Impaired SIRT1 Promotes The Migration Of Vascular Smooth Muscle Cell-derived Foam Cells

Background and Objectives:Atherosclerosis (AS) 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 AS. 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 AS. Besides, atherosclerotic lesions also contain abundant foam cells with vascular smooth muscle cell (VSMC) identity, especially in advanced atherosclerotic lesions. 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.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.Herein, the present study was designed to test the VSMC migratory capability in the process of foam cell formation induced by oxLDL.SIRT1 (Sirtuinl) in mammals, the closest homolog of yeast Silent information regulator 2 (Sir2) protein, is a member of the highly conserved sirtuin family of nicotinamide adenine dinucleotide (NAD+)-dependent histone deacetylases. SIRT1 can target numerous proteins including peroxisome proliferator-activated receptor (PPAR)-γ,PPAR-y coactivator (PGC)-1α, uncoupling protein 2 (UCP2), liver X receptors (LXRs) and nuclear factor (NF)-κB and thus regulate various pathological and physiological processes.For instance, deacetylation of LXR by SIRT1 up-regulates its activity and promotes reverse cholesterol transport to remove cholesterol from cells, ultimately inhibits the foam cell formation. SIRT1 has also been found to exert salutary actions against endothelial dysfunction through inhibiting premature senescence. Upregulating endothelial nitric oxide synthase (eNOS) expression by SIRT1 rescued endothelium dependent vasorelaxation in vivo impaired by high fat diet. Besides, the positive effects of SIRT1 on stabilizing plaques,decreasing cholesterol uptake, inhibiting macrophage foam cell formation and relieving inflammatory raise the prospect of SIRT1 as a promising therapeutic target for AS treatment.However, rare studies have reported the potential role of SIRT1 in the migration during the process of VSMC foam cell formation, which was tested in the present study. Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that degrade various components of the extracellular matrix (ECM). MMP-9, also known as gelatinase B,is found to be highly expressed in unstable plaque in AS. 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. 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.Transient receptor potential vanilloid type 1 (TRPV1) has been reported to contribute to improving cardiovascular and cerebrovascular diseases (C-CeVD). Activation of TRPV1 reduced the accumulation of lipids of VSMCs to inhibit the foam cell formation via regulating metabolic pathways and inducing autophagy. We have found previously that activation of TRPV1 suppressed the proliferation of VSMCs in spontaneously hypertensive rats. However, the potential role of TRPV1 in VSMC migration is poorly understood.Herein, we examined the effect of TRPV1 on VSMC-derived foam cell migration and MMP-9 expression, and tested whether SIRT1 involed in this effect, hoping to raise the prospect of TRPV1 in prevention and treatment of AS.Materials and Methods:1. VSMCs were isolated from the thoracic aorta of C57BL/6J wild-type (WT) mice using the explant technique. The cultured VSMCs were verified through immunofluorescence and flow cytometry to test a-smooth muscle actin (a-SMA).2. VSMC-derived foam cells by oxLDL were verified by Oil Red O staining and quantified by intracellular total cholesterol content.3. Cell migration was examined by Transwell assay.4. SIRT1 agonist SRT1720 (SRT) and antagonist nicotinamide (Nic) were used to modulate the expression of SIRT1 pharmacologically.5. TRPV1 agonist capsaicin (Caps) and TRPV1 antagonist capsazepin (Capz) were used to influence the expression of TRPV1.6. Western blot analysis and immunofluorescence were applied to detect the expressions of proteins quantitatively and qualitatively, respectively.Results:1. VSMC foam cell formation was paralleled with enhanced migration and increased MMP-9 expression.In the present study, oxLDL (80μg/mL) promoted the accumulation of red lipid droplets in cultured VSMC, as visualized by oil-red stain. Consistently, the total cholesterol level within the oxLDL-stimulated VSMC increased significantly, which collectively with the gathered lipid droplets indicated the foam cell formation of VSMCs in response to oxLDL challenge. Meanwhile, compared with the control group, oxLDL-stimulated VSMC showed marked increased migratory capability, detected by Transwell assay. In addition,exposure to oxLDL for 24 hours (h) increased the expression of MMP-9 in VSMCs.2. OxLDL impaired SIRT1 expression which is responsible for upregulated MMP-9 in process of VSMC foam cell formation.Exposure to oxLDL in VSMCs for 24h reduced the expression of SIRT1. SIRT1 agonist SRT and antagonist Nic were respectively used to pharmacologically modulate the expression of SIRT1, in order to identify the effect of SIRT1 on MMP-9 expression. As expected, SRT upregulated the SIRT1 expression both in control and oxLDL-stimulated VSMCs, while Nic at 100μM markedly downregulated the SIRT1 expression in VSMCs.Furthermore, SRT significantly counteracted the increased MMP-9 expression induced by oxLDL,which was diminished by Nic stimulation. Consistently, SRT impeded the oxLDL-induced VSMC migration, which was also diminished by Nic.3. SIRT1 might downregulate MMP-9 expression through inhibiting NF-κB pathway in oxLDL-challenged VSMCs.OxLDL treatment increased the expression of phosphorylated IκBa (p-IκBα) and nuclear NF-κB p65 in VSMCs. SIRT1 activation by SRT significantly decreased oxLDL-induced p-IκBα and nuclear NF-κB p65 expression, which was reversed by SIRT1 antagonist Nic. Incubation with selective NF-κB inhibitor BAY 11-7082 significantly attenuated the increased MMP-9 in VSMCs stimulated by oxLDL.4. Activation of TRPV1 by Caps inhibited the overexpression of MMP-9 and migration in oxLDL-treated VSMCs.Caps and Capz were respectively used to activate and inhibit TRPV1 in VSMCs. Caps upregulated the TRPV1 expression in a concentration-dependent manner. Caps also decreased the oxLDL-induced MMP-9 expression concentration dependently. Also, Caps significantly inhibited the upregulated MMP-9 induced by oxLDL, while TRPV1 inhibitor Capz diminished the effect of Caps. Consistently, Caps inhibited the oxLDL-induced VSMC migration, which was impeded markedly by Capz.5. Activation of TRPV1 by Caps upregulated SIRT1 and inhibited NF-κB expression.It was shown that activation of TRPV1 by Caps increased the level of SIRT1 protein and green fluorescent protein (GFP)-tagged SIRT1 in VSMCs, while Capz inhibited the increase of SIRT1 by Caps. TRPV1 activation also rescued the impaired SIRT1 expression by oxLDL. Furthermore, TRPV1 activation by Caps significantly decreased oxLDL-induced p-IκBα and nuclear NF-κB p65 expression, which was reversed by TRPV1 inhibitor Capz.Conclusions: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 NF-κB activity. Activating TRPV1 by Caps inhibits VSMC foam cell formation and the accompanied migration through rescuing the SIRT1 and suppressing NF-κB signaling. Foam cell formation and phenotypic modulation of VSMCs are two key processes in the development of AS. The present study provides evidence that SIRT1 may be a promising intervention target of AS, and raises the prospect of TRPV1 in prevention and treatment of AS. Some SIRT1 activators, especially resveratrol,have performed the protective roles in atherosclerotic animals and cells in preclinical studies. Although the clinical results are not robust and consistent, strategies to maintain a higher level of SIRT1 may provide useful treatment for AS.