The Polarized Secretion of Endothelial Proteins in Pro-Atherogenic Condition.

Abstract

A single layer of cells called endothelial cells lines the blood vessels and separates the blood from the surrounding tissues. Although the body's largest secretory organs are endothelial cells, little is known about the secretory function of these cells in abnormal conditions. Utilizing a proteomic analysis, our Lab were able to investigate the release of components into the vascular wall and circulation through the investigation of polarized endothelial secretion. A group of 14 proteins whose secretion is elevated in response to lipoprotein was found previously in our lab through performing a proteomic investigation of endothelial secretion in reaction to LDL and oxidized LDL. Our Lab has found that lipoproteins increased and directed the secretion of endothelial FN and CCN1 using Transwell assay. Two targets, fibronectin and MMP1, were the subjects of my detailed investigation. According to my research and using Transwell assay, LDL or oxLDL lipoproteins cause an increase of MMP1 release. Our lab has found that this happened in the absence of an alteration in mRNA expression, indicating post-translational control of these proteins' secretion. In my research, I employed the Transwell experiment to examine how lipoproteins polarized the atherogenic protein MMP1, and I discovered that this polarization was elevated and directed toward LDL and oxLDL applied to either the basal or apical endothelial membranes. The silence of well-known LDL and oxLDL receptors (LDLR, ALK1, CD36, SRB1, and LOX1) did not stop the secretion of MMP1, FN, and CCN1 in the presence of lipoproteins, which suggests the actions of other receptors to regulate endothelial secretion. This finding helped me to think and search for the receptors that detect lipoproteins for endothelial secretion. I performed a proteome analysis and surficial biotinylating test for surficial endothelial proteins to find more lipoprotein receptors. Among the 541 proteins I found, Scavenger receptor class F (SCARF1) was surprisingly elevated with LDL and oxLDL in comparison to control cells. Remarkably, in the Transwell assay, inhibition of SCARF1 dramatically decreased the amount of polarized endothelium fibronectin following treatment with LDL and oxLDL, indicating that SCARF1 is the primary receptor for FN secretion in atherogenic conditions. I discovered that endothelial cells treated with oxLDL had higher cholesterol loading. Most of the cholesterol is kept in vesicles with the late endosome characteristic CD63+. Since it has been demonstrated that the SNARE proteins Syntaxin-4 and Syntaxin-6 react to cholesterol, I ii investigated the potential function of these polarized secretion regulators in these proatherogenic conditions. I discovered that silencing Syntaxin-4 and Syntaxin-6 dramatically reduces the amount of fibronectin secreted in response to LDL or oxLDL as compared to the controls. With LDL and oxLDL, confocal microscopy has shown that syntaxin-6 is dispersed throughout the cytoplasm of endothelial cells, while syntaxin-4 translocate from the endothelial cytoplasm (endoplasmic reticulum) to plasma membranes. Overall, this research has demonstrated that Syntaxin-4 and Syntaxin-6 mediate the response to polarized secretion driven by LDL and oxLDL via cholesterol. Fibronectin and MMP1 are two examples of the proteins released in response to LDL that have been shown to play roles in the development of atherosclerosis. Additionally, SCARF1 is the main receptor to detect lipoproteins for fibronectin secretion. My research reveals a unique secretory response to lipoprotein that triggers the release of pro-inflammatory and pro-atherogenic regulators.