Autophagy In Vascular Smooth Muscle Cells Research Articles

ADORA3: A Key Player in the Pathogenesis of Intracranial Aneurysms and a Potential Diagnostic Biomarker.

The effects of genes on the development of intracranial aneurysms (IAs) remain to be elucidated, and reliable blood biomarkers for diagnosing IAs are yet to be established. This study aimed to identify genes associated with IAs pathogenesis and explore their diagnostic value by analyzing IAs datasets, conducting vascular smooth muscle cells (VSMC) experiments, and performing blood detection. IAs datasets were collected and the differentially expressed genes were analyzed. The selected genes were validated in external datasets. Autophagy was induced in VSMC and the effect of selected genes was determined. The diagnostic value of selected gene on the IAs were explored using area under curve (AUC) analysis using IAs plasma samples. Analysis of 61 samples (32 controls and 29 IAs tissues) revealed a significant increase in expression of ADORA3 compared with normal tissues using empirical Bayes methods of "limma" package; this was further validated by two external datasets. Additionally, induction of autophagy in VSMC lead to upregulation of ADORA3. Conversely, silencing ADORA3 suppressed VSMC proliferation and autophagy. Furthermore, analysis of an IAs blood sample dataset and clinical plasma samples demonstrated increased ADORA3 expression in patients with IA compared with normal subjects. The diagnostic value of blood ADORA3 expression in IAs was moderate when analyzing clinical samples (AUC: 0.756). Combining ADORA3 with IL2RB or CCR7 further enhanced the diagnostic ability for IAs, with the AUC value over 0.83. High expression of ADORA3 is associated with IAs pathogenesis, likely through its promotion of VSMC autophagy. Furthermore, blood ADORA3 levels have the potential to serve as an auxiliary diagnostic biomarker for IAs.

Resveratrol Inhibits Abdominal Aortic Aneurysm Progression by Reducing Extracellular Matrix Degradation, Apoptosis, Autophagy, and Inflammation of Vascular Smooth Muscle Cells via Upregulation of HMOX1.

This study aimed to explore the therapeutic effect of resveratrol (RES) against abdominal aortic aneurysm (AAA) and the role of HMOX1 underlying this effect. Vascular smooth muscle cells (VSMCs) were induced by angiotensin II (Ang II) to construct the microenvironment of AAA. HMOX1 expression was downregulated by the short hairpin ribonucleic acid (RNA) specific to HMOX1 in RES-pretreated VSMCs. The levels of matrix metalloproteinase (MMP)-2, MMP-9, and elastin were measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot. Apoptosis rate was detected. The levels of apoptosis-related proteins (caspase-3 and Bax/Bcl-2), inflammatory cytokines (interleukin [IL]-6, tumor necrosis factor [TNF]-α, and IL-1β), and autophagy-related proteins (Beclin 1, light chain 3 [LC3] II/I, and p62) were detected by western blot. The secretion of inflammatory factors in cell supernatant was detected by enzyme-linked immunosorbent assay (ELISA). The number of autophagic vesicles in VSMCs was observed and analyzed by transmission electron microscopy. A rat model of pancreatic elastase-induced AAA was established to verify the effect and action mechanism of RES. Stimulation of Ang II increased the messenger RNA (mRNA) and protein levels of MMP-2 and MMP-9, decreased elastin expression, and enhanced apoptosis, secretion of inflammatory factors, and autophagy in VSMCs, whereas RES pretreatment ameliorated Ang II-induced VSMC dysfunction. In addition, HMOX1 mRNA and heme oxygenase-1 (HO-1) protein levels were significantly increased in VSMCs pretreated with RES compared with Ang II treatment alone. Silencing of HMOX1 abolished the effects of RES on VSMC dysfunction. Consistently, RES suppressed the development of AAA in rats by increasing the expression of HMOX1. Resveratrol protects against AAA by inhibiting extracellular matrix degradation, apoptosis, autophagy, and inflammation of VSMCs via HMOX1 upregulation. Our study found that angiotensin II (Ang II) stimulated increased the levels of MMP-2 and MMP-9 in vascular smooth muscle cells (VSMCs), decreased elastin expression, and promoted apoptosis, autophagy occurrence, and secretion of inflammatory factors, while resveratrol (RES) pretreatment improved this effect. In addition, downregulation of HMOX1 expression eliminated the effect of RES on the function of VSMCs. Our study elucidates that RES improves AAA progression through HMOX1 at both cellular and animal levels. This work can help doctors better understand the pathological mechanism of the occurrence and development of AAA, and provide a theoretical basis for clinicians to find better treatment options.

