Activation of TRPV1 reduces vascular lipid accumulation and attenuates atherosclerosis (original) (raw)

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Center for Hypertension and Metabolic Diseases, Daping Hospital, Third Military Medical University, Chongqing Institute of Hypertension

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Department of Hypertension and Endocrinology

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Center for Hypertension and Metabolic Diseases, Daping Hospital, Third Military Medical University, Chongqing Institute of Hypertension

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Chongqing 400042

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Department of Hypertension and Endocrinology

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Center for Hypertension and Metabolic Diseases, Daping Hospital, Third Military Medical University, Chongqing Institute of Hypertension

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Chongqing 400042

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People

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s Republic of China

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Department of Hypertension and Endocrinology

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Center for Hypertension and Metabolic Diseases, Daping Hospital, Third Military Medical University, Chongqing Institute of Hypertension

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Chongqing 400042

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People

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s Republic of China

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Department of Hypertension and Endocrinology

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Center for Hypertension and Metabolic Diseases, Daping Hospital, Third Military Medical University, Chongqing Institute of Hypertension

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Chongqing 400042

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People

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s Republic of China

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Department of Hypertension and Endocrinology

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Center for Hypertension and Metabolic Diseases, Daping Hospital, Third Military Medical University, Chongqing Institute of Hypertension

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Chongqing 400042

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People

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s Republic of China

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Department of Hypertension and Endocrinology

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Center for Hypertension and Metabolic Diseases, Daping Hospital, Third Military Medical University, Chongqing Institute of Hypertension

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Chongqing 400042

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People

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s Republic of China

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Department of Hypertension and Endocrinology

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Center for Hypertension and Metabolic Diseases, Daping Hospital, Third Military Medical University, Chongqing Institute of Hypertension

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Chongqing 400042

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These authors contributed equally to this work and are co-first authors.

Author Notes

Revision received:

25 August 2011

Accepted:

06 September 2011

Published:

09 September 2011

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Liqun Ma, Jian Zhong, Zhigang Zhao, Zhidan Luo, Shuangtao Ma, Jing Sun, Hongbo He, Tianqi Zhu, Daoyan Liu, Zhiming Zhu, Martin Tepel, Activation of TRPV1 reduces vascular lipid accumulation and attenuates atherosclerosis, Cardiovascular Research, Volume 92, Issue 3, 1 December 2011, Pages 504–513, https://doi.org/10.1093/cvr/cvr245
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Abstract

Aims

Activation of transient receptor potential vanilloid type-1 (TRPV1) channels may affect lipid storage and the cellular inflammatory response. Now, we tested the hypothesis that activation of TRPV1 channels attenuates atherosclerosis in apolipoprotein E knockout mice (ApoE−/−) but not ApoE−/−TRPV1−/− double knockout mice on a high-fat diet.

Methods and results

Both TRPV1 mRNA and protein expression were identified in vascular smooth muscle cells (VSMC) and in aorta from C57BL/6J mice using RT–PCR, immunoblotting, and immunohistochemistry. In vitro, activation of TRPV1 by the specific agonists capsaicin and resiniferatoxin dose-dependently increased cytosolic calcium and significantly reduced the accumulation of lipids in VSMC from C57BL/6J mice but not from TRPV1−/− mice. TRPV1 activation increased ATP-binding cassette transporter A1 (ABCA1) expression and reduced low-density lipoprotein-related protein 1 (LRP1) expression in VSMC by calcium-dependent and calcineurin- and protein kinase A-dependent mechanisms. These results showed increased cellular cholesterol efflux and reduced cholesterol uptake. In vivo, long-term activation of TRPV1 by capsaicin for 24 weeks increased ABCA1 and reduced LRP1 expression in aorta from ApoE−/− mice on a high-fat diet. Long-term activation of TRPV1 significantly reduced lipid storage and atherosclerotic lesions in the aortic sinus and in the thoracoabdominal aorta from ApoE−/− mice but not from ApoE−/−TRPV1−/− mice on a high-fat diet. These findings indicated that TRPV1 activation ameliorates high-fat diet-induced atherosclerosis.

Conclusion

Activation of TRPV1 may be a novel therapeutic tool to attenuate atherosclerosis caused by a high-fat diet.

