Ivan F. McMurtry

Cellular and molecular basis of pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is caused by functional and structural changes in the pulmonary vasculature, leading to increased pulmonary vascular resistance. The process of pulmonary vascular remodeling is accompanied by endothelial dysfunction, activation of fibroblasts and smooth muscle cells, crosstalk between cells within the vascular wall, and recruitment of circulating progenitor cells. Recent findings have reestablished the role of chronic vasoconstriction in the remodeling process. Although the pathology of PAH in the lung is well known, this article is concerned with the cellular and molecular processes involved. In particular, we focus on the role of the Rho family guanosine triphosphatases in endothelial function and vasoconstriction. The crosstalk between endothelium and vascular smooth muscle is explored in the context of mutations in the bone morphogenetic protein type II receptor, alterations in angiopoietin-1/TIE2 signaling, and the serotonin pathway. We also review the role of voltage-gated K(+) channels and transient receptor potential channels in the regulation of cytosolic [Ca(2+)] and [K(+)], vasoconstriction, proliferation, and cell survival. We highlight the importance of the extracellular matrix as an active regulator of cell behavior and phenotype and evaluate the contribution of the glycoprotein tenascin-c as a key mediator of smooth muscle cell growth and survival. Finally, we discuss the origins of a cell type critical to the process of pulmonary vascular remodeling, the myofibroblast, and review the evidence supporting a contribution for the involvement of endothelial-mesenchymal transition and recruitment of circulating mesenchymal progenitor cells.

Inhibition of hypoxic pulmonary vasoconstriction by calcium antagonists in isolated rat lungs.

The role of a transmembrane calcium influx in hypoxic pulmonary vasoconstriction was studied in isolated, blood perfused, rat lungs. We reasoned that, if the influx of extracellular calcium mediated the hypoxic mechanism, pressor responses to alveolar hypoxia (2.5percent; O2) would be susceptible to inhibition by the calcium antagonists verapamil (2 x 10−5 to 2 x 10−1 mM) and SKF 525 A (2.6 to 260 mM). Susceptibility of hypoxic pressor responses to inhibition by these calcium antagonists was contrasted to that of pressor responses elicited by the humoral vasoconstrictors angiotensin II (1 or 0.5 μ) and prostaglandin F2αto (10 μg). Since neither saralasin (0.5 μM), a competitive antagonist of angiotensin II, nor meclofenamate (6.8 μM), an inhibitor of prostaglandin synthesis, depressed hypoxic pressor responses, it was concluded that these humoral transmitters were not directly involved in the hypoxic mechanism, and therefore served as independent reference agonists. The order of susceptibility of pulmonary pressor responses to inhibition by verapamil was hypoxia > angiotensin II > prostaglandin F2α. SKF 525A also reduced pressor responses to hypoxia more readily than those to angiotensin II. The greater inhibition of hypoxic pulmonary vasoconstriction by both calcium antagonists suggested that the hypoxic mechanism was critically dependent on the transmembrane influx of extracellular calcium. Mediation of the hypoxic response by this type of excitation-contraction coupling is consistent with the idea that hypoxia has a direct depolarizing effect on the vascular smooth muscle. It also provides a unifying explanation for inhibition of the hypoxic mechanism by various agents that have depressant or stabilizing actions on membranes in addition to other pharmacological effects.

Reperfusion after acute coronary occlusion in dogs impairs endothelium-dependent relaxation to acetylcholine and augments contractile reactivity in vitro.

Endothelial injury may contribute to the augmented coronary vascular tone seen in myocardial ischemia by impairing endothelial production or release of vasodilators. In vitro reactivity of arterial rings was studied after 60 min of coronary occlusion and 60 min of reperfusion in anesthetized dogs. Ischemia without reperfusion blunted contractile reactivity to potassium chloride (KCl), whereas ischemia plus reperfusion augmented contractile responses to both KCl and ergonovine. The response to acetylcholine, an endothelium-dependent vasodilator, was abolished in reperfused arteries, whereas the response to nitroprusside, an endothelium-independent vasodilator, was intact. Verapamil pretreatment restored KCl contractile responses to normal in reperfused coronary rings and partially restored endothelium-dependent relaxation. Electron microscopy revealed a nondenuding epicardial coronary endothelial injury in reperfused arteries. These data support the hypothesis that reperfusion of ischemic myocardium augments reactivity to vasoconstrictor agents by causing endothelial cell damage, excessive calcium influx, and loss of modulating vasodilator function.

