Neprilysin Regulates Mouse Pulmonary Artery Smooth Muscle Cell Phenotype

Abstract

Rationale: Neprilysin (NEP) is a cell surface zinc metallopeptidase, which cleaves and inactivates peptides important in regulating pressor responses in the vasculature, and migration and proliferation of cancer cells. Loss of NEP in mice increases vascular remodeling and pulmonary hypertension in response to hypoxia. Understanding how NEP affects pathways that contribute to vascular cell migration and proliferation could provide a better understanding of the role of NEP in the pulmonary circulation. This study was performed to determine whether increased medial thickening observed in pulmonary arteries (PA) of hypoxia-exposed NEP-/mice is due to differences in migration and proliferation of smooth muscle cells (SMC). Methods and Results: PASMCs isolated from NEP+/+ and NEP-/mice were used to measure migration and proliferation in response to serum (0.2%) and PDGF-BB (10ng/ml). Our results show that PASMCs from NEP-/mice exhibit enhanced migration and proliferation which is attenuated by adding back NEP. NEP-/cells had decreased levels of smooth muscle (SM) contractile proteins, α-SM-actin, calponin and SM-22 which was restored by expression of lentivirus expressing full length human NEP. Inhibition of NEP in NEP+/+ cells by phosphoramidon (10μM) or knock down by siRNA resulted in an increase in basal migration and proliferation and a decrease in expression of SM-contractile proteins. To understand how loss of NEP results in altered PASMC phenotype we examined the interaction of NEP with the PDGF receptor (PDGFR). In NEP+/+ cells, NEP, PDGFR and PTEN were present in caveoli. Interaction of PTEN with NEP stabilized its expression and limited PDGFR signaling, maintaining PASMC in a differentiated state. Further analysis showed that loss of NEP led to increase in PDGF receptor associated src kinase activity and inactivation of PTEN by phosphorylation resulting in its constitutive activation. This was accompanied by a decrease in expression of serum response factor (SRF), a transcription important in regulation of smooth muscle genes. Inhibition of PDGFR with a tyrosine kinase inhibitor attenuated the increased migration and proliferation and restored levels of SRF and α SMactin. Conclusion: Loss of NEP increases migration and proliferation of PASMC in response to PDGF and induces a dedifferentiated phenotype. Increased src kinase associated with PDGFR in NEP-/cells results in its constitutive activation resulting in increased migration and proliferation. The results may help explain how increased pulmonary vascular remodeling occurs in the hypoxia-exposed NEP-/mice