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. 2013:2013:925171.
doi: 10.1155/2013/925171. Epub 2013 Jun 26.

Activation of TRPV1 prevents OxLDL-induced lipid accumulation and TNF-α-induced inflammation in macrophages: role of liver X receptor α

Jin-Feng Zhao  1 , Li-Chieh Ching, Yu Ru Kou, Shing-Jong Lin, Jeng Wei, Song-Kun Shyue, Tzong-Shyuan Lee
Affiliations

Activation of TRPV1 prevents OxLDL-induced lipid accumulation and TNF-α-induced inflammation in macrophages: role of liver X receptor α

Jin-Feng Zhao et al. Mediators Inflamm. 2013.

Abstract

The transient receptor potential vanilloid type 1 (TRPV1) is crucial in the pathogenesis of atherosclerosis; yet its role and underlying mechanism in the formation of macrophage foam cells remain unclear. Here, we show increased TRPV1 expression in the area of foamy macrophages in atherosclerotic aortas of apolipoprotein E-deficient mice. Exposure of mouse bone-marrow-derived macrophages to oxidized low-density lipoprotein (oxLDL) upregulated the expression of TRPV1. In addition, oxLDL activated TRPV1 and elicited calcium (Ca(2+)) influx, which were abrogated by the pharmacological TRPV1 antagonist capsazepine. Furthermore, oxLDL-induced lipid accumulation in macrophages was ameliorated by TRPV1 agonists but exacerbated by TRPV1 antagonist. Treatment with TRPV1 agonists did not affect the internalization of oxLDL but promoted cholesterol efflux by upregulating the efflux ATP-binding cassette (ABC) transporters ABCA1 and ABCG1. Moreover, the upregulation of ABC transporters was mainly through liver X receptor α-(LXRα-) dependent regulation of transcription. Moreover, the TNF-α-induced inflammatory response was alleviated by TRPV1 agonists but aggravated by the TRPV1 antagonist and LXR α siRNA in macrophages. Our data suggest that LXR α plays a pivotal role in TRPV1-activation-conferred protection against oxLDL-induced lipid accumulation and TNF-α-induced inflammation in macrophages.