1. Introduction

Atherosclerosis is considered to be an inflammatory process consisting largely of the accumulation of lipids within the artery wall.1,2 Vascular smooth muscle cells (VSMC) have been demonstrated to express a variety of cholesterol uptake receptors and reverse cholesterol transporters, including low-density lipoprotein (LDL) receptor, LDL receptor-related protein 1 (LRP1), and ATP-binding cassette transporter A1 (ABCA1).3,4 These studies suggest that lipid accumulation in VSMC contributes to atherosclerosis development. Recent studies showed that transient receptor potential vanilloid type-1 (TRPV1) channels are expressed in vessels.5,6 TRPV1 channels are activated by the specific agonist, capsaicin, the ‘hot’ component of chili peppers.7,8 Activation of TRPV1 regulates the expression of endothelial cell-derived calcitonin gene-related peptide, which causes protective effects on vascular endothelial cells.6 We recently showed that chronic TRPV1 activation by dietary capsaicin increases the phosphorylation of protein kinase A (PKA) and endothelial nitric oxide (NO) synthase (eNOSser1177) and thus the production of NO in endothelial cells.9 Furthermore, our previous work indicated that activation of TRPV1 by capsaicin also affects lipid metabolism and prevents obesity in male mice.10 From these results, the hypothesis arises that activation of TRPV1 channels may attenuate atherosclerosis. We investigated this hypothesis in apolipoprotein E knockout mice (ApoE−/−) and ApoE/TRPV1 double knockout mice (ApoE−/−TRPV1−/−) on a high-fat diet. In vitro, activation of TRPV1 significantly reduced the accumulation of lipids in VSMC due to an increased cholesterol efflux and reduced cholesterol uptake. In vivo, long-term activation of TRPV1 significantly reduced lipid storage and atherosclerotic lesions in the aortic sinus and in the thoracoabdominal aorta from ApoE−/− but not from ApoE−/−TRPV1−/− mice on a high-fat diet. These findings indicated that TRPV1 activation ameliorates a high-fat diet-induced atherosclerosis.

2. Methods

Methods for cellular total cholesterol analysis, PCR, transient siRNA transfection, biochemical analyses, immunohistochemistry, evaluation of atherosclerotic lesions, and histological analysis are available in the Supplementary material online.

2.1 Genetic mouse models

The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and was approved by the Experimental Animal Ethics Committee of Daping Hospital. ApoE-deficient mice (ApoE−/−) and TRPV1-deficient mice (TRPV1−/−) were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). The ApoE−/−TRPV1−/− mice were generated by breeding TRPV1−/− mice (on C57 BL/6 background) with ApoE−/− mice. The resulting heterozygous progeny were interbred with each other to produce homozygote ApoE−/−TRPV1−/− mice. PCR was used to identify the ApoE genotype and TRPV1 genotype. The expression level of TRPV1 protein was determined by western blot analysis.

To identify the ApoE genotype, the following set of primers was used: F: 5′-GCCTAGCCGAGGGAGAGCCG-3′; R(WT): 5′-TGTGACTTGGGAGCTCTGCAGC-3′ and R(knock out): 5′-GCCGCCCCGACTGCATCT-3′. For genotyping TRPV1, following three primers were used: F: 5′-CACGAGACTAGTGAGACGTG-3′; F(WT): 5′-CCTGCTCAACATGCTCATTG-3′; R(knock out): 5′-TCCTCATGCACTTCAGGA AA-3′.

The male ApoE−/− and ApoE−/− TRPV1−/− mice were randomized into four groups: one group received standard laboratory chow, one group received a high-fat diet, one group received standard laboratory chow plus 0.01% capsaicin (Sigma-Aldrich), and one group received a high-fat diet plus 0.01% capsaicin for 24 weeks. The high-fat diet was supplied with crude protein (18.8%), crude fat (16.2%), crude ash (5.2%), crude fibre (3.98%), nitrogen-free extract (45.2%), calcium (1.24%), phosphorus (0.83%), lysine (1.38%), and methionine/cystine (0.78%) (Shanghai Slac Laboratory Animal Co., Ltd, Shanghai, China). Food intake of all mice was measured. Mice were anaesthetized adequately by inhalation of isoflurane (5% for induction; 1% for maintenance) for blood collection and sacrificed by CO2 inhalation for isolation of the aorta. The adequacy of anaesthesia was monitored by testing tactile stimulus response and forelimb or hindlimb pedal withdrawal reflex, and continual observation of respiratory pattern, mucous membrane colour, and responsiveness to manipulations throughout all the procedure.

2.2 Cell culture

VSMC were obtained from thoracic aorta of mice and cultured by the tissue explant method as described.11 VSMC were maintained in Dulbecco's modified Eagle's medium supplemented with 10% foetal calf serum (HyClone) containing 100 µg/mL penicillin and 100 µg/mL streptomycin (GIBCO, USA). Cultured VSMC were plated and grew at 37°C in a humidified atmosphere of 95% air/5% CO2. To verify that cultured cells were VSMC, immunocytochemical localization of smooth muscle-specific α-actin was performed using anti-smooth muscle α-actin monoclonal antibody (Santa Cruz Biotechnology, USA). VSMC between Passages 2 and 6 were used. Quiescent VSMC were obtained by incubation with serum-free medium for 12 h before all the in vitro experimental procedures were performed.

