Histochem Cell Biol DOI 10.1007/s00418-016-1408-9
ORIGINAL PAPER
Impaired SIRT1 promotes the migration of vascular smooth muscle cell‑derived foam cells Ming‑Jie Zhang1 · Yi Zhou1 · Lei Chen1 · Xu Wang1 · Yan Pi1 · Chun‑Yan Long1 · Meng‑Jiao Sun1 · Xue Chen1 · Chang‑Yue Gao1 · Jing‑Cheng Li1 · Li‑Li Zhang1
Accepted: 10 January 2016 © Springer-Verlag Berlin Heidelberg 2016
Abstract The formation of fat-laden foam cells, contributing to the fatty streaks of the plaques of atheroma, is the critical early process in atherosclerosis. The previous study demonstrated that vascular smooth muscle cells (VSMCs) contain a much larger burden of the excess cholesterol in comparison with monocyte-derived macrophages in human coronary atherosclerosis, as the main origin of foam cells. It is noteworthy that VSMC-derived foam cells are deposited in subintima but not media, where VSMCs normally deposit in. Therefore, migration from media to intima is an indispensable step for a VSMC to accrue neutral lipids and form foam cell. Whether this migration occurs paralleled with or prior to the formation of foam cell is still unclear. Herein, the present study was designed to test the VSMC migratory capability in the process of foam cell formation induced by oxidized low-density lipoprotein (oxLDL). In conclusion, we provide evidence that oxLDL induces the VSMC-derived foam cells formation with increased migration ability and MMP-9 expression, which were partly attributed to the impaired SIRT1 and enhanced nuclear factor-kappa B (NF-κB) activity. As activation of transient receptor potential vanilloid type 1 (TRPV1)
Electronic supplementary material The online version of this article (doi:10.1007/s00418-016-1408-9) contains supplementary material, which is available to authorized users. * Jing‑Cheng Li [email protected] * Li‑Li Zhang [email protected] 1
Department of Neurology, Institute of Surgery Research, Daping Hospital, Third Military Medical University, 10 Changjiang Branch Road, Yuzhong District, Chongqing 400042, People’s Republic of China
has been reported to have anti-atherosclerotic effects, we investigated its role in oxLDL-treated VSMC migration. It is found that activating TRPV1 by capsaicin inhibits VSMC foam cell formation and the accompanied migration through rescuing the SIRT1 and suppressing NF-κB signaling. The present study provides evidence that SIRT1 may be a promising intervention target of atherosclerosis, and raises the prospect of TRPV1 in prevention and treatment of atherosclerosis. Keywords Vascular smooth muscle cell · Foam cell · Migration · SIRT1 · MMP-9 · TRPV1
Introduction Atherosclerosis is the leading cause of death worldwide through its manifestations including ischemic heart disease, stroke and peripheral vascular diseases. The formation of fat-laden foam cells, contributing to the fatty streaks of the plaques of atheroma, is the critical early process in atherosclerosis. Within vascular cells, lipids accumulate as cytoplasmic droplets of cholesterol esters and triglycerides; cells with an abundance of these droplets are termed foam cells. Macrophages are considered the primary source of foam cells in atherosclerosis. Besides, atherosclerotic lesions also contain abundant foam cells with vascular smooth muscle cell (VSMC) identity, especially in advanced atherosclerotic lesions (Rosenfeld and Ross 1990). Oxidized low-density lipoprotein (oxLDL) is the strongest atherogenic factor, which has been proved to promote the foam cell formation both in vivo and in cultured VSMCs. It is noteworthy that VSMC-derived foam cells are deposited in subintima but not media, where VSMCs
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normally deposit in. Therefore, migration from media to intima is an indispensable step for a VSMC to accrue neutral lipids and form foam cell. Whether this migration occurs paralleled with or prior to the formation of foam cell is still unclear. Herein, the present study was designed to test the VSMC migratory capability in the process of foam cell formation induced by oxLDL. Matrix metalloproteinases (MMPs) are a family of zincdependent endopeptidases that degrade various components of the extracellular matrix (ECM) (Visse and Nagase 2003). MMP-9, also known as gelatinase B, is found to be highly expressed in unstable plaque in atherosclerosis. Previous studies have shown that MMP-9 overexpression can enhance VSMC migration into an arterial matrix, and VSMC migration and arterial lesion growth are significantly retarded in MMP-9-deficient arteries after injury (Mason et al. 1999; Cho 2002). Therefore, the MMP-9 expression and its potential regulatory factors were also detected in the present study, in an effort to provide insight into the mechanisms of migration altering during VSMC foam cell formation.
