no differences in lung eNOS protein levels when compared with chronically hypoxic sham surgery controls (P > 0.05). We conclude that reduction of pulmonary blood flow decreases eNOS mRNA and protein expression in normoxic adult rat lungs, and that hypoxia increases eNOS expression independently of changes in hemodynamics. These findings demonstrate that hemodynamic forces maintain eNOS content in the normoxic pulmonary circulation of the adult rat, and suggest that chronic hypoxia increases eNOS expression independently of changes in hemodynamics.

Upregulation of nitric oxide synthase in mice with severe hypoxia-induced pulmonary hypertension

BackgroundThe importance of nitric oxide (NO) in hypoxic pulmonary hypertension has been demonstrated using nitric oxide synthase (NOS) knockout mice. In that model NO from endothelial NOS (eNOS) plays a central role in modulating pulmonary vascular tone and attenuating hypoxic pulmonary hypertension. However, the normal regulation of NOS expression in mice following hypoxia is uncertain. Because genetically engineered mice are often utilized in studies of NO, we conducted the present study to determine how hypoxia alters NOS expression in wild-type mice.MethodMice were exposed to sea level, ambient conditions (5280 feet) or severe altitude (17,000 feet) for 6 weeks from birth, and hemodynamics and lung NOS expression were assessed.ResultsHypoxic mice developed severe pulmonary hypertension (right ventricular systolic pressure [RVsP] 60 mmHg) as compared with normoxic mice (27 mmHg). Using quantitative reverse-transcription PCR, it was found that expressions of eNOS and inducible NOS (iNOS) increased 1.5-fold and 3.5-fold, respectively, in the lung. In addition, the level of lung eNOS protein was increased, neuronal NOS (nNOS) protein was unchanged, and iNOS was below the limit of detection. Immunohistochemistry demonstrated no change in lung iNOS or nNOS staining in either central or peripheral areas, but suggested increased eNOS in the periphery following hypoxia.ConclusionIn mice, hypoxia is associated with increases in lung eNOS, possibly in iNOS, but not in nNOS; this suggests that the pattern of lung NOS expression following hypoxia must be considered in studies using genetically engineered mice.

Spontaneous hypoxaemia and right ventricular hypertrophy in fast growing broiler chickens reared at sea level

1. At 6 weeks of age, the time of most rapid body growth, fast growing broiler chickens showed more right ventricular hypertrophy than slower growing chickens. 2. The degree of right ventricular hypertrophy was directly related to blood haematocrit and indirectly related to arterial oxygen saturation (estimated in the chickens using an ear oximeter designed for man). 3. When oxygen saturation was estimated sequentially from 6 to 17 weeks of age, mean oxygenation improved with age, partly due to the death of the chickens with the worst saturations, but also because of an improvement in oxygenation of the survivors.

Right ventricular angiotensin converting enzyme activity and expression is increased during hypoxic pulmonary hypertension.

OBJECTIVE To determine whether local cardiac angiotensin converting enzyme (ACE) expression is upregulated during the development of hypoxia-induced right ventricular hypertrophy. METHODS ACE activity was measured in membrane preparations from the right ventricle and left ventricle plus septum in normoxic rats and animals exposed to chronic hypoxia for 8 and 14 days. Local cardiac ACE expression was studied by immunohistochemistry using a monoclonal antibody to ACE (9B9). RESULTS In the normal rat heart, ACE expression was confined to vascular endothelium, the valvular endocardium, and localized regions of parietal endocardium. We found that the development of pulmonary hypertension and right ventricular hypertrophy were associated with 2.6- and 3.4-fold increases in membrane-bound right ventricular ACE activity by 8 and 14 days of hypoxia, respectively. Right ventricular ACE activity was positively correlated with the degree of right ventricular hypertrophy (r = 0.83, P < 0.001). In contrast, left ventricular plus septal ACE activity was significantly reduced by approximately 40 and 60% by 8 and 14 days of hypoxia, respectively, compared to controls. In the right ventricle of chronically hypoxic rats, immunohistochemistry demonstrated increased ACE expression in areas of myocardial fibrosis. Interestingly, increased ACE expression was noted in the right ventricular epicardium in chronically hypoxic rats. In the free wall of the left ventricle there was a significant reduction in the number of myocardial capillaries which expressed ACE in chronically hypoxic rats. CONCLUSION Chronic hypoxia has a differential effect on left and right ventricular ACE activity and that the sites of altered ACE expression are highly localized. We speculate that locally increased right ventricular ACE activity and expression may play a role in the pathogenesis of right ventricular hypertrophy secondary to hypoxic pulmonary hypertension.

Improved arterial oxygenation with feed restriction in rapidly growing broiler chickens

1. Rapidly growing broiler chickens fed ad lib. until 56 days, but feed restricted until 60 days of age, had higher arterial oxygen saturations, lower respiratory frequencies, total ventilations that were not different, and higher tidal volumes compared to those fully fed for 56 days. 2. Arterial oxygen saturation correlated negatively with respiratory frequency, but was not related to total ventilation or tidal volume. 3. Hypoventilation appeared not to be the cause of arterial oxygen desaturation. 4. Arterial oxygen desaturation correlated with the degree of right ventricular hypertrophy.

Effect of K ATP + channel inhibition on total and regional vascular resistance in guinea pig pregnancy

Decreased vascular resistance and vasoconstrictor response during pregnancy enables an increase in cardiac output and regional blood flow to the uterine circulation. We sought to determine whether inhibition of vascular smooth muscle ATP-sensitive potassium ([Formula: see text]) channel activity during pregnancy increased systemic and/or regional vascular resistance and resistance response to ANG II. A total of 32 catheterized, awake, pregnant or nonpregnant guinea pigs were treated with either the [Formula: see text]channel inhibitor glibenclamide (3.5 mg/kg) or vehicle (DMSO) ( n = 8/group). In nonpregnant and pregnant animals, glibenclamide raised blood pressure and systemic, uterine, and coronary vascular resistance, diminishing cardiac output and organ blood flow. Glibenclamide produced a greater rise in coronary vascular resistance in the pregnant than nonpregnant groups and increased renal and cerebral vascular resistance in the pregnant animals only. ANG II infusion raised blood pressure and systemic and renal vascular resistance and lowered cardiac output and renal blood flow in vehicle-treated animals. Glibenclamide augmented ANG II-induced systemic vasoconstriction in the nonpregnant and pregnant groups and the rise in uteroplacental vascular resistance in the pregnant animals. We concluded that [Formula: see text] channel activity likely modulates systemic, uterine, and coronary vascular resistance and opposes ANG II-induced systemic vasoconstriction in nonpregnant and pregnant guinea pigs. Pregnancy augments[Formula: see text] channel activity in the uterine, coronary, renal, and cerebral vascular beds and the uteroplacental circulation during ANG II infusion. Thus increased[Formula: see text] channel activity appears to influence regional control of vascular resistance during guinea pig pregnancy but cannot account for the characteristic decrease in systemic vascular resistance and ANG II-induced systemic vasoconstrictor response.

Exaggerated Pulmonary Vasoconstrictor Response to Acute Hypoxia in the Endothelin B Receptor Deficient Rat

Exaggerated Pulmonary Vasoconstrictor Response to Acute Hypoxia in the Endothelin B Receptor Deficient Rat