2.3 Western blotting

Aortae were homogenized and cells were lysed in high-salt extraction buffer (0.5 mol/L Tris, 1% NP-40, 1% Triton X-100, 1 g/L sodium dodecyl sulfate (SDS), 1.5 mol/L NaCl, 0.2 mol/L EDTA, and 0.01 mol/L EGTA) plus 0.2 mmol/L protease inhibitor, placed at −20°C for 20 min, and centrifuged at 12 000 g at 4°C for 20 min to remove insoluble material. Protein concentrations were determined using a DC protein assay kit. Twenty micrograms of lysates were separated by SDS–polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes, and probed with antibodies for TRPV1 (Alomone Labs, Jerusalem, Israel), LRP1, ABCA1, GAPDH, and β-actin (Santa Cruz Biotechnology, USA), respectively. After incubation with secondary antibodies for 2 h, the proteins were detected by enhanced chemiluminescence and quantified using a Gel Doc 2000 Imager (Bio-Rad, CA, USA). Each sample was processed at least three times.

2.4 Measurement of intracellular calcium

Cytosolic calcium concentration was measured in cultured VSMC using the fluorescent dye technique as described by our group.12 VSMC grown on glass cover slips were loaded with 2 μmol/L fura 2-AM and 0.025% Pluronic F-127 for 60 min at room temperature in physiological saline solution (PSS, containing in mmol/L, NaCl 135, KCl 5, CaCl2 1.8, MgCl2 1, d-glucose 11, and HEPES 10, pH 7.4) and then washed three times with PSS to remove extracellular fura 2-AM. Individual cells were defined as the region of interest and average fluorescence was measured using the PTI Fluorescence Master Systems (Photon Technology International, NJ, USA).

Fluorescence was measured at 510 nm with excitation wavelengths of 340 and 380 nm. Images and F340 nm/F380 nm ratio were acquired and analysed using the Felix32 Software.

2.5 Statistics

All values reported are mean ± SEM. Comparisons between groups were analysed using Student's _t_-test or one-way ANOVA with Bonferroni's multiple comparison post hoc test as appropriate (Graph Pad Prism; LaJolla CA, USA). A two-sided _P_-value of <0.05 was considered to indicate statistical significance.

3. Results

3.1 Expression and function of TRPV1 in VSMC and aorta

TRPV1 mRNA (Figure 1 A) and protein expression (Figure 1B) could be detected in aortic tissue and cultured VSMC from wild-type mice but not from TRPV1−/− mice. A positive control was obtained from the brain tissue of wild-type mice. However, neither TRPV1 mRNA nor TRPV1 protein was detected in a mouse macrophage cell line, RAW264.7 (Figure 1A and B). Using immunohistochemistry, we confirmed that the presence of TRPV1 in the muscle layer of the aorta removed the endothelium and adventitia from C57BL/6J mice (Figure 1C). We also proved the presence of TRPV1 in VSMC (Figure 1D). The specific TRPV1 agonist, capsaicin (1 µmol/L), significantly increased the TRPV1 protein expression in VSMC (Figure 1E). The specific TRPV1 agonists, capsaicin (Figure 1F and G) and resiniferatoxin (RTX; see Supplementary material online, Figure S1A and B), dose-dependently increased cytosolic calcium concentrations in VSMC from C57BL/6J mice. Furthermore, capsaicin-induced calcium increase was significantly blocked in VSMC from TRPV1−/− mice or in the presence of the antagonist capsazepine (1 µmol/L) and 5′-indo-RTX (1 µmol/L) (Figure 1H and I). These results indicated the presence and functional integrity of TRPV1 channels in VSMC, extending the observations in recent literature.13

Figure 1

Expression and function of TRPV1 on calcium influx in VSMC. (A) RT–PCR showing the expression of TRPV1 mRNA in the brain, aorta, and primarily cultured VSMC from C57BL/6J wild-type mice but not in those from TRPV1−/− mice and in a mouse macrophage cell line, RAW264.7. Predicted product size was 435 bp. (B) Immunoblotting showing TRPV1 protein expression in the brain, aorta, and VSMC from C57BL/6J wild-type mice, but not in those from TRPV1−/− mice and in RAW264.7. (C and D) Immunohistochemistry showing TRPV1 in the aorta (C) and VSMC (D) from C57BL/6J mice. Scale bar = 50 µm. (E) Effect of TRPV1 agonist capsaicin (Caps) on TRPV1 protein expression, VSMC were stimulated with 1 µmol/L Caps for 24 h and TRPV1 protein were analysed. Each n = 6; *P < 0.05. (F and G) Dose-dependent capsaicin induced calcium influx into VSMC from C57BL/6J mice. Representative fluorescence tracings (F) and summary of the data (G) are shown. Each n = 4–6; *P < 0.05; **P < 0.01. (H and I) Caps (1 µmol/L) induced calcium influx into VSMC from C57BL/6J mice (Control), but not in VSMC from TRPV1−/− mice or after pre-treatment with capsazepine (1 µmol/L, Capz) or 5′-iodo-RTX (1 µmol/L) for 10 min. Representative fluorescence tracings (H) and summary of the data (I) are shown. Each n = 4–6; *P < 0.05; **P < 0.01.

