Membranous Expression of Integrin α5β1 is Elevated in Endothelial Cells of Pulmonary Arterial Hypertension

Abstract

Pulmonary Arterial Hypertension (PAH) is an incurable pathology characterized by remodeling of small pulmonary arteries leading to increased pulmonary vascular resistance and right ventricular heart failure. The endothelial dysfunction and endothelial-to-mesenchymal transition (EndoMT) observed in PAH contribute to the formation of plexiform lesions. Fibronectin is a commonly used molecular marker of EndoMT and is abundantly present in the plexiform lesions observed in human PAH. We have previously observed that pulmonary artery endothelial cells (PAECs) from the lesion forming Sugen/hypoxia rat model of PAH demonstrate increased proliferation and angiogenesis compared to PAECs from normoxic control animals. As the fibronectin receptor integrin α5β1 promotes endothelial proliferation and angiogenesis, we aimed to determine whether the expression of integrin α5β1 was elevated in PAH PAECs. Sugen/hypoxia rat PAECs (PAH PAECs) or normoxic control cells were received from the University of South Alabama. Initially, we observed an increase in mRNA expression of integrins α5, αV and β1 in PAH PAECs compared to control cells using a PCR screening array for molecular markers of epithelial-to-mesenchymal transition. Increased protein expression of the integrin α5, αV and β1 monomers were also observed in membrane fractions of PAH PAECs via western blot, compared to the cell membranes of control PAECs following a cell fractionation assay. To confirm the presence of the integrin α5β1 heterodimer at the membranes of PAH PAECs, co-immunoprecipitation of integrins α5 and β1 was performed from the membrane fractions of PAH and control PAECs. Following immunoprecipitation, western blot analysis demonstrated a strong coprecipitation of integrin α5 and β1 in membrane fractions of PAH PAECs, compared to weak coprecipitation in control PAECs. To confirm the increased membranous expression of integrin α5β1 in human tissue, human pulmonary endothelial cells from patients with idiopathic PAH (IPAH), familial PAH (FPAH) and control cells from failed donors (FD) were analyzed with an integrin-mediated cell adhesion array for cell surface expression of alpha and beta integrins. Human cells were provided by PHBI under the Pulmonary Hypertension Breakthrough Initiative (PHBI)*. Monomeric α5- and β1-mediated adhesion and heterodimeric α5β1-mediated adhesion were increased in IPAH and FPAH cells compared to FD control pulmonary endothelial cells. Collectively, these data strongly indicate that protein levels of the integrin α5β1 fibronectin receptor are increased at the cell membranes of pulmonary endothelial cells in PAH.