TRPV1 Activation Reduces Vascular Lipid Accumulation And Attenuates Atherosclerosis

Background and Objectives: Atherosclerosis is the major factor for developing cardiovascular disease. The process of atherogenesis has been considered to be an inflammatory disease which consists largely of the accumulation of lipids within the artery wall. The key initiating process in atherogenesis is the subendothelial retention of lipoproteins. Although the majority of foam cells in the atherosclerotic lesion are thought to be derived from macrophages, vascular smooth muscle cells (VSMCs) also give rise to a significant number of lipid laden cells. VSMCs from humans, rats, and rabbits have been demonstrated to express a variety of cholesterol uptake receptors and reverse cholesterol transporters, including the LDL receptor, LDL receptor–related protein 1 (LRP1), the ATP binding cassette (ABC) transporter, ABCA1. These studies suggest that lipid accumulation in VSMCs may contribute to atherosclerosis development. However, the mechanism of lipid accumulation in the intima and its uptake by VSMCs was not fully elucidated.Recent studies showed that transient receptor potential vanilloid type-1 (TRPV1) channels are expressed in the vasculature. TRPV1 channels are activated by the specific agonist, capsaicin, the "hot" component of chili peppers. Activation of TRPV1 provokes several effects in the cardiovascular system. Activation of TRPV1 regulates the expression and secretion of endothelial cell-derived calcitonin gene-related peptide, which affords protective effects on vascular endothelial cells. Activation of TRPV1 channels mediate endothelium-dependent vasodilation in coronary arteries. Furthermore, previous work from our group indicated that activation of TRPV1 by capsaicin affects lipid metabolism, prevents the adipogenesis in stimulated preadipocytes, and prevents obesity in male mice. Obesity is a well-known risk factor for atherosclerotic vascular diseases. From these results the hypothesis arises that activation of TRPV1 channels may prevent vascular lipid storage and finally atherosclerosis.To test the above hypothesis, In the present study, we first confirmed the expression and distribution of TRPV1 in aorta and VSMCs, and detected the TRPV1-dependent change of intracellular free calcium ([Ca²⁺]i). Apolipoprotein E knockout (ApoE-/-) mice are most useful and valid models for studying atherosclerosis and exploring effective therapeutic approaches. we validate the hypothesis in ApoE-/- and TRPV1-ApoE double knockout (TRPV1-/-/ApoE-/-) mice on normal diet and high-fat diet. Then we investigated whether PVAT plays a potential role via the TRPV1,AMPK/mTOR pathway in obesity-related vascular dysfunction .In vivo,we examined the accumulation of lipids in VSMCs; In vitro, we examined serum parameters and atherosclerotic lesions in the aorta of mice fed with or without capsaicin. To further study the underlying mechanism, we detected a variety of inflammatory cytokines, cholesterol uptake receptors and reverse cholesterol transporters in aorta and examined the effect of intracellular calcium on the expression of these molecules in VSMCs.Materials and Methods:The present study includes in vivo and in vitro experiments. In vivo models include ApoE-/- mice and TRPV1-/-/ApoE-/- mice fed on normal diet and high fat diet with or without capsaicin, TRPV1-/-/ApoE-/- mice and their ApoE-/- littermates fed with normal diet were also observed, and Wistar rats fed on normal diet and high fat diet. In vitro models were VSMCs primarily cultured from aortas of C57BL/6J wild-type mice and TRPV1-/- mice.1. Expression of TRPV1 protein and mRNA in cells and VSMCs were detected by immunoblotting and RT-PCR respectively. Distribution of TRPV1 in VSMCs and arota was shown by immunofluorescence.2. Capsaicin induced [Ca²⁺]i change was examined by PTI systems.3. Lipid drop in VSMCs was observed using oil-red staining. Concentrations of total cholesterol and triglycerides were determined in plasma with an enzymatic assay according to the diagnostic kits.4. TRPV1-/-/ApoE-/- mice were constructed by breeding TRPV1-/- mice (on C57 BL/6J background) with ApoE-/- mice.5. Serum parameters like triglycerides, total cholesterol, glucose, insulin, TNF-α, IL-6 and hs-CRP were examined with special kits.6. The thoracic aortas, spanning from the left subclavian artery to the renal arteries, were stained for lipid deposition with Oil-red O. Cryostat sections (8um) were obtained from the aortic sinus. Lipid deposition was shown with Oil-red O, collagen was detected using Sirius red, and immunohistochemicalanalysis was performed