The Role Of Junctional Adhesion Molecule-Like Protein In The Initiation And Progression Of Atherosclerosis

Objective:Atherosclerosis (AS) is a pathological basis of many cardiovascular disease, threatening the health of people seriously. Its pathogenesis is complex and is not fully clear. An increasing number of studies have found that AS is an immune inflammatory process occurs late in the damaged vessel intima. Junctional Adhesion Molecule-like protein (JAML), as a recently discovered member of JAM family, belongs to the secreted and type I transmembrane glycoprotein. Previous studies have showed that JAML is mainly distributed in neutrophils, endothelial γδT cells, memory T cells, monocytes and macrophages, playing important roles in immunity, inflammation and tissue homeostasis through regulating leukocyte-endothelial interactions, inflammatory and growth fator secretion in γδT cells. However, it is not clear that whether JAML participates in the pathology of AS. This subject explored the role of JAML in the initiation and progression of Atherosclerosis and the regulation of macrophages’ function in vivo and vitro. This will provide a new idea for the new drug research of AS.Methods:1. Detection of JAML expression level in atherosclerotic plaque in vivo.Coronary arteries with mild or severe stenosis from cases with coronary atherosclerotic heart disease were immunostained with anti-JAML antibody to detect the distribution and the expression level of JAML in the atherosclerotic plaque. Confocal immunofluorescence was used to detect the location in particular cells. Immunoblot was used to measure the expression of JAML in the aorta of C57BL/6 mice fed standard chow and ApoE-/- mice fed high-fat diet.2. To explore the role of JAML in the initation and progression of AS, the regulation of inflammatory responses and the activation of NF-κB in atherosclerotic plaque.Experimental animal models of the early and advanced atherosclerosis were built in ApoE-/- mice. All the animals were transfected with lentivirus-mediated scrambled shRNA or JAML shRNA. Body weight and serum lipid levels at the end of the experiments in ApoE-/- mice was measured with the enzymatic method; General oil 0-staining in aorta stem was used to measure the plaque area in aorta; Frozen section tissues in ApoE-/- mice was used to analyze the plaque area, contents of lipid, macrophages, smooth muscle cells and the type I collagen fiber by staining with H&E, oil red O, MOMA-2, α-SMA and Sirus red. We also calculate the plaque instability index by using the formula:(oil red O+ area+MOMA-2+ area)/(α-SMA+ area+collagen I+ area)×100%. Immunohistochemistry was used to detect the expression level of IL-6 and TNF-α in aortic roots; Immunoblot was used to detect the activation of NF-κB in aorta.3. Verify the role of JAML in oxLDL-induced macrophages and the regulation of NF-κB signaling pathway.Reverse transcriptase-PCR (RT-PCR) was used to analyse the JAML mRNA levels in selected representative cells including murine macrophage, SMCs and human umbilical vein ECs and monocytes separated from human peripheral blood; Westernblot was used to detect the expression level of JAML in RAW264.7 cells activated by oxLDL, LPS and TNF-α. RAW264.7 cells were transfected with scrambled siRNA or JAML siRNA, then stimulated by oxLDL respectively. The expression levels of inflammatory factors were detected by real time RT-PCR and the activation of p65 were measured by Western blot.Results:1. In vivo, the expression of JAML was significantly elevated in atherosclerotic arteries; Immunofluorescence and confocal images manifested that JAML was strongly expressed in the macrophage of atherosclerotic plaque, to a lesser extent endothelial cells and smooth muscle cells. The same results were confirmed in the aorta of C57BL/6 with normal diet and ApoE-/- mice with high fat diet.2. In atherosclerotic mice, there was no difference in body weight and serum lipid between JAML deficiency groups and control groups after feded with high fat diet; JAML deficiency reduced lesion area in early atherosclerosis, reduced instability index in early and advanced atherosclerosis; JAML deficiency also decreased inflammatory responses and attenuated the activation of NF-κB in atherosclerotic mice.3. In vitro, the expression of JAML was significantly elevated in oxLDL or TNF-a stimulated macrophages but was not significantly changed in LPS treated cells; JAML deficiency also decreased inflammatory responses and attenuated the activation of NF-κB in oxLDL-induced macrophages.Conclusion and Innovation:1. We first detected the expression and the location of JAML in atherosclerotic plaque. It was confirmed that the expression of JAML is elevated in atherosclerotic plaque. JAML is strongly expressed in the macrophage of atherosclerotic plaque, to a lesser extent in endothelial cells and smooth muscle cells and this gives us a hint as to the major role of JAML in the atherosclerostic cells.2. We first confirmed that JAML promoted the initiation and progression of AS. JAML deficiency can inhibit the formation of atherosclerotic plaque, reduce the inflammatory responses and increase the stability of atherosclerotic plaque.3. We also explored the mechanism of JAML promoting the initiation and progression of atherosclerosis and confirmed that JAML can aggravate the dysfunction of macrophages, induce the secretion of inflammatory factors and activate the NF-κB pathway.In conlusion, on the one hand, this subject will serve a new target for anti-AS drugs; on the other hand, JAML, as a secreting protein, will be an important indicator to diagnose and monitor AS.