The roles of METTL3 on autophagy and proliferation of vascular smooth muscle cells are mediated by mTOR rather than by CDK1

BackgroundAberrant proliferation of vascular smooth muscle cells (VSMCs) is the cause of neointima formation followed by vascular injury. Autophagy is involved in this pathological process, but its function is controversial. Recently, we found that methyltransferase like 3 (METTL3) inhibited VSMC proliferation by activating autophagosome formation. Moreover, we also demonstrated that METTL3 reduced the levels of phosphorylated mammalian target of rapamycin (p-mTOR) and cyclin dependent kinase 1 (p-CDK1/CDC2), which were critical for autophagy and proliferation regulation. However, whether mTOR and CDK1 mediated the function of METTL3 on autophagy and proliferation in VSMCs remains unknown.ResultsWe showed that the activator of mTOR, MHY1485 largely reversed the effects of METTL3 overexpression on VSMC autophagy and proliferation. Rapamycin, the inhibitor of mTOR, obviously nullified the pro-proliferation effects of METTL3 knockdown by activating autophagy in VSMCs. Unexpectedly, mTOR did not contribute to the impacts of METTL3 on migration and phenotypic switching of VSMCs. On the other hand, by knockdown of CDK1 in VSMC with METTL3 deficiency, we demonstrated that CDK1 was involved in METTL3-regulated proliferation of VSMCs, but this effect was not mediated by autophagy.ConclusionsWe concluded that mTOR but not CDK1 mediated the role of METTL3 on VSMC proliferation and autophagy.

Hsa_circ_0001402 alleviates vascular neointimal hyperplasia through a miR-183-5p-dependent regulation of vascular smooth muscle cell proliferation, migration, and autophagy

IntroductionVascular neointimal hyperplasia, a pathological process observed in cardiovascular diseases such as atherosclerosis and pulmonary hypertension, involves the abundant presence of vascular smooth muscle cells (VSMCs). The proliferation, migration, and autophagy of VSMCs are associated with the development of neointimal lesions. Circular RNAs (circRNAs) play critical roles in regulating VSMC proliferation and migration, thereby participating in neointimal hyperplasia. However, the regulatory roles of circRNAs in VSMC autophagy remain unclear. ObjectivesWe aimed to identify circRNAs that are involved in VSMC autophagy-mediated neointimal hyperplasia, as well as elucidate the underlying mechanisms. MethodsDual-luciferase reporter gene assay was performed to validate two competing endogenous RNA axes, hsa_circ_0001402/miR-183-5p/FKBP prolyl isomerase like (FKBPL) and hsa_circ_0001402/miR-183-5p/beclin 1 (BECN1). Cell proliferation and migration analyses were employed to investigate the effects of hsa_circ_0001402, miR-183-5p, or FKBPL on VSMC proliferation and migration. Cell autophagy analysis was conducted to reveal the role of hsa_circ_0001402 or miR-183-5p on VSMC autophagy. The role of hsa_circ_0001402 or miR-183-5p on neointimal hyperplasia was evaluated using a mouse model of common carotid artery ligation. ResultsHsa_circ_0001402 acted as a sponge for miR-183-5p, leading to the suppression of miR-183-5p expression. Through direct interaction with the coding sequence (CDS) of FKBPL, miR-183-5p promoted VSMC proliferation and migration by decreasing FKBPL levels. Besides, miR-183-5p reduced BECN1 levels by targeting the 3′-untranslated region (UTR) of BECN1, thus inhibiting VSMC autophagy. By acting as a miR-183-5p sponge, overexpression of hsa_circ_0001402 increased FKBPL levels to inhibit VSMC proliferation and migration, while simultaneously elevating BECN1 levels to activate VSMC autophagy, thereby alleviating neointimal hyperplasia. ConclusionHsa_circ_0001402, acting as a miR-183-5p sponge, increases FKBPL levels to inhibit VSMC proliferation and migration, while enhancing BECN1 levels to activate VSMC autophagy, thus alleviating neointimal hyperplasia. The hsa_circ_0001402/miR-183-5p/FKBPL axis and hsa_circ_0001402/miR-183-5p/BECN1 axis may offer potential therapeutic targets for neointimal hyperplasia.

