TLR4 Induces Inflammatory Responses And Lipid Accumulation In Vascular Smooth Muscle Cells By Down-regulating ABCG1 Expression Through PPARγ

Background and Objectives:Atherosclerosis(AS) is a chronic inflammatory process that induced by cholesterol and lipoproteins accumulated in the vessel wall. Atherosclerosis formation results from interactions of circulating factors and various cell types in the vessel wall, including endothelial cells, lymphocytes, monocytes, and smooth muscle cells(SMCs). SMCs play an important role in the process of AS. SMCs are the major producers of extracellular matrix within the vessel wall. SMCs can modify the type of matrix proteins produced in response to atherogenic stimulation. SMCs can be converted to other cell type functions,like macrophages. SMCs also express a variety of lipid uptake receptors to form foam cells same as endothelial cells. SMCs express a variety of adhesion molecules which can adhere and migrate into the vessel wall.Toll like receptor 4(TLR4) plays an important role in the atherosclerotic process, and widely distributed in macrophages, endothelial cells, vascular smooth muscle cells and fat cells. Ox LDL-induced inflammatory response involves a series of mechanisms. Recent studies have found that TLR4 high expressed in lipid-rich atherosclerotic plaques, while in TLR4 deficient mice atherosclerosis lesion reduced significantly.Peroxisome proliferator activated receptor γ(PPARγ), which can regulate transcriptional function of cell differentiation and lipid metabolism, is a member of the nuclear receptor superfamily. ATP-binding cassette transporter G1(ABCG1) plays an important role in cholesterol efflux. In the existing research, ABCG1 plays an critical role in anti-apoptotic and anti-inflammatory by regulating cholesterol balance. PPARγ agonists can modulate the expression of ABCG1 in macrophages.Thus, we speculate that TLR4 is essential for inflammatory response and lipid accumulation in VSMCs. More importantly, we found that the underlying molecular mechanisms involves TLR4 down-regulating ABCG1 expression through PPARγ.Methods:1. The laboratory mouse genotypes were identified by agarose gel electrophoresis.2. The primary VSMCs were cultured by tissue explant method.3. The primary VSMCs and TLR4/PPARγ,TLR4/ABCG1 co-location in VSMCs were identified by immunofluorescence method.4. The mRNA expression of TNF-α、IL-6、MCP-1、VCAM-1 were measured by real-time PCR.5. The protein expression of TLR4、PPARγ、ABCG1 were measured by western blot analysis.6. Lipid droplets in VSMCs were detected by bodipy staining, total cholesterol in VSMCs quantitated by enzymatic colorimetric method.7. Rosiglitazone activate PPARγ signaling pathways in WT-VSMCs.8. PPARγ-si RNA transfected with WT-VSMCs, induced PPARγ gene silencingResults:1. We successfully identified mouse genotypes, and cultured and identified the primary VSMCs in vitro.2. TLR4/PPARγ, TLR4/ABCG1 were co-location in WT-VSMCs assessed by immunofluorescence method.3. Ox LDL stimulated WT-VSMCs and activated TLR4 signaling, upregulated the expression of TLR4. Therefore it promote TNF-α, IL-6, MCP-1 and VCAM-1 secretion. The inflammatory cytokines above secretion were inhibited when TLR4 knockout.4. Ox LDL stimulation activated TLR4 pathway and increased lipid deposition significantly in WT-VSMCs, while TLR4 knockout lipid deposition reduced in VSMCs.5. TLR4 activation down-regulated PPARγ and ABCG1 expression, but it would rescue when TLR4 knockout.6. Rosiglitazone increased ABCG1 expression, reduced inflammation and lipid deposition significantly in WT-VSMCs after ox LDL stimulation.7. PPARγ gene silencing inhibited ABCG1 expression, induced inflammation worse and increased lipid deposition in oxLDL-stimulated WT-VSMCs.Conclusions: TLR4 down-regulates ABCG1 expression and induces inflammation and lipid accumulation in VSMCs via PPARγ.