3.2 Activation of TRPV1 by capsaicin reduces accumulation of lipids in VSMC

There is indirect evidence linking TRPV1 with lipid storage and lipid metabolism.10,14 Therefore, we investigated the effects of TRPV1 activation on lipid accumulation in VSMC. Oil red O staining of intracellular lipid droplets showed that the administration of 50 µg/mL oxidized LDL (oxLDL) for 72 h significantly increased lipid accumulation in cultured VSMC from C57BL/6J mice by about 154 ± 13% from n = 6 separate experiments (Figure 2A). As indicated in Figure 2B, the administration of the TRPV1 agonist capsaicin (1 µmol/L) significantly reduced intracellular lipid droplets in oxLDL-stimulated VSMC from C57BL/6J mice but not TRPV1−/− mice. The inhibition of capsaicin on intracellular lipid droplets was reversed by TRPV1 antagonist capsazepine in a concentration-dependent manner (see Supplementary material online, Figure S2). The total cholesterol level of VSMC from C57BL/6J mice was significantly reduced by capsaicin (control, 0.31 ± 0.05 µg/106 cells; capsaicin, 0.17 ± 0.02 µg/106 cells; each n = 5–6, P < 0.05 by ANOVA; Figure 2C), which was more pronounced when VSMC were cultured in the presence of 50 µg/mL oxLDL (Figure 2D).

Figure 2

Activation of TRPV1 reduces accumulation of lipids in VSMC. (A) Representative pictures of oil red O staining of intracellular lipid droplets in cultured VSMC from C57BL/6J mice without (Control) and with administration of 50 µg/mL oxLDL for 72 h. Scale bar = 10 µm. (B) Oil red O staining of intracellular lipid droplets in oxLDL-stimulated VSMC from C57BL/6J mice or from TRPV1−/−mice in the absence (Control) and presence of Caps or Capz. VSMC were cultured in the presence of 50 µg/mL oxLDL for 3 days when images of intracellular lipid droplets were obtained. Representative pictures of three independent experiments are shown. Scale bar = 50 µm. (C and D) Bar graphs showing total cholesterol levels in VSMC from C57BL/6J and TRPV1−/− mice under routine culturing conditions (C) and after culturing in the presence of 50 µg/mL oxLDL for 3 days (D). Total intracellular cholesterol was measured in VSMC from C57BL/6J and TRPV1−/− mice cultured in the absence (Control) or presence of Caps or Capz. Data are mean ± SEM from five to six independent experiments. *P < 0.05 compared with Control.

3.3 Activation of TRPV1 affects cholesterol transporters in VSMC

After we identified the regulatory effect of TRPV1 on total cellular cholesterol level, we investigated whether TRPV1 activation may directly affect cholesterol transporters that are associated with intracellular cholesterol accumulation. First, we showed that both ABCA1 and LRP1 are colocalized with TRPV1 on VSMC (Figure 3A and B). Furthermore, oil red O staining of intracellular lipid droplets showed that RNA interference knockdown of ABCA1 reversed capsaicin-induced reduction in lipid accumulation in cultured VSMC from C57BL/6J mice (see Supplementary material online, Figure S3A), and RNA interference knockdown of LRP1 had the synergistic effect with capsaicin in attenuated lipid accumulation in VSMC (see Supplementary material online, Figure S3B). The activation of TRPV1 by the specific agonist capsaicin significantly increased the expression of ABCA1 from 1.00 ± 0.06 to 2.18 ± 0.20 (n = 3, P < 0.05; Figure 3C), whereas it significantly reduced the expression of LRP1 from 1.00 ± 0.20 to 0.53 ± 0.08 (n = 6, P < 0.05; Figure 3D). However, these effects of capsaicin disappeared in VSMC from TRPV1−/− mice (Figure 3C and D).

Figure 3

Activation of TRPV1 affects cholesterol transporters ABCA1 and LRP1 in VSMC. (A and B) Immunofluorescence showing the colocalization of ABCA1 (A) and LRP1 (B) with TRPV1 on the surface of VSMC from C57BL/6J mice. ABCA1, LRP1, and TRPV1 were identified using specific primary antibodies and fluorescence-labelled secondary antibodies. 4,6-Diamidino-2-phenylindol (DAPI) was used for nuclear counterstaining (blue fluorescence). Negative controls (Control) were performed with phosphate-buffered saline (PBS) instead of primary antibodies. Scale bar = 50 µm. (C and D) Representative immunoblottings and summary data showing the effect of capsaicin on the expression of ABCA1 (C) and LRP1 (D) in VSMC from C57BL/6J wild-type mice and TRPV1−/− mice. VSMC were cultured in the absence (Control) or presence of 1 µmol/L Caps for 24 h. Data are mean ± SEM, each n = 3–6; *P < 0.05.

