Abstract Body (Do not enter title and authors here): Background
Disruption of microvascular endothelial integrity significantly contributes to ischemic cardiovascular injury. PLVAP is an endothelial-specific integral membrane glycoprotein localized to caveolae and fenestrae, predominantly expressed in cardiac microvascular endothelial cells (CMVECs). The precise role of PLVAP and its regulatory mechanisms remain unclear. We hypothesize that Aryl Hydrocarbon Receptor Nuclear Translocator (ARNT) modulates PLVAP to regulate cardiac barrier function.
Methods and Results
We utilized primary CMVECs isolated from inducible endothelial-specific ARNT knockout (KO) mouse hearts and human CMVECs treated with siRNA. ARNT deletion resulted in reduced PLVAP expression at both protein and mRNA levels (P < 0.0001). Chromatin immunoprecipitation followed by qPCR (ChIP-qPCR) confirmed that ARNT binds to the PLVAP promoter region, indicating transcriptional regulation of PLVAP by ARNT. Promoter assays further demonstrated that ARNT regulates PLVAP expression (P < 0.0001). Immunofluorescence staining with wheat germ agglutinin (WGA) revealed a significant reduction in the glycocalyx layer in PLVAP-deleted human microvascular endothelial cells, alongside decreased expression of endothelial junctional proteins VE-Cadherin and Occludin. Conversely, activating ARNT/HIF1α heterodimers using a prolyl-4 hydroxylase inhibitor (DMOG) or overexpressing ARNT restored the endothelial glycocalyx, permeability, and VE-Cadherin expression in LPS-stimulated cells. Furthermore, ARNT activation increased the size of plasmalemma vesicle pores and heightened endothelial permeability in human primary endothelial cells.
Conclusions This study highlights the vital role of the endothelial ARNT in regulating PLVAP which controls vascular permeability. These findings provide insights into the molecular mechanisms of endothelial permeability and identify potential therapeutic targets for treating vascular and cardiac complications.