The Potential Role of Connexins in the Pathogenesis of Atherosclerosis.

Connexins (Cx) are members of a protein family which enable extracellular and intercellular communication through hemichannels and gap junctions (GJ), respectively. Cx take part in transporting important cell-cell messengers such as 3',5'-cyclic adenosine monophosphate (cAMP), adenosine triphosphate (ATP), and inositol 1,4,5-trisphosphate (IP3), among others. Therefore, they play a significant role in regulating cell homeostasis, proliferation, and differentiation. Alterations in Cx distribution, degradation, and post-translational modifications have been correlated with cancers, as well as cardiovascular and neurological diseases. Depending on the isoform, Cx have been shown either to promote or suppress the development of atherosclerosis, a progressive inflammatory disease affecting large and medium-sized arteries. Cx might contribute to the progression of the disease by enhancing endothelial dysfunction, monocyte recruitment, vascular smooth muscle cell (VSMC) activation, or by inhibiting VSMC autophagy. Inhibition or modulation of the expression of specific isoforms could suppress atherosclerotic plaque formation and diminish pro-inflammatory conditions. A better understanding of the complexity of atherosclerosis pathophysiology linked with Cx could result in developing novel therapeutic strategies. This review aims to present the role of Cx in the pathogenesis of atherosclerosis and discusses whether they can become novel therapeutic targets.

C/EBPα-Mediated Transcriptional Activation of PIK3C2A Regulates Autophagy, Matrix Metalloproteinase Expression, and Phenotypic of Vascular Smooth Muscle Cells in Aortic Dissection.

Purpose To investigate the function of C/EBPα in the development of aortic dissection (AD) and the underlying mechanism. Methods Aortic vascular smooth muscle cells (VSMCs) were isolated, cultured, and identified from AD rats. Then, C/EBPα and PIK3C2A were knockdown or overexpressed by siRNA or plasmid transfection, respectively. Rapamycin or 3-MA was utilized to stimulate and restrain autophagy of VSMCs, respectively. Western blot was used to evaluate the expression levels of C/EBPα, PIK3C2A, LC3, Beclin-1, p62, MMP-2, MMP-9, α-SMA, SM-MHC, and OPN. The pathological status of aortic ring was evaluated by stretch stress, and ChIP assay was used to analyze the binding between C/EBPα and PIK3C2A. C/EBPα shRNA was injected into tail vein to observe the effect of C/EBPα knockdown in vivo on phenotype, autophagy of aortic vascular tissue by immunohistochemical staining and Western blot. Results The protein levels of C/EBPα, PIK3C2A, MMP-2, MMP-9, and LC3 in the aorta of AD rats were all upregulated significantly. C/EBPα and rapamycin promoted notable upregulation of the synthesized proteins (OPN), PIK3C2A, matrix metalloproteinases, LC3, and Beclin-1 in VSMCs, while suppressed contractile proteins (α-SMA and SM-MHC) and p62. The opposite results were observed in the C/EBPα-knockdown VSMCs, PIK3C2A-knockdown VSMCs, or VSMCs treated with 3-MA. C/EBPα, PIK3C2A, and LC3 were dramatically upregulated by the stimulation of 3 g and 5 g stretch stress. The downregulated contractile proteins, upregulated synthetic proteins, activated autophagy, and aggravated pathological state in 5 g stretch stress-treated aortic rings were significantly reversed by the knockdown of C/EBPα. ChIP results indicated that there was a binding site for C/EBPα in the promoter of PIK3C2A. C/EBPα also downregulated α-SMA level and upregulated OPN levels in AD rats in vivo. Conclusion Our data indicated that during the development of AD, C/EBPα regulated the transition of VSMC phenotype and extracellular matrix remodeling by activating autophagy through regulating the transcriptional activity of PIK3C2A promoter.

Hsa_circRNA_0008028 Deficiency Ameliorates High Glucose-Induced Proliferation, Calcification, and Autophagy of Vascular Smooth Muscle Cells via miR-182-5p/TRIB3 Axis.