3.4 TRPV1 activation regulates ABCA1/LRP1 expression by calcium-evoked calcineurin- and PKA-dependent mechanisms in VSMC

For further understanding of the possible molecular mechanisms of ABCA1 and LRP1 expression regulated by TRPV1 activation, we detected the role of PKA and calcineurin in the expression of ABCA1 and LRP1 stimulated by capsaicin. Cyclosporin A (CsA), a specific inhibitor of calcineurin, had an inhibitory tendency on capsaicin-activated ABCA1 expression (Figure 4A), and blocked the effects of TRPV1 activation on LRP1 expression (0.57 ± 0.06 vs.1.02 ± 0.08; n = 3, P < 0.05; Figure 4B). As shown in Supplementary material online, Figure S4, capsaicin increased the expression of phospho-PKA. KT5720, a specific inhibitor of PKA, blocked the effects of TRPV1 activation on ABCA1 (1.35 ± 0.06 vs.0.83 ± 0.07; n = 3, P < 0.01; Figure 4C) and LRP1 expression (0.34 ± 0.05 vs.0.71 ± 0.10; n = 3, P < 0.05; Figure 4C). As expected, trapping of intracellular calcium by BAPTA blocked the effects of capsaicin on ABCA1 and LRP1 expression (Figure 4D). However, peroxisome proliferator activated receptor gamma (PPARγ) antagonist, GW9662, did not affect ABCA1 expression after activation of TRPV1 (see Supplementary material online, Figure S5). These results in vitro indicated that TRPV1 activation increased ABCA1 expression and reduced LRP1 expression, leading to increased cholesterol efflux and reduced cholesterol uptake of VSMC, through calcium-dependent calcineurin signal and PKA phosphorylation mechanisms.

Figure 4

TRPV1 activation regulates ABCA1/LRP1 expression by calcium evoked calcineurin- and PKA-dependent mechanisms. (A and B) Representative immunoblottings and summary data showing the effect of the calcineurin inhibitor CsA on the expression of ABCA1 (A) and LRP1 (B) in VSMC from C57BL/6J wild-type mice. VSMC were treated without (Control) or with 1 µmol/L Caps for 24 h in the presence of 1 µmol/L CsA (Caps + CsA). Data are mean ± SEM, each n = 3–5; *P < 0.05; **P < 0.01. (C) Representative immunoblottings and summary data showing the effect of PKA inhibitor KT5720 (10 µmol/L) on the expression of ABCA1 and LRP1 in VSMC from C57BL/6J wild-type mice. VSMC were cultured in the absence (Control) or presence of 1 µmol/L Caps for 24 h without or with KT5720. Data are mean ± SEM, each n = 3–6; *P < 0.05; **P < 0.01. (D) Representative immunoblottings and summary data showing the effect of BAPTA (10 µmol/L) on the expression of ABCA1 and LRP1 in VSMC from C57BL/6J mice. VSMC were cultured in the presence of BAPTA (10 µmol/L) for 2 h. Data are mean ± SEM, each n = 3.

3.5 In vivo activation of TRPV1 increases ABCA1 expression and reduces LRP1 expression in aorta from ApoE−/− mice

To confirm these effects in vivo, we evaluated the effects of TRPV1 activation on cholesterol transporters in ApoE−/− mice. Table 1 showed the characteristics of ApoE−/− mice that were randomly allocated to a normal diet, a high-fat diet, normal diet plus capsaicin, or a high-fat diet plus capsaicin, respectively. Compared with mice on normal diet, administration of a high-fat diet significantly increased plasma triglycerides by 300% and total cholesterol by 72% in ApoE−/− mice (P < 0.01; Table 1). Compared with mice on a high-fat diet, the mice on a high-fat diet plus capsaicin showed significantly lower plasma triglycerides (2.39 ± 0.50 vs. 4.17 ± 0.74 mmol/L, P < 0.05 Table 1) and significantly lower total cholesterol (13.75 ± 2.17 vs. 18.62 ± 1.54 mmol/L, P < 0.05; Table 1). In ApoE−/− mice on a high-fat diet, capsaicin did not affect fasting plasma glucose or insulin (Table 1). No difference in plasma lipids was shown in ApoE−/−TRPV1−/− mice fed with different diets (see Supplementary material online, Figure S6). Using immunofluorescence, we confirmed the expression of TRPV1, ABCA1, and LRP1 in the aorta from ApoE−/− mice (Figure 5A and B). Compared with ApoE−/− mice on a high-fat diet, the ApoE−/− mice on a high-fat diet plus capsaicin showed significantly higher expression of ABCA1 in the aorta (5.60 ± 1.62 vs. 1.32 ± 1.21, n = 3, P < 0.05; Figure 5C) and reduced expression of LRP1 in the aorta (0.55 ± 0.04 vs. 0.81 ± 0.04; n = 3, P < 0.05; Figure 5D), and similar changes of ABCA1 and LRP1 in the aorta between ApoE−/− mice on normal diet and ApoE−/− mice on normal diet plus capsaicin were also observed (Figure 5C and D). On the other hand, long-term administration of capsaicin did not affect the expressions of other cholesterol transporters and receptors, such as scavenger receptor type A (SR-A), ATP-binding cassette subfamily G member 1 (ABCG1), lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), and caveolin-1 (Cav-1) in ApoE−/− mice (see Supplementary material online, Figures S7 and S8).

