Omkar Paudel

TRPV4 channel contributes to serotonin-induced pulmonary vasoconstriction and the enhanced vascular reactivity in chronic hypoxic pulmonary hypertension

Transient receptor potential vanilloid 4 (TRPV4) is a mechanosensitive channel in pulmonary arterial smooth muscle cells (PASMCs). Its upregulation by chronic hypoxia is associated with enhanced myogenic tone, and genetic deletion of trpv4 suppresses the development of chronic hypoxic pulmonary hypertension (CHPH). Here we further examine the roles of TRPV4 in agonist-induced pulmonary vasoconstriction and in the enhanced vasoreactivity in CHPH. Initial evaluation of TRPV4-selective antagonists HC-067047 and RN-1734 in KCl-contracted pulmonary arteries (PAs) of trpv4(-/-) mice found that submicromolar HC-067047 was devoid of off-target effect on pulmonary vasoconstriction. Inhibition of TRPV4 with 0.5 μM HC-067047 significantly reduced the sensitivity of serotonin (5-HT)-induced contraction in wild-type (WT) PAs but had no effect on endothelin-1 or phenylephrine-activated response. Similar shift in the concentration-response curve of 5-HT was observed in trpv4(-/-) PAs, confirming specific TRPV4 contribution to 5-HT-induced vasoconstriction. 5-HT-induced Ca(2+) response was attenuated by HC-067047 in WT PASMCs but not in trpv4(-/-) PASMCs, suggesting TRPV4 is a major Ca(2+) pathway for 5-HT-induced Ca(2+) mobilization. Nifedipine also attenuated 5-HT-induced Ca(2+) response in WT PASMCs but did not cause further reduction in the presence of HC-067047, suggesting interdependence of TRPV4 and voltage-gated Ca(2+) channels in the 5-HT response. Chronic exposure (3-4 wk) of WT mice to 10% O2 caused significant increase in 5-HT-induced maximal contraction, which was partially reversed by HC-067047. In concordance, the enhancement of 5-HT-induced contraction was significantly reduced in PAs of CH trpv4(-/-) mice and HC-067047 had no further effect on the 5-HT induced response. These results suggest unequivocally that TRPV4 contributes to 5-HT-dependent pharmaco-mechanical coupling and plays a major role in the enhanced pulmonary vasoreactivity to 5-HT in CHPH.

Classical transient receptor potential 1 and 6 contribute to hypoxic pulmonary hypertension through differential regulation of pulmonary vascular functions

Hypoxic pulmonary hypertension is characterized by increased vascular tone, altered vasoreactivity, and vascular remodeling, which are associated with alterations in Ca2+ homeostasis in pulmonary arterial smooth muscle cells. We have previously shown that classical transient receptor potential 1 and 6 (TRPC1 and TRPC6) are upregulated in pulmonary arteries (PAs) of chronic hypoxic rats, but it is unclear whether these channels are essential for the development of pulmonary hypertension. Here we found that pulmonary hypertension was suppressed in TRPC1 and TRPC6 knockout (Trpc1−/− and Trpc6−/−) mice compared with wild-type after exposure to 10% O2 for 1 and 3 weeks. Muscularization of pulmonary microvessels was inhibited, but rarefaction was unaltered in hypoxic Trpc1−/− and Trpc6−/− mice. Small PAs of normoxic wild-type mice exhibited vasomotor tone, which was significantly enhanced by chronic hypoxia. Similar vasomotor tone was found in normoxic Trpc1−/− PAs, but the hypoxia-induced enhancement was blunted. In contrast, there was minimal vascular tone in normoxic Trpc6−/− PAs, but the hypoxia-enhanced tone was preserved. Chronic hypoxia caused significant increase in serotonin-induced vasoconstriction; the augmented vasoreactivity was attenuated in Trpc1−/− and eliminated in Trpc6−/− PAs. Moreover, the effects of 3-week hypoxia on pulmonary arterial pressure, right ventricular hypertrophy, and muscularization of microvessels were further suppressed in TRPC1-TRPC6 double-knockout mice. Our results, therefore, provide clear evidence that TRPC1 and TRPC6 participate differentially in various pathophysiological processes, and that the presence of TRPC1 and TRPC6 is essential for the full development of hypoxic pulmonary hypertension in the mouse model.