Background It is well-known that dysfunctions of vascular smooth muscle cells (VSMCs) act an essential part in vascular complications of diabetes. Studies have shown that circular RNAs (circRNAs) and microRNAs (miRNAs) play a crucial role in regulating cell functions. However, their influence on the proliferation, calcification, and autophagy of VSMCs remains to be further explored. Therefore, this study elucidates the role and mechanism of hsa_circRNA_0008028 in high glucose- (HG-, 30 mM) treated VSMCs in vitro. Methods Quantitative real-time polymerase chain reaction (qRT-PCR) was chosen to detect the levels of hsa_circRNA_0008028, miR-182-5p, and tribble 3 (TRIB3). Then, dual-luciferase reporter and RNA immunoprecipitation (RIP) assays were used to predict and verify the binding relationship between miR-182-5p and hsa_circRNA_0008028 or TRIB3. Cell counting kit-8 assay, 5-ethynyl-2′-deoxyuridine (EdU) staining, corresponding commercial kits, and western blotting were used to measure indexes reflecting cell viability, proliferation, calcification, and autophagy of VSMCs, respectively. Results In HG-induced VSMCs, hsa_circRNA_0008028 and TRIB3 were highly expressed, whereas miR-182-5p decreased. Meanwhile, cell proliferation, calcification, and autophagy could be repressed by silencing of hsa_circRNA_0008028. However, these effects can be eliminated by miR-182-5p inhibition. Furthermore, it was demonstrated that hsa_circRNA_0008028 could promote the expression of TRIB3, a target of miR-182-5p, by directly sponging miR-182-5p. The expression of TRIB3 was suppressed by hsa_circRNA_0008028 knockout, which was rescued by miR-182-5p inhibition. Conclusion This study reveals that hsa_circRNA_0008028 can act as a sponge of miR-182-5p and promote HG-induced proliferation, calcification, and autophagy of VSMCs partly by regulating TRIB3.

Extracellular vesicle miR-32 derived from macrophage promotes arterial calcification in mice with type 2 diabetes via inhibiting VSMC autophagy

BackgroundThe development of diabetes vascular calcification (VC) is tightly associated with the inhibition of vascular smooth muscle cell (VSMC) autophagy. Previously, our team found that miR-32-5p (miR-32) promotes macrophage activation, and miR-32 is expressed at higher level in the plasma of patients with coronary calcification. However, whether miR-32 mediates the function of macrophages in type 2 diabetes (T2D) VC is still unclear.MethodsWild-type (WT) and miR-32−/− mice were used in this study. qRT-PCR and western blotting were used to analyze gene expression. Flow cytometry was used to analyze the influence of glucose concentration on macrophage polarization. Nanoparticle tracking analysis (NTA), transmission electron microscopy, and confocal microscopy were used to identify macrophage extracellular vehicles (EVs). Immunofluorescence, in situ hybridization (ISH), immunohistochemistry, and alizarin red staining were used to analyze the influence of macrophage EVs on autophagy and calcification of the aorta of miR-32−/− mice. A luciferase assay was used to analyze the effect of miR-32 on myocyte enhancer factor 2D (Mef2d) expression. Co-IP combined with mass spectrometry (MS) and transcriptome sequencing was used to analyze the signalling pathway by which Mef2d acts in VSMC autophagy.ResultsWe found that high glucose conditions upregulate miR-32 expression in macrophages and their EVs. Importantly, macrophages and their EVs promote VSMC osteogenic differentiation and upregulate miR-32 expression in VSMCs. Moreover, miR-32 mimics transfection promoted osteogenic differentiation and inhibited autophagy in VSMCs. In vitro and in vivo experiments showed that Mef2d is the key target gene of miR-32 that inhibits VSMC autophagy. Furthermore, MS and transcriptome sequencing found that cGMP-PKG is an important signalling pathway by which Mef2d regulates VSMC autophagy. In addition, after T2D miR-32−/− mice were injected with macrophage EVs via the caudal vein, miR-32 was detected in aortic VSMCs of miR-32−/− mice. Moreover, autophagy was significantly inhibited, and calcification was significantly enhanced in aorta cells.ConclusionsThese results reveal that EVs are the key pathway by which macrophages promote T2D VC, and that EVs miR-32 is a key cause of autophagy inhibition in VSMCs.

