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Sildenafil inhibits hypoxia-induced transient receptor potential canonical protein expression in pulmonary arterial smooth muscle via cGMP-PKG-PPAR? axis.

ABSTRACT: Transient receptor potential canonical (TRPC) proteins play important roles in chronically hypoxic pulmonary hypertension (CHPH). Previous results indicated that sildenafil inhibited TRPC1 and TRPC6 expression in rat distal pulmonary arteries (PAs). However, the underlying mechanisms remain unknown. We undertook this study to investigate the downstream signaling of sildenafil's regulation on TRPC1 and TRPC6 expression in pulmonary arterial smooth muscle cells (PASMCs). Hypoxia-exposed rats (10% O2 for 21 d) and rat distal PASMCs (4% O2 for 60 h) were taken as models to mimic CHPH. Real-time PCR, Western blotting, and Fura-2-based fluorescent microscopy were performed for mRNA, protein, and Ca(2+) measurements, respectively. The cellular cyclic guanosine monophosphate (cGMP) analogue 8-(4-chlorophenylthio)-guanosine 3',5'-cyclic monophosphate sodium salt (CPT-cGMP) (100 ?M) inhibited TRPC1 and TRPC6 expression, store-operated Ca(2+) entry (SOCE), and the proliferation and migration of PASMCs exposed to prolonged hypoxia. The inhibition of CPT-cGMP on TRPC1 and TRPC6 expression in PASMCs was relieved by either the inhibition or knockdown of cGMP-dependent protein kinase (PKG) and peroxisome proliferator-activated receptor ? (PPAR?) expression. Under hypoxic conditions, CPT-cGMP increased PPAR? expression. This increase was abolished by the PKG antagonists Rp8 or KT5823. PPAR? agonist GW1929 significantly decreased TRPC1 and TRPC6 expression in PASMCs. Moreover, hypoxia exposure decreased, whereas sildenafil treatment increased, PKG and PPAR? expression in PASMCs ex vivo, and in rat distal PAs in vivo. The suppressive effects of sildenafil on TRPC1 and TRPC6 in rat distal PAs and on the hemodynamic parameters of CHPH were inhibited by treatment with the PPAR? antagonist T0070907. We conclude that sildenafil inhibits TRPC1 and TRPC6 expression in PASMCs via cGMP-PKG-PPAR?-dependent signaling during CHPH.

SUBMITTER: Wang J

PROVIDER: S-EPMC3824028 | biostudies-literature | 2013 Aug

REPOSITORIES: biostudies-literature

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Sildenafil inhibits hypoxia-induced transient receptor potential canonical protein expression in pulmonary arterial smooth muscle via cGMP-PKG-PPARγ axis.

Wang Jian J Yang Kai K Xu Lei L Zhang Yi Y Lai Ning N Jiang Hua H Zhang Yajie Y Zhong Nanshan N Ran Pixin P Lu Wenju W

American journal of respiratory cell and molecular biology 20130801 2

Transient receptor potential canonical (TRPC) proteins play important roles in chronically hypoxic pulmonary hypertension (CHPH). Previous results indicated that sildenafil inhibited TRPC1 and TRPC6 expression in rat distal pulmonary arteries (PAs). However, the underlying mechanisms remain unknown. We undertook this study to investigate the downstream signaling of sildenafil's regulation on TRPC1 and TRPC6 expression in pulmonary arterial smooth muscle cells (PASMCs). Hypoxia-exposed rats (10% ...[more]

