The siRNA targeting MMP‐9 mitigates Homocysteine induced dysruption of barrier integrity in Human intestinal microvascular cells

Abstract

Homocysteine (Hcy), a sulfur containing non-protein amino acid causes loss of vascular integrity by disorganizing several junctional proteins that contribute to the maintenance and regulation of the endothelial barrier, causing leaky vasculature. However, Hcy mediated response in gut-specific microvascular endothelial cell permeability is not yet established. We hypothesize if Hcy induces disruption of junctional protein in human intestinal microvascular endothelial cells (HIMEC), through MMP-9 activation. And, if siRNA for MMP-9 can mitigate this altered permeability. HIMEC leakage was evaluated by in-vitro transwell system using FITC-BSA. In addition, we used the Electric Cell-Substrate Impedance Sensing technique to monitor cell adhesion/spreading and junctional integrity, results demonstrated lower intercellular resistance in Hcy treated HIMEC. Interestingly we observed restoration of barrier functions in cells treated with Hcy using MMP-9 siRNA. Hcy instigated cell monolayer permeability and siRNA for MMP-9 mitigated this permeability. The permeability was caused by disruption of VE-Cadherin and Z0-1 in HIMEC mediated through MMP-9 activation as observed in immunoblot and confocal analysis. These results indicates that siRNA for MMP-9 alleviated Hcy induced junctional disruption which shows Hcy induced leaky barrier was mediated in part by MMP-9 activation.