Histochem Cell Biol
Protocol approval was obtained from the Animal Research Committee of the Third Military Medical University. Cell culture VSMCs were isolated from the thoracic aorta of WT mice at 8–10 weeks of age using an explant technique described previously (McMurray et al. 1991). Briefly, all the mice were killed by neck breaking and the thoracic aorta were removed under aseptic condition. After extraneous tissue, adventitia and endothelial layer were all stripped, and the aortic media was cut into 1 × 1 mm2 pieces. These pieces were then transferred to a culture flask containing with DMEM supplemented with 20 % FBS, 100 U/mL penicillin and 100 μg/mL streptomycin in the incubator containing 5 % CO2 and 95 % air at 37 °C. When the cells formed a confluent monolayer (10–14 days), they were passaged by trypsin and cultured by DMEM supplemented with 20 % FBS, 100 U/mL penicillin and 100 μg/mL streptomycin. The cultured VSMCs were verified through immunofluorescence and flow cytometry to test α-SMA. We used cells in the 8–10 passages for experiments.
Materials and methods Flow cytometry Reagents Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), tripsin and phosphate buffer saline (PBS) were purchased from Hyclone (Logan, UT, USA). Oil Red O, capsaicin and capsazepine were obtained from Sigma-Aldrich (St. Louis, MO, USA). SRT1720 was obtained from Selleck Chemicals (Houston, TX, USA). Nicotinamide was obtained from Supelco (Bellefonte, PA, USA). oxLDL was purchased from Yiyuan Biotechnologies (Guangzhou, China). SDSPAGE prepare kit, BCA protein assay kit and reagents for immunofluorescence were from Beyotime (Jiangsu, China). Antibodies targeting TRPV1 (1:500), SIRT1 (1:600), nuclear factor-kappa B p65 (NF-κB, 1:500), phosphorylated IκBα (p-IκBα, 1:100) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibody against MMP-9 (1:1000), α-smooth muscle actin (α-SMA for flow cytometry, 1:50) and isotype control (1:4000) were from Abcam (Burlingame, CA, USA). α-SMA for immunofluorescence was from Beyotime (Jiangsu, China). Antibody targeting β-actin (1:2000) and secondary antibodies (1:3000) were purchased from ZSGB-Bio (Beijing, China). Animals The C57BL/6J wild-type (WT) mice were purchased at 8–10 weeks of age from Jackson Laboratory (Bar Harbor, Maine, USA). Animal care and procedures conformed to the Guide for the Care and Use of Laboratory Animals.
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Cultured mouse VSMCs in a 6-well plate were trypsinized, harvested, washed twice with ice-cold PBS and centrifuged at 700×g for 5 minutes (min). Single-cell suspensions were fixed with immuno-staining fix solution for 30 min and broken by 0.3 % Triton X-100 for 10 min. Non-specific proteins were blocked with 1 % bovine serum albumin (BSA) for 30 min. After blocking, cells were grouped and stained with appropriate amount of anti-mouse α-SMA, corresponding antibody isotype controls and PBS, respectively, according to the manufacturer’s instructions. FITC-labeled secondary antibody was then added after the cells were washed with PBS for twice. Samples were analyzed by flow cytometry (Beckman Coulter, Miami, FL, USA), and the data were analyzed with the FlowJo software (Tree Star, Inc., USA). Assay for foam cell formation Foam cells were verified by Oil Red O staining and quantified by intracellular total cholesterol content. Cultured VSMCs were plated on 12-well plates and treated with control (no oxLDL) or oxLDL (80 μg/mL) in serum-free DMEM for 48 hours (h). After that, the cells were washed with PBS, fixed with 4 % paraformaldehyde and stained with 0.3 % Oil Red O. Foam cells were photographed under a microscope (Leica, German) at 400× magnification. Intracellular total cholesterol content was detected by isopropanol extraction. After Oil Red O staining for foam cells, the wells plated by cultured VSMCs were added
Histochem Cell Biol
Fig. 1 VSMC foam cell formation was paralleled with enhanced migration and increased MMP-9 expression. Primary VSMCs from WT mice increased lipid droplet accumulation and total cholesterol level at 48 h post-oxLDL (80 μg/mL) challenge than that with serumfree medium (Control), which were verified by Oil Red O staining (a) and quantified by intracellular total cholesterol content (b). Primary VSMCs from WT mice were allowed to migrate in Transwell chambers for 24 h in response to serum-free medium (Control) and
oxLDL (80 μg/mL). Migratory cells were stained with crystal violet and counted from high-power (×200) fields. Compared with the control group, oxLDL-stimulated VSMC showed marked increased migratory capability (c). Exposure to oxLDL (80 μg/mL) for 24 h increased the expression of MMP-9 in VSMCs than that in control (d). *p