Table 1

Biochemical characteristics of ApoE−/− mice after 24 weeks of treatment

ND, normal diet; NCaps, normal diet plus capsaicin; HD, high-fat diet; HCaps, high-fat diet plus capsaicin; HDL-C, high-density lipoprotein-cholesterol; LDL-C, low-density lipoprotein-cholesterol. Values are expressed as mean ± SEM for 15–20 animals.

**P < 0.01 vs. ND group.

#P < 0.05 vs. HD group.

&&P < 0.05 vs. NCaps group.

Table 1

Biochemical characteristics of ApoE−/− mice after 24 weeks of treatment

ND, normal diet; NCaps, normal diet plus capsaicin; HD, high-fat diet; HCaps, high-fat diet plus capsaicin; HDL-C, high-density lipoprotein-cholesterol; LDL-C, low-density lipoprotein-cholesterol. Values are expressed as mean ± SEM for 15–20 animals.

**P < 0.01 vs. ND group.

#P < 0.05 vs. HD group.

&&P < 0.05 vs. NCaps group.

Figure 5

In vivo activation of TRPV1 affects cholesterol transporters in the aorta. ApoE−/− mice were randomly allocated to four groups, which were fed for 24 weeks with normal diet (ND), normal diet plus capsaicin (NCaps), high-fat diet (HD), or high-fat diet plus capsaicin (HCaps), respectively. (A and B) Immunofluorescence showing the colocalization of ABCA1 (A) and LRP1 (B) with TRPV1 on the surface of the aorta from C57BL/6J mice. ABCA1, LRP1, and TRPV1 were identified using specific primary antibodies and fluorescence-labelled secondary antibodies. Negative controls (Control) were performed with PBS instead of primary antibodies. Scale bar = 200 µm. (C and D) Representative immunoblottings and summary data showing the in vivo effect of capsaicin on the expression of ABCA1 (C) and LRP1 (D) in the aorta from ApoE−/− mice. Data are mean ± SEM of aortas from ApoE−/− mice randomly allocated to ND, NCaps, HD, or HCaps for 24 weeks, respectively. Each n = 3; *P < 0.05 by ANOVA.

3.6 TRPV1 reduces lipid storage and atherosclerotic lesions in ApoE−/− but not ApoE−/−TRPV1−/− mice on a high-fat diet

To prove that TRPV1 directly affects atherosclerosis, we compared the effects of chronic activation of TRPV1 on atherosclerotic lesions from ApoE−/− and ApoE−/−TRPV1−/− mice. PCR was used to identify the ApoE genotype (Figure 6A) and TRPV1 genotype (Figure 6B). Food intake showed differences between mice fed with and without capsaicin only during the first 4 days, then mice fed with capsaicin recovered to normal eating (Figure 6C and D). As shown in Supplementary material online, Figure S9A and Figure 6E, lipid storage in atherosclerotic lesions of the descending thoracoabdominal aorta was significantly lower in ApoE−/− mice on a high-fat diet plus capsaicin compared with ApoE−/− mice on a high-fat diet (0.52 ± 0.27 vs. 1.00 ± 0.29; n = 6, P < 0.05). Furthermore, ApoE−/−TRPV1−/− mice on a high-fat diet plus capsaicin had similar atherosclerotic lesions compared with ApoE−/−TRPV1−/− mice on a high-fat diet (0.99 ± 0.06 vs. 1.00 ± 0.13; n = 6, P = NS). Similar findings were obtained in histomorphological analyses of atherosclerotic lesions in the aortic sinus. The lesions in the aortic sinus was significantly decreased in ApoE−/− mice on a high-fat diet plus capsaicin compared with ApoE−/− mice on a high-fat diet (0.52 ± 0.09 vs. 1.00 ± 0.19; n = 8–10, P < 0.05; see Supplementary material online, Figure S9B and Figure 6F). There were no significant differences in lesions in ApoE−/−TRPV1−/− mice on a high-fat diet plus capsaicin compared with ApoE−/−TRPV1−/− mice on a high-fat diet (see Supplementary material online, Figure S9B and Figure 6F). Moreover, immunohistochemical analysis and sirius red staining of atherosclerotic lesions in the aortic sinus showed that no significant differences were observed in the composition of atherosclerotic lesions in all ApoE−/− mice groups (see Supplementary material online, Figure S10). These results confirmed our experimental evidence that long-term activation of TRPV1 ameliorates high-fat-induced atherosclerosis.