with specific primary antibodies including ABCA1, LRP1, anti mouse macrophage, anti–α-actin for smooth muscle cells, and anti-CD3 T lymphocytes.7. Intercostal artery were stained with Victoria blue and Ponceau S.Periaortic adipose tissue stained with hematoxylin and eosin.Vascular reactivity in the isolated mesenteric artery rings was measured using an isometric myograph (Danish Myotech Technology, Denmark). Expression of TRPV1, eNOS, AMPK,mTOR, PI3Kand AKT were detected by immunoblotting.8. Expression of inflammatory cytokines, cholesterol uptake receptors and reverse cholesterol transporters including ABCA1, LRP1, CD36, SR-A, TNF-α, CRP, MCP-1, ICAM-1, VCAM-1 and P-selectin were detected by immunoblotting.9. Expression of NFAT3 in VSMCs from TRPV1 knock out mice and wild type littermates with capsaicin intervention in the presence or absence of TRPV1 specific blocker was detected by immunoblotting.Results:1. TRPV1 existed in VSMCs and arota of mice. Expression of TRPV1 protein was up-regulated by capsaicin or RTX treatment.2. Capsaicin or RTX dose-dependently increased [Ca2+]i in VSMCs, while this effect can be inhibited in TRPV1-/- mice or in the presence of capsazepine and 5'indo-RTX.3. Administration of the TRPV1 agonist capsaicin but not the TRPV1 antagonist capsazepine significantly reduced intracellular lipid droplets in ox-LDL-stimulated VSMC from C57BL/6J mice. The total cholesterol level in VSMC was significantly reduced by capsaicin, but not in the presence of capsazepine or in TRPV1-/- VSMC.4. Chronic capsaicin administration significantly reduced plasma levels of triglycerides and total cholesterol in ApoE-/- mice but with little effect on food intake. Diatery capsaicin also reduced serum levels TNF-α,IL-6 and hs-CRP. However, capsaicin had no effect on TRPV1-/-ApoE-/- mice. TRPV1-/-ApoE-/- mice were successfully constructed. Compared to ApoE-/- littermates, serum levels of triglycerides, total cholesterol ,TNF-α,IL-6 and hs-CRP were significantly higher in TRPV1-/-ApoE-/- mice. 5. Long-term activation of TRPV1 markedly reduced lipid storage and atherosclerotic lesions of descending thoracoabdominal aorta and the aortic sinus in ApoE-/- mice but not ApoE-/-TRPV1-/- mice on high fat diet. Atherosclerotic lesions in TRPV1-/-ApoE-/- mice were significantly more than ApoE-/- littermates.6. No significant differences were observed in the composition of atherosclerotic lesions in ApoE-/- or TRPV1-/-ApoE-/- mice. In addition, activation of TRPV1 increased expression of ABCA1, CD3 and decreased expression of LRP1 in atherosclerotic lesions from ApoE-/- mice on a high fat diet. Compared to ApoE-/- littermates, ABCA1 and CD3 was down-regulated and LRP1 was up-regulated in TRPV1-/-ApoE-/- mice.7. Obesity-related impairment of endothelium-dependent relaxation of the aorta was markedly attenuated by temporary periaortic fat removal while obesity-related enhancement of contractile performance was unaffected. Moreover, an HFD led to a downregulation of TRPV1,AMPK and eNOS in the aorta.8. Activation of TRPV1 increased the expression of ABCA1 and decreased the expression of LRP1 in aortas from ApoE-/- mice on a high-fat diet and in wild-type VSMCs, but not in aortas from TRPV1-/- ApoE-/- mice or in TRPV1-/- VSMCs.9. Activation of TRPV1 reduced expression of adhesion molecules and inflammatory cytokines in aortas from ApoE-/- mice on a high-fat diet and also in wild-type VSMCs, but not in TRPV1-/- VSMCs.10. Administration of the A23187 significantly increased the expression of ABCA1 and decreased the expression of LRP1 in VSMCs, while this effect can be inhibited in TRPV1-/- VSMCs or in the presence of BAPTA.Conclusions:1. TRPV1 was expressed in VSMCs and aortas from mice. Capsaicin and RTX induced TRPV1-dependent changes of intracellular free calcium in VSMCs.2. Long-term activation of TRPV1 markedly reduced lipid storage, atherosclerotic lesions, blood levels of lipids and inflammatory factors in ApoE-/- mice on high-fat diet. Compared to ApoE-/- littermates, double knockout of TRPV1 and ApoE significantly increased atherosclerotic lesions and blood levels of lipids and inflammatory factors.3. PVAT likely impacts obesity-related vascular dysfunction and remodelling through reduction of TRPV1 expression, eNOS-mediated vasodilatation and the AMPK/mTOR pathway.4. Activation of TRPV1 affected cholesterol transporters and NFAT3 through increase of intracellular calcium concentration. It indicates that the beneficial effects of TRPV1 activation on prevention of atherosclerosis are due both to its effects on cholesterol transport and on the expressionof inflammatory cytokines.