Abstract 207: Autophagy Is Differentially Regulated In Leukocyte And Nonleukocyte Foam Cells During Atherosclerosis

Atherosclerosis is characterized by a build-up of foam cells in the arterial wall, resulting from excess cholesterol uptake and accumulation of cytosolic lipid droplets (LDs). Autophagy has been shown to be atheroprotective in part by promoting the catabolism of LDs which liberates free cholesterol for efflux out of foam cells to cholesterol acceptors (ApoA-I or HDL) for removal from the body. Apart from macrophages (MΦ), vascular smooth muscle cells (VSMCs) comprise 50-70% of foam cells in the plaques. Unlike MΦ, the capacity of VSMC foam cells to metabolize cholesterol via autophagy is unknown. Here, we performed a comparative analysis of the autophagic capacity and cholesterol efflux of arterial foam cell subtypes of the atherosclerotic plaque. Atherosclerosis was induced in hypercholesterolemic autophagy reporter mice (GFP-LC3 mice receiving PCSK9-AAV and fed a Western diet). Autophagic flux in aortic digests was assessed by quantifying GFP-LC3 fluorescence after ex vivo treatment with the autophagy inhibitor Bafilomycin A1 or vehicle. MΦ foam cells displayed functional autophagy as shown by an accumulation of GFP-LC3 upon autophagy inhibition. In contrast, VSMC foam cells did not similarly accumulate GFP-LC3 upon bafilomycin treatment, suggesting dysfunction autophagy in these cells. Additionally, immunostaining of late-stage aortic roots showed MΦ, but not VSMC, foam cells induction of the active autophagy marker pATG16L1. Cell culture studies of lipid loaded MΦ and VSMC corroborated this inability for VSMCs to initiate autophagy in vivo . In vitro , MΦ foam cells effluxed cholesterol to ApoA-I (14%) and HDL (50%), whereas VSMC foam cells minimally effluxed cholesterol to HDL (7%) but not apoA-I. However, unlike MΦ foam cells, VSMC efflux was pharmacologically induced by treatment with metformin. Our data therefore demonstrates a lack of functional autophagy in VSMC, as compared to MΦ foam cells, which impairs their ability to perform cholesterol efflux. This autophagy defect in VSMC foam cells can be increased by autophagy activation using metformin, highlighting both the importance of understanding cholesterol metabolism in all foam cell populations and a new avenue to treat atherosclerosis.

Abstract 102: Autophagy Is Differentially Regulated In Leukocyte And Nonleukocyte Foam Cells During Atherosclerosis

Atherosclerosis is characterized by a build-up of foam cells in the arterial wall, resulting from excess cholesterol uptake and accumulation of cytosolic lipid droplets (LDs). Autophagy has been shown to be atheroprotective in part by promoting the catabolism of LDs which liberates free cholesterol for efflux out of foam cells to cholesterol acceptors (ApoA-I or HDL) for removal from the body. Apart from macrophages (MΦ), vascular smooth muscle cells (VSMCs) comprise 50-70% of foam cells in the plaques. Unlike MΦ, the capacity of VSMC foam cells to metabolize cholesterol via autophagy is unknown. Here, w e performed a comparative analysis of the autophagic capacity and cholesterol efflux of arterial foam cell subtypes of the atherosclerotic plaque . Atherosclerosis was induced in hypercholesterolemic autophagy reporter mice (GFP-LC3 mice receiving PCSK9-AAV and fed a Western diet). Autophagic flux in aortic digests was assessed by quantifying GFP-LC3 fluorescence after ex vivo treatment with the autophagy inhibitor Bafilomycin A1 or vehicle. MΦ foam cells displayed functional autophagy as shown by an accumulation of GFP-LC3 upon autophagy inhibition. In contrast, VSMC foam cells did not similarly accumulate GFP-LC3 upon bafilomycin treatment, suggesting dysfunction autophagy in these cells. Additionally, immunostaining of late-stage aortic roots showed MΦ, but not VSMC, foam cells induction of the active autophagy marker pATG16L1. Cell culture studies of lipid loaded MΦ and VSMC corroborated this inability for VSMCs to initiate autophagy in vivo . In vitro , MΦ foam cells effluxed cholesterol to ApoA-I (14%) and HDL (50%), whereas VSMC foam cells minimally effluxed cholesterol to HDL (7%) but not apoA-I. However, unlike MΦ foam cells, VSMC efflux was pharmacologically induced by treatment with metformin. Our data therefore demonstrates a lack of functional autophagy in VSMC, as compared to MΦ foam cells, which impairs their ability to perform cholesterol efflux. This autophagy defect in VSMC foam cells can be increased by autophagy activation using metformin, highlighting both the importance of understanding cholesterol metabolism in all foam cell populations and a new avenue to treat atherosclerosis.

