The Effects For Inhibition Of Galectin-3and RAGE On The Initiation And Development Of Atherosclerois In ApoE-deficient Mice

It has been accepted that atherosclerosis is a chronic inflammatory disease. Inflammation not only contributes to the formation of the atherosclerotic plaques, but also plays a predominant role in lesion destabilization. Many atherogenic factors, such as oxidative stress, advanced glycation end products, hypertention and smoking contribute to endothelial dysfunction. Injured endothelium facilitates the internalization of circulating lipids, the adhesion and differentiation of monocytes, and the released of cytokines. Monocytes are the primary inflammatory cell type that infiltrates early atherosclerotic plaques. Their recruitment into plaques drives disease progression. The recruitment of monocytes into the dysfunctional endotheliums is an early and critical event in atheroma initiation and development. Local inflammation is attributed to various factors produced by leukocytes, including ICAM-1, VCAM-1, MCP-1, which facilitate the monocytes transendothelial migration and then differentrate into macrophages. The activated macrophages synthesize proteolytic enzymes, such as interstitial collagenase and gelatinase, which degrade collagen, elastin and other extracellular matrix proteins, ultimately induce plaque destabilization.Recently, receptor of advanced glycation end products (RAGE) has been implicated in age related diseases including atherosclerosis, diabetes and Alzheimer’s disease. It is considered that RAGE is a crucial regulator in expression of adhesion molecules and chemokines, and plays a pivotal role in promoting inflammatory processes. Accumulating evidence suggested that RAGE, when binding to its ligand such as AGE, could activate NF-κB and result in the up-regulation of MMP-2and MMP-9, which weaken the fibrous caps and accelerate the disruption of the plaques. RAGE, therefore, acts as a linker between inflammatory cytokines and the progression of atherosclerosis. Moreover, the AGE-RAGE interaction induces the cellular activation of signal transduction cascades that generate reactive oxygen species (ROS) which in turn trigger inflammation. Oxidative stress has been suggested to play a critical role in the progression of atherosclerosis and vascular inflammation. Oxidative stress leads to increased expression of pro-inflammatory cytokines and adhesion molecules by activation of redox-sensitive transcription nuclear factor NF-κB, which was involved in lesion development and destabilization. Thus, blockade of ROS generation and inflammatory cytokine expression may be useful as a therapeutic approach to stabilize plaques. Previous studies showed that both AMPK activator WS070117and NAC act as the antioxidant. Thereby we try to demonstrate that the antioxidant is able to affect the RAGE axis and associated inflammatory factors.Monocyte adhesion to endothelium plays the initial role in progression of atherosclerosis. It is well known that the adhesion molecules expressed on the endothelium is response for the interaction, then the monocytes are retained in the extracellular matrix and differentiate into macrophages, thereby the retention of monocytes to the ECM is also critical in the progress of atherosclerosis. Galectin-3is a modulator of cell adhesion, which facilitates the metastatic cells adhesion to the endothelium. Several mechanisms are involved in this interaction. Firstly, the extracellular matrices such as laminin, fibronectin and MAC-2binding protein, act as the ligands for galectin-3. Secondly, galectin-3mediates the cross-link of CD98expressed on the activated monocytes and macrophages, and then induces the adhesion of integrin to extracellular matrix. The ligation between integrins of actived monocytes, especially the αMβ2integrin, and galectin-3promotes the adhesion of monocytes to the sub-endothelial ECM, therefor strengthens the monocyte binding to ECM. Galectin-3could also induce the recognizatin of monocyte to the endothelium directly. Previous studies indicated that the modified citrus pectin (MCP) that specifically combining with the carbohydrate-binding domain of galectin-3inhibites the adhesion of several tumors to the endothelium. However, it is not clear whether the MCP can suppress the adhesion of monocytes to endothelium.The study aims at:1) demonstrating that antioxidant WS070117and NAC inhibit the progression of atherosclerosis in apoE-/-mice via (or at least partly) the suppression of RAGE axis and subsequent decrease the adhesion moleculars, chemokines and MMPs;2) investigating the role of galectin-3in the initiation of atherosclerosis and the interaction between monocytes and endothelium and whether the MCP can suppress the adhesion of monocytes to endothelium.Part1The effects of antioxidant WS070117and N-acetylcysteine on progression of atherosclerotic plaque in apoE-deficient miceAims AMPK activator WS070117and N-acetylcysteine (NAC) have antioxidant and anti-inflammatory properties. To explore the effects of WS070117and NAC on the progression of atherosclerosis, we examined the changes of the expressions of receptor of advanced glycation end products (RAGE), adhesion molecules, chemokines, matrix metalloproteinases (MMPs) and the activation of nuclear factor kappa B (NF-κB) in atherosclerotic plaques of apoE-deficient mice administered with WS070117and NAC.Methods and results WS070117(5mg/kg/day) and NAC (200mg/kg/day) were separately administered to10-week-old apoE-/-mice fed with atherogenetic diet. After treated with the drugs for8weeks, apoE-/-mice displayed reduced serum malondialdehyde (MDA) level and impaired reactive oxygen species (ROS) generation in aortic root and left common carotid artery. Although there were no differences in serum levels of glucose and lipids in each group, the antioxidants exhibited a significant decrease in the amounts of macrophages, lipid deposition, but not smooth muscle cells. WS070117dramatically reduced plaque volume while the NAC increased collagen content in atherosclerotic lesions when the plaque composition and size were analysed histologically in these apoE-/-mice. Moreover, we found that both antioxidants have the ability to inhibit the activation of NF-κB and reduced expression of RAGE as well as down-regulated the expression of vascular cell adhesion molecule-1(VCAM-1), monocyte chemoattractant protein-1(MCP-1), or MMP2and MMP9separately, accompanied by inhibition of activation of ERK1/2, p38MAPK and JNK, as shown by immunofluorescent staining, immunohistochemistry and Western blot.Conclusions In the present study, we show novel data to suggest that WS070117and NAC inhibit the progression of atherosclerotic plaques through suppression of RAGE and NF-κB pathways in apoE-/-mice. Part2The inhibition of atherosclerosis in apoE-deficient-mice by oral intake of modified citrus pectinAims This study aims at investigating whether galectin-3is involved in the adhesion between endothelium and monocytes. We studied the effects of a high PH-and temperature-modified citrus pectin (MCP), a nondigestible, water-soluble polysaccharide fiber derived from citrus fruit that specifically inhibits the carbohydrate-binding protein galectin-3, on the progression of atherosclerosis in apoE-deficient-mice and its interfere with interaction between endothelium and monocytes.Methods and results The apoE-/-mice were fed with an atherogenetic diet and the1%MCP in drinking water for8weeks or10%MCP were gavaged for4weeks. The sections from BCA were evaluated by pathological methods. The results indicat that although there were no differences in serum levels of glucose and lipids, the area of atherosclerotic plaques and the amount of monocytes/macrophages in the BCA of apoE-/-mice were effectively reduced after treatment with MCP.Conclusions In the study, we found that MCP, given orally in drinking water or gavaged for4weeks, could inhibit the adhesion of monocytes/macrophages with endothelium in atherosclerois in apoE-/-mice, suggesting that the galectin-3could be used as a target for the prevention or treatment of the atherosclerosis.