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Figures

Figure 1
Figure 1
Expression of TRPV1 is increased in atherosclerotic lesions of ApoE−/− mice. (a) Western blot analysis of protein expression of TRPV1 in aortas from 5-month-old ApoE−/− and wild-type (WT) mice. α-Tubulin was a normalization control. Data are mean ± SD from 6 animals. *P < 0.05 versus WT mice. (b) Immunohistochemical staining for TRPV1, F4/80 (macrophage marker), and α-actin (smooth-muscle-cell marker) in atherosclerotic lesions of aortas from 5-month-old ApoE−/− mice. Specificity of immunostaining was confirmed with an IgG-negative control. Hematoxylin was used as counterstaining. Magnification = 100 x. (c) Western blot analysis of protein expression of TRPV1 induced by oxLDL (50 μg/mL) relative to that induced by vehicle (PBS) for 0–24 h. Data are mean ± SD from 5 independent experiments. *P < 0.05 versus vehicle-treated group. α-Tubulin was a normalization control.
Figure 2
Figure 2
Treatment with oxLDL upregulates and activates TRPV1 in macrophages. (a) Intracellular levels of Ca2+ ([Ca2+]i) in response to incubation with oxLDL (50 μg/mL) or native LDL (50 μg/mL). [Ca2+]i was quantified by measuring the intensity of Ca2+-sensitive Fluo-8 fluorescence. (b) [Ca2+]i level at 30 sec and 240 min after incubation with oxLDL in BMDMs pretreated or not with capsazepine (CPZ; 10 μM). Data are mean ± SD from 5 independent experiments. *P < 0.05 versus vehicle, # P < 0.05 versus 30 sec/oxLDL, and $ P < 0.05 versus 240 min/oxLDL. (c) Representative microscopy images of Ca2+-binding Fluo-8 fluorescence at 240 min after incubation with or without oxLDL in BMDMs pretreated or not with capsazepine. (d) [Ca2+]i level at 30 sec and 240 min after incubation with evodiamine (Evo; 0.5 μM) or capsaicin (Cap; 10 μM) in BMDMs pretreated or not with capsazepine. Data are mean ± SD from 5 independent experiments. *P < 0.05 versus LDL-treated group or vehicle, # P < 0.05 versus 30 sec/Evo- or Cap-treated group, and $ P < 0.05 versus 240 min/Evo- or Cap-treated group.
Figure 3
Figure 3
Activation of TRPV1 by agonists alleviates oxLDL-induced foam-cell formation.Cells were incubated with vehicle (DMSO), evodiamine (0.5 μM), capsaicin (10 μM), or capsazepine (10 μM) with or without oxLDL (50 μg/mL). (a) Representative microscopy images of cells with intracellular lipids stained by Oil-red O. Hematoxylin was used as counterstaining. Magnification  =  400 x. ((b) and (c)) Intracellular levels of cholesterol (b) and triglycerides (c) were extracted by use of hexane/isopropanol (3/2, v/v) and analyzed by colorimetric assay kits. Data are mean ± SD from 5 independent experiments. *P < 0.05 versus vehicle-treated cells, # P < 0.05 versus oxLDL-treated cells.
Figure 4
Figure 4
TRPV1 activation by agonists promotes apoAI- and HDL-dependent cholesterol efflux in macrophages. (a) For Dil-oxLDL binding assay, BMDMs were treated with vehicle, evodiamine (125, 250, 500, 500 nM), or capsaicin (2.5, 5, 10 μM) for 12 h and then incubated with 10 μg/mL Dil-oxLDL at 4°C for 4 h. Cellular lysates were analyzed by fluorimetry. ((b) and (c)) BMDMs were treated with indicated concentrations of evodiamine (125, 250, 500, 500 nM) or capsaicin (2.5, 5, 10 μM) for 12 h, followed by NBD-cholesterol (1 μg/mL) for another 6 h in the presence of (b) apoAI (10 μg/mL) or (c) HDL (50 μg/mL). The medium and cell lysates were collected for the measurement of fluorescence. Cholesterol efflux was defined as fluorescence in the medium relative to total amount of fluorescence. Data are mean ± SEM from 5 independent experiments. *P < 0.05 versus vehicle treatment.
Figure 5
Figure 5
Effect of TRPV1 activation on expression of SR-A, CD36, SR-BI, ABCA1, and ABCG1 in macrophages. BMDMs were incubated with vehicle, (a) evodiamine (125, 250, 500 nM), or (b) capsaicin (2.5, 5, 10 μM) for 24 h. Western blot analysis of protein levels of SR-A, CD36, ABCA1, ABCG1, SR-BI, and α-tubulin. Data are mean ± SD from 5 independent experiments. *P < 0.05 versus vehicle-treated cells.
Figure 6
Figure 6
Treatment with TRPV1 agonists increases the activation of LXRα in macrophages. (a) BMDMs were pretreated with capsazepine (10 μM) for 1 h then incubated with evodiamine (500 nM), capsaicin (10 μM), or T0901317 (10 μM) for 6 h. Western blot analysis of nuclear protein level of LXRα and Histone H1 as a normalization control. (b) Macrophages were treated with vehicle, evodiamine (500 nM), capsaicin (10 μM), or T0901317 for 6 h, then immunoprecipitated with anti-LXRα or rabbit IgG. PCR amplification involved specific primers for the ABCA1 gene promoter. The amplified DNA products were separated by electrophoresis with 2% agarose gel. (c) Macrophages were transfected with plasmid phABCA1-Luc or phABCA1-DR4 m-Luc for 24 h, treated with vehicle, evodiamine (500 nM), capsaicin (10 μM), or TO901317 (10 μM) for 24 h, then lysed for Luc activity assays with renilla activity as an internal control. Data are mean ± SD from 5 independent experiments. *P < 0.05 versus vehicle-treated cells, # P < 0.05 versus phABCA1-Luc-transfected cells with evodiamine or capsaicin treatment.
Figure 7
Figure 7
Knockdown of LXRα abolishes the protein expression of ABCA1 and ABCG1 and attenuates lipid accumulation by TRPV1 agonists. BMDMs were preincubated with control siRNA (50 nmol/L) or LXRα siRNA (50 nmol/L) for 24 h, followed by evodiamine or capsaicin treatment for additional 24 h. (a) Western blot analysis of protein expression of LXRα. (b) RT-PCR analysis of mRNA expression of ABCA1 and ABCG1. (c) ApoAI- and HDL-dependent cholesterol efflux was evaluated by use of NBD-cholesterol. Data are mean ± SD from 5 independent experiments. *P < 0.05 versus control siRNA-treated cells, # P < 0.05 versus control siRNA-treated cells with evodiamine or capsaicin treatment.
Figure 8
Figure 8
Knockdown of LXRα diminishes the protective effect of TRPV1 agonists against TNF-α-induced inflammation in macrophages. (a) BMDMs were pretreated with capsazepine (10 μM) for 1 h or (b) transfected with control siRNA (50 nmol/L) or LXRα siRNA (50 nmol/L) for 24 h, then incubated with vehicle (DMSO), evodiamine (500 nM), and capsaicin (10 μM) with or without TNF-α (10 ng/mL) for an additional 18 h. ELISA of levels of MCP-1, IL-6, and MIP-2 in the culture medium. Data are mean ± SD from 5 independent experiments. *P < 0.05 versus vehicle-treated cells, and # P < 0.05 versus TNF-α-treated cells, and & P < 0.05 versus TNF-α-treated group with evodiamine or capsaicin treatment.
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