Alteration of Pulmonary Artery Integrin Levels in Chronic Hypoxia and Monocrotaline-Induced Pulmonary Hypertension

Background: Pulmonary hypertension is associated with vascular remodeling and increased extracellular matrix (ECM) deposition. While the contribution of ECM in vascular remodeling is well documented, the roles played by their receptors, integrins, in pulmonary hypertension have received little attention. Here we characterized the changes of integrin expression in endothelium-denuded pulmonary arteries (PAs) and aorta of chronic hypoxia as well as monocrotaline-treated rats. Methods and Results: Immunoblot showed increased α1-, α8- and αv-integrins, and decreased α5-integrin levels in PAs of both models. β1- and β3-integrins were reduced in PAs of chronic hypoxia and monocrotaline-treated rats, respectively. Integrin expression in aorta was minimally affected. Differential expression of α1- and α5-integrins induced by chronic hypoxia was further examined. Immunostaining showed that they were expressed on the surface of PA smooth muscle cells (PASMCs), and their distribution was unaltered by chronic hypoxia. Phosphorylation of focal adhesion kinase was augmented in PAs of chronic hypoxia rats, and in chronic hypoxia PASMCs cultured on the α1-ligand collagen IV. Moreover, α1-integrin binding hexapeptide GRGDTP elicited an enhanced Ca2+ response, whereas the response to α5-integrin binding peptide GRGDNP was reduced in CH-PASMCs. Conclusion: Integrins in PASMCs are differentially regulated in pulmonary hypertension, and the dynamic integrin-ECM interactions may contribute to the vascular remodeling accompanying disease progression.

CD38 Mediates Angiotensin II–Induced Intracellular Ca2+ Release in Rat Pulmonary Arterial Smooth Muscle Cells

CD38 is a multifunctional enzyme that catalyzes the formation of the endogenous Ca(2+)-mobilizing messengers cyclic ADP-ribose (cADPR) and nicotinic acid adenosine dinucleotide phosphate (NAADP) for the activation of ryanodine receptors (RyRs) of sarcoplasmic reticulum and NAADP-sensitive Ca(2+) release channels in endolysosomes, respectively. It plays important roles in systemic vascular functions, but there is little information on CD38 in pulmonary arterial smooth muscle cells (PASMCs). Earlier studies suggested a redox-sensing role of CD38 in hypoxic pulmonary vasoconstriction. This study sought to characterize its roles in angiotensin II (Ang II)-induced Ca(2+) release (AICR) in PASMCs. Examination of CD38 expression in various rat arteries found high levels of CD38 mRNA and protein in pulmonary arteries. The Ang II-elicited Ca(2+) response consisted of extracellular Ca(2+) influx and intracellular Ca(2+) release in PASMCs. AICR activated in the absence of extracellular Ca(2+) was reduced by pharmacological or siRNA inhibition of CD38, by the cADPR antagonist 8-bromo-cADPR or ryanodine, and by the NAADP antagonist Ned-19 or disruption of endolysosomal Ca(2+) stores with the vacuolar H(+)-ATPase inhibitor bafilomycin A1. Suppression of AICR by the inhibitions of cADPR- and NAADP-dependent pathways were nonadditive, indicating interdependence of RyR- and NAADP-gated Ca(2+) release. Furthermore, AICR was inhibited by the protein kinase C inhibitor staurosporine, the nonspecific NADPH oxidase (NOX) inhibitors apocynin and diphenyleneiodonium, the NOX2-specific inhibitor gp91ds-tat, and the scavenger of reactive oxygen species (ROS) tempol. These results provide the first evidence that Ang II activates CD38-dependent Ca(2+) release via the NOX2-ROS pathway in PASMCs.

