Pulmonary Artery Smooth Muscle Cell Hif-1 Alpha Regulates Endothelin Expression Via Microrna-543

Pulmonary artery smooth muscle cells (PASMCs) express endothelin (ET-1). which modulates the pulmonary vascular response to hypoxia. Although cross-talk between hypoxia-inducible factor-1 alpha (HIF-1 alpha), an O-2-sensitive transcription factor, and ET-1 is established, the cell-specific relationship between HIF-1 alpha and ET-1 expression remains incompletely understood. We tested the hypotheses that in PASMCs 1) HIF-1 alpha expression constrains ET-1 expression. and 2) a specific microRNA (miRNA) links HIF-1 alpha and ET-1 expression. In human (h)PASMCs, depletion of HIF-la with s1RNA increased ET-1 expression at both the mRNA and protein levels (P < 0.01). In HIF-1 alpha(-/-) murine PASMCs, ET-1 gene and protein expression was increased (P < 0.0001) compared with HIF-1 alpha(+/+) cells. miRNA profiles were screened in hPASMCs transfected with siRNA-HIF-1 alpha, and RNA hybridization was performed on the Agilent (Santa Clara, CA) human miRNA microarray. With HIF-1 alpha depletion. miRNA-543 increased 2.4-fold (P < 0.01). In hPASMCs, miRNA-543 overexpression increased ET-1 gene (P < 0.01) and protein (P < 0.01) expression, decreased TWIST gene expression (P < 0.05), and increased ET-1 gene and protein expression, compared with nontargeting controls (P < 0.01). Moreover, we evaluated low passage hPASMCs from control and patients with idiopathic pulmonary arterial hypertension (IPAH). Compared with controls, protein expression of HIF-1 alpha and Twist-related protein-1 (TWIST1) was decreased (P < 0.05), and miRNA-543 and ET-1 expression increased (P < 0.001) in hPASMCs from patients with IPAH. Thus, in PASMCs, loss of HIF-1 alpha increases miRNA-543, which decreases Twist expression, leading to an increase in PASMC ET-1 expression. This previously undescribed link between HIF-1 alpha and ET-1 via miRNA-543 mediated Twist suppression represents another layer of molecular regulation that might determine pulmonary vascular tone.