ORIGINAL PAPER
Impaired SIRT1 promotes the migration of vascular smooth muscle cell‑derived foam cells Ming‑Jie Zhang1 · Yi Zhou1 · Lei Chen1 · Xu Wang1 · Yan Pi1 · Chun‑Yan Long1 · Meng‑Jiao Sun1 · Xue Chen1 · Chang‑Yue Gao1 · Jing‑Cheng Li1 · Li‑Li Zhang1
Accepted: 10 January 2016 © Springer-Verlag Berlin Heidelberg 2016
Abstract The formation of fat-laden foam cells, contributing to the fatty streaks of the plaques of atheroma, is the critical early process in atherosclerosis. The previous study demonstrated that vascular smooth muscle cells (VSMCs) contain a much larger burden of the excess cholesterol in comparison with monocyte-derived macrophages in human coronary atherosclerosis, as the main origin of foam cells. It is noteworthy that VSMC-derived foam cells are deposited in subintima but not media, where VSMCs normally deposit in. Therefore, migration from media to intima is an indispensable step for a VSMC to accrue neutral lipids and form foam cell. Whether this migration occurs paralleled with or prior to the formation of foam cell is still unclear. Herein, the present study was designed to test the VSMC migratory capability in the process of foam cell formation induced by oxidized low-density lipoprotein (oxLDL). In conclusion, we provide evidence that oxLDL induces the VSMC-derived foam cells formation with increased migration ability and MMP-9 expression, which were partly attributed to the impaired SIRT1 and enhanced nuclear factor-kappa B (NF-κB) activity. As activation of transient receptor potential vanilloid type 1 (TRPV1)
Electronic supplementary material The online version of this article (doi:10.1007/s00418-016-1408-9) contains supplementary material, which is available to authorized users. * Jing‑Cheng Li [email protected] * Li‑Li Zhang [email protected] 1
Department of Neurology, Institute of Surgery Research, Daping Hospital, Third Military Medical University, 10 Changjiang Branch Road, Yuzhong District, Chongqing 400042, People’s Republic of China
has been reported to have anti-atherosclerotic effects, we investigated its role in oxLDL-treated VSMC migration. It is found that activating TRPV1 by capsaicin inhibits VSMC foam cell formation and the accompanied migration through rescuing the SIRT1 and suppressing NF-κB signaling. The present study provides evidence that SIRT1 may be a promising intervention target of atherosclerosis, and raises the prospect of TRPV1 in prevention and treatment of atherosclerosis. Keywords Vascular smooth muscle cell · Foam cell · Migration · SIRT1 · MMP-9 · TRPV1
Introduction Atherosclerosis is the leading cause of death worldwide through its manifestations including ischemic heart disease, stroke and peripheral vascular diseases. The formation of fat-laden foam cells, contributing to the fatty streaks of the plaques of atheroma, is the critical early process in atherosclerosis. Within vascular cells, lipids accumulate as cytoplasmic droplets of cholesterol esters and triglycerides; cells with an abundance of these droplets are termed foam cells. Macrophages are considered the primary source of foam cells in atherosclerosis. Besides, atherosclerotic lesions also contain abundant foam cells with vascular smooth muscle cell (VSMC) identity, especially in advanced atherosclerotic lesions (Rosenfeld and Ross 1990). Oxidized low-density lipoprotein (oxLDL) is the strongest atherogenic factor, which has been proved to promote the foam cell formation both in vivo and in cultured VSMCs. It is noteworthy that VSMC-derived foam cells are deposited in subintima but not media, where VSMCs
13
normally deposit in. Therefore, migration from media to intima is an indispensable step for a VSMC to accrue neutral lipids and form foam cell. Whether this migration occurs paralleled with or prior to the formation of foam cell is still unclear. Herein, the present study was designed to test the VSMC migratory capability in the process of foam cell formation induced by oxLDL. Matrix metalloproteinases (MMPs) are a family of zincdependent endopeptidases that degrade various components of the extracellular matrix (ECM) (Visse and Nagase 2003). MMP-9, also known as gelatinase B, is found to be highly expressed in unstable plaque in atherosclerosis. Previous studies have shown that MMP-9 overexpression can enhance VSMC migration into an arterial matrix, and VSMC migration and arterial lesion growth are significantly retarded in MMP-9-deficient arteries after injury (Mason et al. 1999; Cho 2002). Therefore, the MMP-9 expression and its potential regulatory factors were also detected in the present study, in an effort to provide insight into the mechanisms of migration altering during VSMC foam cell formation.