PMID: 23526219

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Project description:ObjectivePrevious studies showed the benefit of canonical transient receptor potential 6 (TRPC6) channel deficiency in promoting endothelial healing of arterial injuries in hypercholesterolemic animals. Long-term studies utilizing a carotid wire-injury model were undertaken in wild-type (WT) and TRPC6-/- mice to determine the effects of TRPC6 on phenotypic modulation of vascular smooth muscle cells (SMC) and neointimal hyperplasia. We hypothesized that TRPC6 was essential in the maintenance or reexpression of a differentiated SMC phenotype and minimized luminal stenosis following arterial injury.MethodsThe common carotid arteries (CCA) of WT and TRPC6-/- mice were evaluated at baseline and 4 weeks after wire injury. At baseline, CCA of TRPC6-/- mice had reduced staining of MYH11 and SM22, fewer elastin lamina, luminal dilation, and wall thinning. After carotid wire injury, TRPC6-/- mice developed significantly more pronounced luminal stenosis compared with WT mice. Injured TRPC6-/- CCA demonstrated increased medial/intimal cell number and active cell proliferation when compared with WT CCA. Immunohistochemistry suggested that expression of contractile biomarkers in medial SMC were essentially at baseline levels in WT CCA at 28 days after wire injury. By contrast, at 28 days after injury medial SMC from TRPC6-/- CCA showed a significant decrease in the expression of contractile biomarkers relative to baseline levels. To assess the role of TRPC6 in systemic arterial SMC phenotype modulation, SMC were harvested from thoracic aortae of WT and TRPC6-/- mice and were characterized. TRPC6-/- SMC showed enhanced proliferation and migration in response to serum stimulation. Expression of contractile phenotype biomarkers, MYH11 and SM22, was attenuated in TRPC6-/- SMC. siRNA-mediated TRPC6 deficiency inhibited contractile biomarker expression in a mouse SMC line.ConclusionsThese results suggest that TRPC6 contributes to the restoration or maintenance of arterial SMC contractile phenotype following injury. Understanding the role of TRPC6 in phenotypic modulation may lead to mechanism-based therapies for attenuation of IH.

Project description:Danshen, the dried root of Salvia miltiorrhiza, is widely used in clinics in China for treating various diseases, including cardiovascular diseases. Sodium tanshinone IIA sulfonate (STS), a water-soluble derivative of tanshinone IIA isolated as the major active component from Danshen, was recently reported to be effective in attenuating the characteristic pulmonary vascular changes associated with chronically hypoxic pulmonary hypertension (CHPH); however, the underlying detailed mechanisms are poorly understood. In this study, we investigated the effects of STS on basal intracellular Ca(2+) concentration ([Ca(2+)](i)) and store-operated Ca(2+) entry (SOCE) in distal pulmonary arterial smooth muscle cells (PASMCs) exposed to prolonged hypoxia or isolated from CHPH rats. SOCE measured by Mn(2+) quenching of Fura-2 fluorescence in PASMCs from rats exposed to chronic hypoxia (10% O(2), 21 d) was increased by 59%, and basal [Ca(2+)](i) was increased by 119%; this effect was inhibited by intraperitoneal injection of STS. These inhibitory effects of STS on hypoxic increases of SOCE and basal [Ca(2+)](i) were associated with reduced expression of canonical transient receptor potential (TRPC)1 and TRPC6 in distal pulmonary arterial smooth muscle and decreases on right ventricular pressure, right ventricular hypertrophy, and peripheral pulmonary vessel thickening. In ex vivo cultured distal PASMCs from normoxic rats, STS (0-25 ?M) dose-dependently inhibited hypoxia-induced cell proliferation and migration, paralleled with attenuation in increases of basal [Ca(2+)](i), SOCE, mRNA, and protein expression of TRPC1 and TRPC6. STS also relieved right ventricular systolic pressure, right ventricular hypertrophy, and TRPC1 and TRPC6 protein expression in distal pulmonary arteries in a monocrotaline-induced rat model of pulmonary arterial hypertension. These results indicate that STS prevents pulmonary arterial hypertension development likely by inhibiting TRPC1 and TRPC6 expression, resulting in normalized basal [Ca(2+)](i) and attenuated proliferation and migration of PASMCs.

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Sildenafil inhibits hypoxia-induced transient receptor potential canonical protein expression in pulmonary arterial smooth muscle via cGMP-PKG-PPAR? axis.

Publications

Sildenafil inhibits hypoxia-induced transient receptor potential canonical protein expression in pulmonary arterial smooth muscle via cGMP-PKG-PPARγ axis.

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