Figure 6

In vivo activation of TRPV1 reduces atherosclerotic lesions in the aorta in ApoE−/− mice but not in ApoE−/−TRPV1−/− mice on a high-fat diet. Genotyping of F2 mice to verify the APOE−/−TRPV1−/− mice by PCR. The predicted products are 245 bp (A) and 600 bp (B). (C and D) Bar graphs showing daily food intake during the first 10 days after the start of treatment in ApoE−/− mice (C) and ApoE−/−TRPV1−/− mice (D). Mice were randomly allocated to groups, which were fed for 24 weeks with normal diet (ND), normal diet plus capsaicin (NCaps), high-fat diet (HD), or high-fat diet plus capsaicin (HCaps), respectively. (E) Summary data of computer-assisted quantitative image analysis of lipid deposition in the descending thoracoabdominal aorta of ApoE−/− mice and ApoE−/−TRPV1−/− mice. Each n = 6; **P < 0.01. (F) Bar graphs showing atherosclerotic lesions in aortic sinus of consecutive sections of the haematoxylin–eosin-stained aortic root quantified by computer-assisted image analysis. Data are mean ± SEM. Each n = 5–10; **P < 0.01.

4. Discussion

Accumulation of vascular lipids is one major event in the pathogenesis of atherosclerosis. ABCA1 is a protein that mediates the cellular efflux of phospholipids and cholesterol to lipid-poor apolipoprotein A1,15 and LRP1 is an α-2-macroglobulin receptor on the cell surface that enhances the cholesterol influx.16 Here, we give experimental evidence that activation of TRPV1 reduces accumulation of lipids in cultured cells by increased cholesterol efflux due to increased ABCA1 expression and reduced lipid uptake due to reduced LRP1 expression. In vivo too, the administration of the TRPV1 agonist capsaicin increased ABCA1 expression, reduced LRP1 expression, and finally reduced lipid storage and atherosclerotic lesions in ApoE−/− mice but not in the aorta from ApoE−/−TRPV1−/− mice. The results of our present study reveal a key contribution of TRPV1 for the prevention of atherosclerosis in mice on a high-fat diet.

Although macrophage-foam cell formation in atherosclerotic lesions has been extensively studied, little is known on how VSMC become foam cells by regulating cholesterol transporter. VSMC have a variety of cholesterol uptake receptors and reverse cholesterol transporters, including the LDL receptor, LRP1, and the ABCA1.2,4,17 LRP1 is highly expressed in the vascular wall and LRP1-mediated, matrix-retained LDL internalization could be crucial for the formation of VSMC-derived foam cell.16 Recently, Judith et al. showed that Ang II upregulates LRP1 receptor expression and LRP1-mediated aggregated LDL uptake in vascular cells.3 With continued lipid loading in a proatherogenic milieu, VSMC go on to downregulate the levels of ABCA1, enhancing foam cell formation.4,15 These studies suggest that lipid accumulation in VSMC may contribute to atherosclerosis development. However, the mechanism of lipid accumulation and its uptake by VSMC was poorly investigated.

It has been well known that TRPV1 channels were expressed in sensory nerves mediating the action of heat, acid, or pain.8 Activation of TRPV1 by capsaicin mediates a painful, burning sensation in the human gut via TRPV1. Acute exposure to capsaicin or chili aggravates abdominal pain in patients with dyspepsia and irritable bowel syndrome. However, chronic ingestion of chili was found to improve functional dyspepsia and gastro-oesophageal reflux disease symptoms in small randomized, controlled studies.18 According to literatures, some effects of capsaicin on carbohydrate metabolism have been reported. Dietary capsaicin may reduce fasting glucose, insulin levels, and affect glucose tolerance in obese mice.19 On the other hand, capsaicin may increase both glucose absorption from the gastrointestinal tract and the release of glucagon.20 However, in the present study, glucose and insulin levels were not significantly different between ApoE−/− mice fed with and without capsaicin. TRPV1 channels have also been demonstrated in vascular system, including endothelial cells and VSMC.7,21 Currently, it is unknown whether TRPV1 channels are uniformly distributed in all cell types from different blood vessels, including the aorta, coronary arteries, or mesenteric arteries. Chronic activation of TRPV1 improves endothelium-dependent vasodilatation through increasing PKA and eNOS phosphorylation.9 Tissue-specific activation of TRPV1 channels may mediate endothelium-dependent vasodilatation or smooth-muscle-associated vasoconstriction.22,23 In the present study, we not only confirmed the expression of TRPV1 mRNA and protein in VSMC and aorta, but also confirmed the expected molecular mass of TRPV1 of 95 kDa and showed that the antibodies were able to identify TRPV1 by immunoblots.