Histochem Cell Biol
Protocol approval was obtained from the Animal Research Committee of the Third Military Medical University. Cell culture VSMCs were isolated from the thoracic aorta of WT mice at 8–10 weeks of age using an explant technique described previously (McMurray et al. 1991). Briefly, all the mice were killed by neck breaking and the thoracic aorta were removed under aseptic condition. After extraneous tissue, adventitia and endothelial layer were all stripped, and the aortic media was cut into 1 × 1 mm2 pieces. These pieces were then transferred to a culture flask containing with DMEM supplemented with 20 % FBS, 100 U/mL penicillin and 100 μg/mL streptomycin in the incubator containing 5 % CO2 and 95 % air at 37 °C. When the cells formed a confluent monolayer (10–14 days), they were passaged by trypsin and cultured by DMEM supplemented with 20 % FBS, 100 U/mL penicillin and 100 μg/mL streptomycin. The cultured VSMCs were verified through immunofluorescence and flow cytometry to test α-SMA. We used cells in the 8–10 passages for experiments.
Materials and methods Flow cytometry Reagents Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), tripsin and phosphate buffer saline (PBS) were purchased from Hyclone (Logan, UT, USA). Oil Red O, capsaicin and capsazepine were obtained from Sigma-Aldrich (St. Louis, MO, USA). SRT1720 was obtained from Selleck Chemicals (Houston, TX, USA). Nicotinamide was obtained from Supelco (Bellefonte, PA, USA). oxLDL was purchased from Yiyuan Biotechnologies (Guangzhou, China). SDSPAGE prepare kit, BCA protein assay kit and reagents for immunofluorescence were from Beyotime (Jiangsu, China). Antibodies targeting TRPV1 (1:500), SIRT1 (1:600), nuclear factor-kappa B p65 (NF-κB, 1:500), phosphorylated IκBα (p-IκBα, 1:100) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibody against MMP-9 (1:1000), α-smooth muscle actin (α-SMA for flow cytometry, 1:50) and isotype control (1:4000) were from Abcam (Burlingame, CA, USA). α-SMA for immunofluorescence was from Beyotime (Jiangsu, China). Antibody targeting β-actin (1:2000) and secondary antibodies (1:3000) were purchased from ZSGB-Bio (Beijing, China). Animals The C57BL/6J wild-type (WT) mice were purchased at 8–10 weeks of age from Jackson Laboratory (Bar Harbor, Maine, USA). Animal care and procedures conformed to the Guide for the Care and Use of Laboratory Animals.
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Cultured mouse VSMCs in a 6-well plate were trypsinized, harvested, washed twice with ice-cold PBS and centrifuged at 700×g for 5 minutes (min). Single-cell suspensions were fixed with immuno-staining fix solution for 30 min and broken by 0.3 % Triton X-100 for 10 min. Non-specific proteins were blocked with 1 % bovine serum albumin (BSA) for 30 min. After blocking, cells were grouped and stained with appropriate amount of anti-mouse α-SMA, corresponding antibody isotype controls and PBS, respectively, according to the manufacturer’s instructions. FITC-labeled secondary antibody was then added after the cells were washed with PBS for twice. Samples were analyzed by flow cytometry (Beckman Coulter, Miami, FL, USA), and the data were analyzed with the FlowJo software (Tree Star, Inc., USA). Assay for foam cell formation Foam cells were verified by Oil Red O staining and quantified by intracellular total cholesterol content. Cultured VSMCs were plated on 12-well plates and treated with control (no oxLDL) or oxLDL (80 μg/mL) in serum-free DMEM for 48 hours (h). After that, the cells were washed with PBS, fixed with 4 % paraformaldehyde and stained with 0.3 % Oil Red O. Foam cells were photographed under a microscope (Leica, German) at 400× magnification. Intracellular total cholesterol content was detected by isopropanol extraction. After Oil Red O staining for foam cells, the wells plated by cultured VSMCs were added
Histochem Cell Biol
Fig. 1 VSMC foam cell formation was paralleled with enhanced migration and increased MMP-9 expression. Primary VSMCs from WT mice increased lipid droplet accumulation and total cholesterol level at 48 h post-oxLDL (80 μg/mL) challenge than that with serumfree medium (Control), which were verified by Oil Red O staining (a) and quantified by intracellular total cholesterol content (b). Primary VSMCs from WT mice were allowed to migrate in Transwell chambers for 24 h in response to serum-free medium (Control) and
oxLDL (80 μg/mL). Migratory cells were stained with crystal violet and counted from high-power (×200) fields. Compared with the control group, oxLDL-stimulated VSMC showed marked increased migratory capability (c). Exposure to oxLDL (80 μg/mL) for 24 h increased the expression of MMP-9 in VSMCs than that in control (d). *p
