Background & Hypothesis:
Excessive proliferation of pulmonary artery smooth muscle cells (PASMCs) and sustained pulmonary vasoconstriction are thought to play critical roles in the development of idiopathic pulmonary arterial hypertension (IPAH). Recently, we demonstrated that upregulation of the canonical transient receptor potential 6 (TRPC6) channel contributes to excessive proliferation of PASMCs isolated from IPAH patients. This study aimed at characterizing whether up-regulated TRPC6 expression affects resting cytosolic [Ca
2+
] ([Ca
2+
]
cyt
) level and Ca
2+
entry in PASMCs of IPAH patients.
Methods & Results:
[Ca
2+
]
cyt
was measured by fluorescence ratio video imaging with the Ca
2+
indicator fura-2. 1-Oleoyl-2-acetyl-sn-glycerol (OAG), a cell-permeable diacylglycerol analog that activates TRPC6 channels, was used to stimulate channel activities in the presence of extracellular Ca
2+
. The resting [Ca
2+
]
cyt
andOAG-mediated increase in [Ca
2+
]cyt were significantly higher in PASMCs from IPAH patients compared to PASMCs isolated from secondary pulmonary artery hypertension and normotensive patients. In PASMCs from IPAH patients, inhibition of TRPC6 expression by adenovirus-mediated siRNA specifically targeted the human TRPC6 gene led to an approximately 90% reduction of TRPC6 mRNA and protein levels and significantly attenuated OAG-mediated increase in [Ca
2+
]
cyt
. Treatment of the IPAH-PASMCs with siRNA also decreased the resting [Ca
2+
]
cyt
and significantly inhibited cell proliferation in comparison to cells treated with scrambled control siRNA. Furthermore, exogenous overexpression of human TRPC6 increased the resting [Ca
2+
]
cyt
and enhanced OAG-mediated Ca
2+
entry in normal PASMCs.
C
onclusions:
These results suggest that upregulation of TRPC6 channels in PASMC from IPAH patients serves as an important pathway for agonist-mediated Ca
2+
entry, mitogen-mediated PASMC proliferation and, ultimately, pulmonary vascular remodeling. Targeting TRPC6 expression and function in PASMCs would help develop novel therapeutic approaches for IPAH patients.
Selectively upregulated microRNAs in pulmonary artery smooth muscle cells from patients with idiopathic pulmonary arterial hypertension
