TRPV1 Activation Impedes Foam Cell Formation By Inducing Autophagy In OxLDL-treated Vascular Smooth Muscle Cells

Background and Objectives:Atherosclerosis is pathologically characterized by the deposition of foam cells in the vessel wall. Increase in foam cells promote the development of the plaque and increase its instability, leading to the occurrence of cardiovascular events, which was reported to be the leading cause of cardiovascular and cerebrovascular disorders. It is well accepted that both vascular smooth muscle cells(VSMCs) and macrophages can be induced to be foam cells. In early stage of the atherosclerotic plaque, the type of foam cell is macrophage-derived. In contrast, up to 45% of the foam cells are VSMC-derived in late stage atherosclerotic lesions, whereas only 30% of foam cells were of macrophage markers. By now, there are amount of studies reporting mechanisms underlying macrophage foam cell formation. However, VSMC-derived foam cells have only been attracted attention in recent years. The specific regulatory mechanism of VSMC foam cell formation is far from being elucidated. Fully elucidating these mechanisms would be helpful for the prevention and treatment of atherosclerosis.Formation of foam cell is mainly due to the accumulation of cytoplasmic droplets of cholesterol esters and triglycerides. Previous studies considered that the metabolism of lipid droplets in the cytoplasm was mainly carried out by the series of fat hydrolase. Recent studies have confirmed that autophagy-mediated lysosomal pathway is another important degradation of lipid droplets. Especially in the environment of atherosclerosis, which suffered consistent lipid stimulation, autophagy-mediated decomposition of lipid droplets may be more important than traditional decomposition pathway. Under this context, the more direct evidence is that from macrophage foam cell. In macrophage foam cell, autophagy regulates cholesterol efflux via lysosomal acid lipase. Inhibition of autophagy can increase, whereas activation of autophagy can reduce intracellular accumulation of lipids. However, rare studies have reported the potential role of autophagy in the formation of VSMC foam cell.Transient receptor potential vanilloid subfamily 1(TRPV1) is activated by capsaicin. It is a nonselective cation channel and plays protective effect against cardiovascular and cerebrovascular diseases. Previous study has shown that activation of TRPV1 by capsaicin could reduce the lipid accumulation in cultured VSMC via increasing cholesterol efflux and reducing lipid uptake. Most recently, it was reported that activation of TRPV1 by capsaicin could induce autophagy in hepatocytes and mouse thymocytes. In addition, activation of TRPV1 was reported to promote the phosphorylation of AMP-activated protein kinase(AMPK), which could further induce autophagy. From these results we assumed that activation of TRPV1 by capsaicin could induce autophagy by activating AMPK, which further inhibited VSMC foam cell formation.To test the above hypothesis, we firstly investigated the role of autophagy in VSMC foam cell formation. Primary VSMCs were treated with oxidized low-density lipoprotein(ox LDL) to induce foam cell formation. Autophagy inhibitor(3-MA) and inducer(rapamycin) were used to explore the role of autophagy during VSMC foam cell formation. Next, we used wild type(WT) and TRPV1 knockout(TRPV1-/-)-VSMC treated with TRPV1 agonist capsaicin and antagonist 5’-iodo-resiniferatoxin(i RTX) to explore the effect of TRPV1 on autophagy-lysosome pathway. To investigate whether activation of TRPV1 by capsaicin inhibits VSMC foam cell formation through autophagy induction, we used autophagy-related gene 7(Atg7) small interfering RNA(si RNA) to knock down Atg7, a key autophagy-related gene. To further investigate the potential mechanisms of TRPV1 in inducing autophagy in VSMCs, we analyzed the activation status of AMPK. Furthermore, we evaluated the involvement of AMPK activation in TRPV1-induced autophagy by using the AMPK inhibitor compound C.Materials and Methods:Both in vivo and in vitro experiments were conducted. In vitro study applied VSMC derived from WT mice and TRPV1-/- mice. In vivo study, both TRPV1-/- mice and WT mice were fed on high fat diet(HFD) or high fat diet with capsaicin(HFD+Cap).1. VSMCs were isolated using an explant technique from the thoracic aorta of mice.2. Ox LDL was used to induce VSMC foam cell formation.3. The LC3II/LC3 I ratio and expression of beclin-1 and p62 were detected to evaluated the status of autophagy.4. Foam cell formation was detected by oil red O staining. Intracellular total cholesterol was detected by enzymatic assay.5. Primary WT and TRPV1-/--VSMC were treated with TRPV1 agonist capsaicin and TRPV1 antagonist. Expression of lysosomal-associated membrane protein 1(LAMP1) in VSMC and aorta were detected by western blot and immunofluorescence.6. Autophagy-related gene 7(Atg7) small interfering RNA(si RNA) was used to knock down Atg7.7. Compound C was used to inhibit AMPK and expression of Atg7 and LC3 were detected by western blot.Results:1. Ox LDL increased lipid accumulation dose-dependently in VSMCs, with an obvious effect of 80μg/ml. Ox LDL(80μg/ml) time-dependently decreased the level of beclin-1 level and the ratio of LC3II/LC3 I in primary VSMCs. 24h-treatment showed an obvious effect. Meanwhile, a significant increase of p62 level was observed 24 h post ox LDL treatment.2. Both oil red O staining and total cholesterol quantification showed that rapamycin inhibited the ox LDL-induced lipid accumulation in VSMCs; in contrast, addition of 3-MA increased the lipid accumulation in VSMCs.3. In ox LDL-stimulated VSMCs from WT mice, capsaicin significantly increased the expression of beclin-1, the ratio of LC3II/LC3 I and GFP-labeled autophagosomes; in contrast, the above results were not observed in VSMCs from TRPV1-/- mice.4. In ox LDL-stimulated VSMCs from WT mice, capsaicin significantly increased the number of lysosomes and the expression of LAMP-1, which was counteracted by i RTX(a TRPV1 inhibitor). In TRPV1-/--VSMCs, there was no detectable change in the expression of LAMP-1.5. In WT mice, capsaicin increased the LAMP-1 expression in the area of smooth muscle cells significantly. In contrast, in TRPV1-/- mice, capsaicin exerted no effect.6. In con siRNA-transfected VSMCs, capsaicin increased the expression of Atg7 and the ratio of LC3II/LC3 I significantly and decreased lipid accumulation. In the contrast, in Atg7 si RNA-transfected VSMCs, capsaicin failed to increase the ratio of LC3II/LC3 I and to inhibit the lipid accumulation.7. In WT-VSMCs, capsaicin increased the expression of p-AMPK(Thr-172) significantly. On the contrary, in TRPV1-/--VSMCs, capsaicin exerted no effect. Compound C significantly inhibited the TRPV1-activated AMPK and attenuated the TRPV1-increased Atg7 level and LC3II/LC3 I ratio.Conclusions:The present study provided evidence that autophagy was inhibited in ox LDL-induced VSMC foam cell formation; modulation of autophagy could regulate VSMC foam cell formation; activation of TRPV1 by capsaicin could inhibit VSMC foam cell formation by rescuing ox LDL-impaired autophagy through AMPK signaling pathway. Thus, our study revealed novel role of autophagy during foam cell formation of VSMC and highlighted that TRPV1 might be served as a promising therapeutic target for atherosclerosis.