The pulmonary circulation of homozygous or heterozygous eNOS-null mice is hyperresponsive to mild hypoxia

Acute hypoxic vasoconstriction and development of hypoxic pulmonary hypertension (PHTN) are unique properties of the pulmonary circulation. The pulmonary endothelium produces vasoactive factors, including nitric oxide (NO), that modify these phenomena. We tested the hypothesis that NO produced by endothelial nitric oxide synthase (eNOS) modulates pulmonary vascular responses to hypoxia using mice with targeted disruption of the eNOS gene (eNOS-/-). Marked PHTN was found in eNOS-/- mice raised in mild hypoxia when compared with either controls or eNOS-/- mice raised in conditions simulating sea level. We found an approximate twofold increase in partially and fully muscularized distal pulmonary arteries in eNOS-/- mice compared with controls. Consistent with vasoconstriction being the primary mechanism of PHTN, however, acute inhalation of 25 ppm NO resulted in normalization of RV pressure in eNOS-/- mice. In addition to studies of eNOS-/- mice, the dose-effect of eNOS was tested using heterozygous eNOS+/- mice. Although the lungs of eNOS+/- mice had 50% of normal eNOS protein, the response to hypoxia was indistinguishable from that of eNOS-/- mice. We conclude that eNOS-derived NO is an important modulator of the pulmonary vascular response to chronic hypoxia and that more than 50% of eNOS expression is required to maintain normal pulmonary vascular tone.

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.

Formation of Plexiform Lesions in Experimental Severe Pulmonary Arterial Hypertension

Background— The plexiform lesion is the hallmark of severe pulmonary arterial hypertension. However, its genesis and hemodynamic effects are largely unknown because of the limited availability of lung tissue samples from patients with pulmonary arterial hypertension and the lack of appropriate animal models. This study investigated whether rats with severe progressive pulmonary hypertension developed plexiform lesions. Methods and Results— After a single subcutaneous injection of the vascular endothelial growth factor receptor blocker Sugen 5416, rats were exposed to hypoxia for 3 weeks. They were then returned to normoxia for an additional 10 to 11 weeks. Hemodynamic and histological examinations were performed at 13 to 14 weeks after the Sugen 5416 injection. All rats developed pulmonary hypertension (right ventricular systolic pressure ≈100 mm Hg) and severe pulmonary arteriopathy, including concentric neointimal and complex plexiform-like lesions. There were 2 patterns of complex lesion formation: a lesion forming within the vessel lumen (stalk-like) and another that projected outside the vessel (aneurysm-like). Immunohistochemical analyses showed that these structures had cellular and molecular features closely resembling human plexiform lesions. Conclusions— Severe, sustained pulmonary hypertension in a very late stage of the Sugen 5416/hypoxia/normoxia-exposed rat is accompanied by the formation of lesions that are indistinguishable from the pulmonary arteriopathy of human pulmonary arterial hypertension. This unique model provides a new and rigorous approach for investigating the genesis, hemodynamic effects, and reversibility of plexiform and other occlusive lesions in pulmonary arterial hypertension.

Role of endothelin-1 in lung disease

Endothelin-1 (ET-1) is a 21 amino acid peptide with diverse biological activity that has been implicated in numerous diseases. ET-1 is a potent mitogen regulator of smooth muscle tone, and inflammatory mediator that may play a key role in diseases of the airways, pulmonary circulation, and inflammatory lung diseases, both acute and chronic. This review will focus on the biology of ET-1 and its role in lung disease.

Therapeutic potential of RhoA/Rho kinase inhibitors in pulmonary hypertension

A burgeoning body of evidence suggests that RhoA/Rho kinase (ROCK) signalling plays an important role in the pathogenesis of various experimental models of pulmonary hypertension (PH), including chronic hypoxia‐, monocrotaline‐, bleomycin‐, shunt‐ and vascular endothelial growth factor receptor inhibition plus chronic hypoxia‐induced PH. ROCK has been incriminated in pathophysiologic events ranging from mediation of sustained abnormal vasoconstriction to promotion of vascular inflammation and remodelling. In addition, the 3‐hydoxy‐3‐methylglutaryl CoA reductase inhibitors, statins, which inhibit activation of RhoA by preventing post‐translational isoprenylation of the protein and its translocation to the plasma membrane ameliorate PH in several different rat models, and may also be effective in PH patients. Also, phosphorylation of RhoA and prevention of its translocation to the plasma membrane are involved in the protective effect of the type 5‐PDE inhibitor, sildenafil, against hypoxia‐ and bleomycin‐induced PH. Collectively, these and other observations indicate that independent of the cause of PH, activation of the RhoA/ROCK pathway serves as a point of convergence of various signalling cascades in the pathogenesis of the disease. We propose that ROCK inhibitors and other drugs that inhibit this pathway might be useful in the treatment of various forms of PH.

