Idiopathic pulmonary arterial hypertension is a progressive, chronic, and usually lethal lung disease of unknown causes with limited therapeutic options. Pulmonary arterial hypertension is characterized by aberrant pulmonary vasoconstriction and abnormal pulmonary vascular remodeling. Several lines of evidence suggest that deregulated GPCRs may represent a key factor in the progression of PAH. Specifically, GPCRs are centrally involved in the pathobiology of PAH. Identifying dysregulated GPCRs in PAH may pave the way to targeting abnormal pulmonary vasoconstriction and remodeling, thereby resulting in options for translational research. Therefore, the purpose of the present study was to identify and analyze both known and orphan GPCRs potentially involved in the development of PAH.An analysis of GPCR expression was performed in laser micro-dissected pulmonary vessels from donors and IPAH patients using a quantitative GPCR high-throughput expression screening system. The quantitative GPCR expression analysis revealed that a large number of GPCRs were upregulated in the pulmonary vasculature of PAH patients, and these included VIPR1, chemokine receptors (CMKLR1, CXCR7, CXCR6), a leukotriene receptor (CYSLTR1), neuropeptide Y receptors (NPY1R, NPY5R), lysophosphatidic acid receptors (LPAR1, LPAR2), an endothelin receptor (EDNRB), a trace amine receptor (TAAR8), a leucine-rich repeat (LGR7), a niacin receptor (GPR109B), and 5 orphan receptors (GPR18, GPR21, GPR97 GPR112, and GPR115). The purinergic receptor (P2Y2) and dopamine receptor (DRD1) were downregulated.Further, the mRNA expression of relevant GPCR hits was analyzed in human PASMCs derived from donors and PAH patients. The qPCR results revealed the upregulation of VIPR1 and LGR7 in PASMCs from IPAH patients. In contrast, P2Y2 and DRD1 were downregulated in the PASMCs of IPAH patients.The potential role of candidate GPCRs in PASMCs was previously determined by siRNA knockdown or ligand stimulation. The proliferation of PASMCs determined using BrdU incorporation assay indicated that the knockdown of GPR115, LGR7, and PTAFR significantly reduced serum-induced PASMC proliferation. On the other hand, the knockdown of P2Y2 did not result in any significant changes in PASMC proliferation. Furthermore, the P2Y2 ligand MRS2768 reduced both PDGF- and serum-induced PASMC proliferation. In addition, MRS2768 limited both serum- and PDGF-induced PASMC migration. The knockdown of P2Y2 by siRNA resulted in a spontaneous increase in PASMC migration under serum-starved conditions. MRS2768 also decreased MMP2 activation in PASMCs. Therefore, MRS2768 and its receptor P2Y2 play significant role in migration. Moreover, upon MRS2768 stimulation, P2Y2 promoted cAMP (a potent vasodilator) production in PASMCs. In addition, MRS2768 activated the cAMP/EPAC-mediated inhibition of RhoA/ROCK1 and reduced the MLC phosphorylation. Due to its role in decreasing MLC phosphorylation, MRS2768 might thereby reduce PASMC asoconstriction.overall, this study implies that more GPCRs are differentially expressed in the pulmonary vasculatures of PAH patients than in those of donors. Most interestingly, P2Y2 might play a key role in pulmonary vascular remodeling by regulating PASMC proliferation and migration.
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