The capsaicin-induced calcium influx through TRPV1 channels has already been reported in HEK293 cells transfected with encoding cDNA of TRPV1.8 Moreover, capsaicin, as a known specific agonist of TRPV1 channels, induced a dose-dependent calcium influx into VSMC.24 As reported, TRPV1 activity is involved in calcineurin pathway.25 A similar mechanism of calcineurin–nuclear factor-activated T cells (NFAT)-dependent activation of TRPC3 and TRPC6 gene expression has also recently reported in myocardial cells.26,27 Similar to those reports, we showed that specific activation of TRPV1 by capsaicin could not only cause transmembrane calcium influx and increase the expression of TRPV1, but also reduce the accumulation of lipids in VSMC. Which mechanisms mediate the reduced intracellular lipid accumulation after TRPV1 activation? Previous reports showed that elevated cytosolic calcium markedly suppressed intracellular lipid accumulation and cholesterol and triglyceride levels in adipocytes.28,29 Similar to that, we showed that capsaicin-induced elevation of cytosolic calcium enhanced cholesterol efflux and reduced cholesterol influx through increasing ABCA1 expression and decreasing LRP1 expression in cultured VSMC, as a result, reduced intracellular lipid droplets and cholesterol levels in VSMC, as well as in the aorta from ApoE−/− mice on a high-fat diet. However, the regulation effects of capsaicin were not observed on other cholesterol transporters and receptors, such as SR-A, ABCG1, LOX-1, and Cav-1 in the aorta, although they were also considered playing roles in intracellular lipid homeostasis in the procedure of atherogenesis.30–33 In recent literature, cholesterol-lowering intervention by simvastatin downregulated the overexpression of vascular LRP1 induced by hypercholesterolaemia and that simvastatin did not influence LRP1 expression beyond its cholesterol-lowering effects in male New Zealand rabbits.34 According to our present data, in vivo in mice and in cultured cells, the observed effects of capsaicin are primarily caused by its activation of TRPV1 channels, rather than indirectly by lowering hypercholesterolaemia, although we observed lower plasma triglycerides and total cholesterol after administration of capsaicin in vivo. It cannot be excluded that several TRPV1-associated mechanisms collaborate in vivo. In vivo, both the TRPV1-associated reduction in plasma cholesterol including the reduction in remnant lipoprotein particles and the TRPV1-associated increase in ABCA1 expression and reduction in LRP1 expression in VSMC will result in prevention of atherosclerosis.

The underlying mechanisms of calcium-dependent regulation of cholesterol transporter are supported by previous studies. PPARγ enhances cholesterol efflux by inducing the transcription of liver-X-receptor α and thus inducing ABCA1 expression.35 Inhibiting calcineurin by special immunosuppressant sirolimus with CsA was reported to downregulate ABCA1 protein expressions.36 Kiss et al.37 and Zhu and Hui38 showed that ABCA1 and LRP1 were regulated by PKA, which is calcium-dependent. Taken together, we supposed that increased cytosolic calcium will modulate PPARγ-, calcineurin-, and PKA-dependent pathways, thus will finally change cholesterol transporters expression. However, our experimental data using PPARγ antagonist showed that the increased ABCA1 expression after activation of TRPV1 may not be explained by PPARγ mechanism. Moreover, our research elucidated that TRPV1 activation increased ABCA1 expression and reduced LRP1 expression through calcineurin- and PKA-dependent mechanisms, which were calcium-evoked, thus leading to increased cholesterol efflux and reduced cholesterol uptake into VSMC. The capsaicin content was found to range from 2.19 to 19.73 mg/g of dry weight of capsicum fruits.39 Moreover, one human study showed that regular consumption of chili (30 g/day; 55% cayenne chili) for 4 weeks may attenuate postprandial hyperinsulinaemia.40 Based on these values, one may estimate that average 18 g dried chili (about 180 mg capsaicin) per day would be beneficial to humans.

In summary, our present study for the first time gives experimental evidence that continuous activation of TRPV1 seems to be a promising novel mechanism to attenuate atherosclerosis evoked by a high-fat diet.

Supplementary material

Supplementary material is available at Cardiovascular Research online.

Funding

This work was supported by grants from natural science foundation of China (no. 81130006, 30900619, and 30890042), clinical medical science grants of Chinese Medical Association (no. 07060700078), and 973 Program of China (no. 2011CB503902 and 2012CB517805).

Conflict of interest: none declared.

Acknowledgments

We would like to thank Lijuan Wang for excellent technical assistance.

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Author notes

†

These authors contributed equally to this work and are co-first authors.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2011. For permissions please email: journals.permissions@oup.com.

Topic:

• aorta
• atherosclerosis
• calcium
• capsaicin
• cholesterol
• diet
• muscle, smooth, vascular
• apolipoprotein e
• lipids
• mice
• abca1 gene
• trpv1 receptor

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