Inhibition of Connexin 43 reverses ox-LDL-mediated inhibition of autophagy in VSMC by inhibiting the PI3K/Akt/mTOR signaling pathway.

BackgroundOxidized low-density lipoproteins (ox-LDL) may induce foam cell formation from the vascular smooth muscle cell (VSMC) by inhibiting VSMC autophagy. This process accelerates the formation of atherosclerosis (AS). Connexin 43 (Cx43), which is the most widely distributed connexin in VSMC is associated with autophagy. However, the mechanism of action and the involvement of Cx43 in ox-LDL-inhibited VSMC autophagy remain unclear.MethodsThe primary VSMC were obtained and identified, before primary VSMC were pretreated with an inhibitor (Cx43-specific inhibitor Gap26 and PI3K inhibitor LY294002) and stimulated with ox-LDL.ResultsOx-LDL not only inhibited autophagy in VSMC via downregulation of autophagy-related proteins (such as Beclin 1, LC3B, p62), but also increased Cx43 protein levels. Then we added Gap26 to VSMC in the ox-LDL+Gap26 group, in which autophagy-related proteins were increased and the accumulation of lipid droplets was reduced. These result suggested that an enhanced level of autophagy and an alleviation of lipid accumulation might be caused by inhibiting Cx43 in VSMC. The phosphorylation levels of PI3K, AKT, mTOR were increased by ox-LDL, thus down-regulating autophagy-related proteins. However, this situation was partially reversed by the Gap26. Moreover, Cx43 expression were decreased by LY294002 in ox-LDL-induced VSMCs.ConclusionInhibiting Cx43 may activate VSMC autophagy to inhibit foam cell formation by inhibiting the PI3K/AKT/mTOR signaling pathway.

Apolipoprotein J Attenuates Vascular Restenosis by Promoting Autophagy and Inhibiting the Proliferation and Migration of Vascular Smooth Muscle Cells.

This research investigated the mechanism of CLU in vascular restenosis by regulating vascular smooth muscle cell (VSMC) proliferation and migration. Firstly, rat models of balloon injury (BI) were established, followed by the assessment of the injury to the common carotid artery. The effect of CLU on the intimal hyperplasia of BI rats was measured after the intervention in CLU, in addition to the evaluation of proliferation, migration, and autophagy of VSMCs. Moreover, the interaction between ATG and LC3 was analyzed, followed by validation of the role of autophagy in CLU's regulation on the proliferation and migration of VSMCs. It was found that CLU was highly expressed in BI rats. Altogether, our findings indicated that CLU was highly expressed in vascular restenosis, and CLU over-expression promoted the binding between ATG3 and LC3, thus facilitating VSMC autophagy and eventually attenuating intimal hyperplasia and vascular restenosis.

Vitamin D receptor deficiency increases systolic blood pressure by upregulating the renin-angiotensin system and autophagy.

The vitamin D receptor (VDR) may regulate blood pressure via multiple pathways. The present study investigated the underlying mechanism by which VDR deficiency increases blood pressure. A total of 16 8-week-old male littermate mice were randomly divided into the VDR knockout and wild-type groups (VDR-/- and VDR+/+, respectively). Blood pressure was measured using a four-channel PowerLab data acquisition and ADI software analysis system. After euthanasia, vascular smooth muscle cells (VSMCs) were isolated from the VDR-/- and VDR+/+ mice. Oxidative stress, renin-angiotensin system (RAS) activation and autophagy markers were measured in the isolated VSMCs using reverse transcription-quantitative PCR (RT-qPCR), western blotting and transmission electron microscopy (TEM) assays. Mean systolic pressure was significantly higher in the VDR-/- mice compared with the VDR+/+ mice. RT-qPCR and western blotting analyses indicated that RAS markers (angiotensin II and II type 1 receptor) were significantly upregulated, oxidative stress was increased (evidenced by reduced superoxide dismutase and peroxiredoxin-4) and autophagy was activated (upregulation of autophagy related protein 7, Beclin 1 and microtubule-associated proteins 1A/1B light chain 3A) in the VDR-/- VSMCs compared with the VDR+/+ VSMCs. TEM demonstrated that there were more autophagy bodies in the VDR-/- VSMCs compared with the VDR+/+ VSMCs. In conclusion, VDR deficiency was associated with high blood pressure. The mechanism underlying the increase in blood pressure caused by VDR deficiency may involve activation of the RAS, as well as increased oxidative stress and autophagy of VSMCs.