Classical Transient Receptor Potential 1 and 6 Contribute to Hypoxic Pulmonary Hypertension Through Differential Regulation of Pulmonary Vascular FunctionsNovelty and Significance

Hypoxic pulmonary hypertension is characterized by increased vascular tone, altered vasoreactivity, and vascular remodeling, which are associated with alterations in Ca2+ homeostasis in pulmonary arterial smooth muscle cells. We have previously shown that classical transient receptor potential 1 and 6 (TRPC1 and TRPC6) are upregulated in pulmonary arteries (PAs) of chronic hypoxic rats, but it is unclear whether these channels are essential for the development of pulmonary hypertension. Here we found that pulmonary hypertension was suppressed in TRPC1 and TRPC6 knockout (Trpc1−/− and Trpc6−/−) mice compared with wild-type after exposure to 10% O2 for 1 and 3 weeks. Muscularization of pulmonary microvessels was inhibited, but rarefaction was unaltered in hypoxic Trpc1−/− and Trpc6−/− mice. Small PAs of normoxic wild-type mice exhibited vasomotor tone, which was significantly enhanced by chronic hypoxia. Similar vasomotor tone was found in normoxic Trpc1−/− PAs, but the hypoxia-induced enhancement was blunted. In contrast, there was minimal vascular tone in normoxic Trpc6−/− PAs, but the hypoxia-enhanced tone was preserved. Chronic hypoxia caused significant increase in serotonin-induced vasoconstriction; the augmented vasoreactivity was attenuated in Trpc1−/− and eliminated in Trpc6−/− PAs. Moreover, the effects of 3-week hypoxia on pulmonary arterial pressure, right ventricular hypertrophy, and muscularization of microvessels were further suppressed in TRPC1-TRPC6 double-knockout mice. Our results, therefore, provide clear evidence that TRPC1 and TRPC6 participate differentially in various pathophysiological processes, and that the presence of TRPC1 and TRPC6 is essential for the full development of hypoxic pulmonary hypertension in the mouse model.

TRPC1 and TRPC6 Contribute to Hypoxic Pulmonary Hypertension through Differential Regulation of Pulmonary Vascular Functions RR

Hypoxic pulmonary hypertension is characterized by increased vascular tone, altered vasoreactivity, and vascular remodeling, which are associated with alterations in Ca2+ homeostasis in pulmonary arterial smooth muscle cells. We have previously shown that classical transient receptor potential 1 and 6 (TRPC1 and TRPC6) are upregulated in pulmonary arteries (PAs) of chronic hypoxic rats, but it is unclear whether these channels are essential for the development of pulmonary hypertension. Here we found that pulmonary hypertension was suppressed in TRPC1 and TRPC6 knockout (Trpc1−/− and Trpc6−/−) mice compared with wild-type after exposure to 10% O2 for 1 and 3 weeks. Muscularization of pulmonary microvessels was inhibited, but rarefaction was unaltered in hypoxic Trpc1−/− and Trpc6−/− mice. Small PAs of normoxic wild-type mice exhibited vasomotor tone, which was significantly enhanced by chronic hypoxia. Similar vasomotor tone was found in normoxic Trpc1−/− PAs, but the hypoxia-induced enhancement was blunted. In contrast, there was minimal vascular tone in normoxic Trpc6−/− PAs, but the hypoxia-enhanced tone was preserved. Chronic hypoxia caused significant increase in serotonin-induced vasoconstriction; the augmented vasoreactivity was attenuated in Trpc1−/− and eliminated in Trpc6−/− PAs. Moreover, the effects of 3-week hypoxia on pulmonary arterial pressure, right ventricular hypertrophy, and muscularization of microvessels were further suppressed in TRPC1-TRPC6 double-knockout mice. Our results, therefore, provide clear evidence that TRPC1 and TRPC6 participate differentially in various pathophysiological processes, and that the presence of TRPC1 and TRPC6 is essential for the full development of hypoxic pulmonary hypertension in the mouse model.

Clinical Management of Acute Interstitial Pneumonia: A Case Report

We describe a 51-year-old woman who was admitted to hospital because of cough and expectoration accompanied with general fatigue and progressive dyspnea. Chest HRCT scan showed areas of ground glass attenuation, consolidation, and traction bronchiectasis in bilateral bases of lungs. BAL fluid test and transbronchial lung biopsy failed to offer insightful evidence for diagnosis. She was clinically diagnosed with acute interstitial pneumonia (AIP). Treatment with mechanical ventilation and intravenous application of methylprednisolone (80 mg/day) showed poor clinical response and thus was followed by steroid pulse therapy (500 mg/day, 3 days). However, she died of respiratory dysfunction eventually. Autopsy showed diffuse alveolar damage associated with hyaline membrane formation, pulmonary interstitial, immature collagen edema, and focal type II pneumocyte hyperplasia.