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.

Variable expression of endothelial NO synthase in three forms of rat pulmonary hypertension

Endothelial nitric oxide (NO) synthase (eNOS) mRNA and protein and NO production are increased in hypoxia-induced hypertensive rat lungs, but it is uncertain whether eNOS gene expression and activity are increased in other forms of rat pulmonary hypertension. To investigate these questions, we measured eNOS mRNA and protein, eNOS immunohistochemical localization, perfusate NO product levels, and NO-mediated suppression of resting vascular tone in chronically hypoxic (3-4 wk at barometric pressure of 410 mmHg), monocrotaline-treated (4 wk after 60 mg/kg), and fawn-hooded (6-9 mo old) rats. eNOS mRNA levels (Northern blot) were greater in hypoxic and monocrotaline-treated lungs (130 and 125% of control lungs, respectively; P < 0.05) but not in fawn-hooded lungs. Western blotting indicated that eNOS protein levels increased to 300 +/- 46% of control levels in hypoxic lungs (P < 0.05) but were decreased by 50 +/- 5 and 60 +/- 11%, respectively, in monocrotaline-treated and fawn-hooded lungs (P < 0.05). Immunostaining showed prominent eNOS expression in small neomuscularized arterioles in all groups, whereas perfusate NO product levels increased in chronically hypoxic lungs (3.4 +/- 1.4 microM; P < 0.05) but not in either monocrotaline-treated (0.7 +/- 0.3 microM) or fawn-hooded (0.45 +/- 0.1 microM) lungs vs. normotensive lungs (0.12 +/- 0.07 microM). All hypertensive lungs had increased baseline perfusion pressure in response to nitro-L-arginine but not to the inducible NOS inhibitor aminoguanidine. These results indicate that even though NO activity suppresses resting vascular tone in pulmonary hypertension, there are differences among the groups regarding eNOS gene expression and NO production. A better understanding of eNOS gene expression and activity in these models may provide insights into the regulation of this vasodilator system in various forms of human pulmonary hypertension.Endothelial nitric oxide (NO) synthase (eNOS) mRNA and protein and NO production are increased in hypoxia-induced hypertensive rat lungs, but it is uncertain whether eNOS gene expression and activity are increased in other forms of rat pulmonary hypertension. To investigate these questions, we measured eNOS mRNA and protein, eNOS immunohistochemical localization, perfusate NO product levels, and NO-mediated suppression of resting vascular tone in chronically hypoxic (3-4 wk at barometric pressure of 410 mmHg), monocrotaline-treated (4 wk after 60 mg/kg), and fawn-hooded (6-9 mo old) rats. eNOS mRNA levels (Northern blot) were greater in hypoxic and monocrotaline-treated lungs (130 and 125% of control lungs, respectively; P < 0.05) but not in fawn-hooded lungs. Western blotting indicated that eNOS protein levels increased to 300 ± 46% of control levels in hypoxic lungs ( P < 0.05) but were decreased by 50 ± 5 and 60 ± 11%, respectively, in monocrotaline-treated and fawn-hooded lungs ( P < 0.05). Immunostaining showed prominent eNOS expression in small neomuscularized arterioles in all groups, whereas perfusate NO product levels increased in chronically hypoxic lungs (3.4 ± 1.4 μM; P < 0.05) but not in either monocrotaline-treated (0.7 ± 0.3 μM) or fawn-hooded (0.45 ± 0.1 μM) lungs vs. normotensive lungs (0.12 ± 0.07 μM). All hypertensive lungs had increased baseline perfusion pressure in response to nitro-l-arginine but not to the inducible NOS inhibitor aminoguanidine. These results indicate that even though NO activity suppresses resting vascular tone in pulmonary hypertension, there are differences among the groups regarding eNOS gene expression and NO production. A better understanding of eNOS gene expression and activity in these models may provide insights into the regulation of this vasodilator system in various forms of human pulmonary hypertension.