Vascular smooth muscle cell-derived hydrogen sulfide promotes atherosclerotic plaque stability via TFEB (transcription factor EB)-mediated autophagy

ABSTRACT Vascular smooth muscle cells (VSMCs) contribute to plaque stability. VSMCs are also a major source of CTH (cystathionine gamma-lyase)-hydrogen sulfide (H2S), a protective gasotransmitter in atherosclerosis. However, the role of VSMC endogenous CTH-H2S in pathogenesis of plaque stability and the mechanism are unknown. In human carotid plaques, CTH expression in ACTA2+ cells was dramatically downregulated in lesion areas in comparison to non-lesion areas. Intraplaque CTH expression was positively correlated with collagen content, whereas there was a negative correlation with CD68+ and necrotic core area, resulting in a rigorous correlation with vulnerability index (r = −0.9033). Deletion of Cth in VSMCs exacerbated plaque vulnerability, and were associated with VSMC autophagy decline, all of which were rescued by H2S donor. In ox-LDL treated VSMCs, cth deletion reduced collagen and heightened apoptosis association with autophagy reduction, and vice versa. For the mechanism, CTH-H2S mediated VSMC autophagosome formation, autolysosome formation and lysosome function, in part by activation of TFEB, a master regulator for autophagy. Interference with TFEB blocked CTH-H2S effects on VSMCs collagen and apoptosis. Next, we demonstrated that CTH-H2S sulfhydrated TFEB at Cys212 site, facilitating its nuclear translocation, and then promoting transcription of its target genes such as ATG9A, LAPTM5 or LDLRAP1. Conclusively, CTH-H2S increases VSMC autophagy by sulfhydration and activation of TFEB, promotes collagen secretion and inhibits apoptosis, thereby attenuating atherogenesis and plaque vulnerability. CTH-H2S may act as a warning biomarker for vulnerable plaque.

TAK1 accelerates transplant arteriosclerosis in rat aortic allografts by inducing autophagy in vascular smooth muscle cells

Background and aimsThe proliferation and migration of vascular smooth muscle cells (VSMCs) are fundamental hallmarks of vasculopathy. Transforming growth factor β-activated kinase-1 (TAK1) plays a crucial role in mediating cellular functions, including autophagy, which has been recently linked to the regulation of VSMC functions and the development of vasculopathy. This study aims to better dissect how TAK1 controls VSMC proliferation and migration. MethodsA rat model of graft arteriosclerosis was employed to explore the influence of TAK1 signaling activation on VSMC proliferation, migration, autophagy, and neointima formation in vivo. Knockdown and pharmacological inhibition of TAK1 were utilized in cultured VSMCs to investigate the mechanisms underlying the progression of VSMC proliferation and migration. ResultsIncreased phosphorylation of TAK1 (Thr-184/Thr-187) was examined in SMα-actin positive cells in the medial and neointimal lesions of aortic allografts. Lentivirus-mediated Tak1 shRNA transfection of aortic allografts robustly suppressed neointimal formation and lumen stenosis, as well as autophagy and cell proliferative responses. In cultured PDGF-BB-incubated VSMCs, genetic and pharmacological inhibition of TAK1 markedly attenuated autophagy activation, and blocked the progression of cell cycle, proliferation, and migration responses. ConclusionsActivation of TAK1 in VSMCs in the setting of aortic transplantation is an early and critical event in VSMC proliferation and migration, as well as neointima formation, because it controls autophagy activation, constituting a potential molecular mechanism and target for preventing transplant vasculopathy.

Galangin inhibits neointima formation induced by vascular injury via regulating the PI3K/AKT/mTOR pathway.

Aims: The proliferation and migration of vascular smooth muscle cells (VSMCs) play vital roles in the pathological process of neointima formation after vascular injury. Galangin, an extract of the ginger plant galangal, is involved in numerous biological activities, including inhibiting the proliferation and migration of tumor cells, but its effect on VSMCs is unknown. This study focused on the role and mechanism of galangin in the neointima formation induced by vascular injury. Methods and results: In this study, we found that galangin restrained the PDGF-BB-induced proliferation, migration and phenotypic switching of VSMCs in a concentration-dependent manner. In vivo, we established a model of carotid artery balloon injury in rats, followed by intragastric administration of galangin (40 mg kg-1 day-1 or 80 mg kg-1 day-1) for 14 or 28 consecutive days. Then, the degree of neointima hyperplasia was evaluated by H&E staining, and the level of relevant protein expression was assessed by immunofluorescence and western blotting. In vitro, we isolated and grew primary rat aortic smooth muscle cells, which were treated with PDGF-BB and different doses of galangin, and then CCK-8 assay, wound healing assay, transwell assay, western blotting and immunofluorescence assays were performed. We found that galangin significantly inhibited PDGF-BB-induced proliferation, migration, and phenotypic switching of VSMCs and promoted autophagy in VSMCs in vitro, and galangin significantly inhibited neointimal hyperplasia after the common carotid artery balloon injury in rats. In terms of mechanisms, galangin inhibited the PI3K/AKT/mTOR pathway, thereby suppressing VSMC's switch from a contractile to a synthetic phenotype, inhibiting VSMC proliferation, migration and phenotypic switching and upregulating the Beclin1 protein expression levels and the ratio of LC3BII/I, promoting VSMC autophagy, and thereby inhibiting neointimal hyperplasia after vascular injury. Conclusion: Our study suggests that galangin inhibits neointimal hyperplasia after vascular injury by inhibiting smooth muscle cell proliferation, migration and phenotypic switching and by promoting autophagy, and that galangin may be a promising drug for the prevention and treatment of vascular restenosis after PCI.

Autophagy Is Involved in Stellate Ganglion Block Reversing Posthemorrhagic Shock Mesenteric Lymph-Mediated Vascular Hyporeactivity.

Objective: The aim of this study was to clarify the role of autophagy in stellate ganglion block (SGB) reversing posthemorrhagic shock mesenteric lymph (PHSML)-mediated vascular hyporeactivity.Methods: Hemorrhagic shock model in conscious rats was employed to observe the effects of SGB (0.2 ml of 0.25% ropivacaine hydrochloride hydrate) and autophagy inhibitor 3-methyladenine (3-MA; 30 mg/kg) on the vascular reactivity of second-order rat mesenteric arteries in vitro, while the effects of PHSML (1 ml/kg) and autophagy agonist rapamycin (Rapa, 10 mg/kg) on the beneficial effect of SGB were investigated. The cellular viability, contractility, and autophagy-related protein expressions in vascular smooth muscle cells (VSMCs) were detected following treatments of PHSML, PHSML obtained from the rats that underwent hemorrhagic shock plus SGB (PHSML-SGB), and PHSML plus 3-MA (5 mM), respectively.Results: Hemorrhagic shock significantly decreased the vascular reactivity to gradient norepinephrine (NE), which is reversed by the SGB treatment and 3-MA administration. On the contrary, PHSML intravenous infusion and Rapa administration inhibited the vascular contractile responses in rats that underwent hemorrhagic shock plus SGB treatment. PHSML treatment significantly inhibited the cellular viability and contractility in VSMCs, increased the expressions of LC3-II and Beclin 1, and decreased the expression of p62, along with opposite appearances in these indices following PHSML-SGB treatment. In addition, 3-MA counteracted the adverse roles of PHSML in these indices in VSMCs.Conclusion: SGB inhibits PHSML-mediated vascular hyporeactivity by reducing the excessive autophagy in VSMCs.

Decreased phosphorylation facilitates the degradation of the endogenous protective molecule c-Ski in vascular smooth muscle cells

The dysfunction of vascular smooth muscle cells (VSMCs) is critical for atherosclerosis (AS) progression. Autophagy is indispensable during phenotypic switching and proliferation of VSMCs, contribute to AS development. Cellular Sloan-Kettering Institute (c-Ski), the repressor of TGF-β signaling, is involved in diverse physiological and pathological processes. We previously defined c-Ski also as an endogenous protective molecule against AS via inhibiting abnormal proliferation and autophagy of VSMCs. However, the endogenous level of c-Ski in VSMCs is markedly decreased during the progression of AS, so that the protective effect is drastically weakened. Elucidating the molecular mechanisms is key to the understanding of AS development and treatment. We determined that oxidized low-density lipoprotein (ox-LDL) and platelet-derived growth factor (PDGF) directly induced the degradation of c-Ski protein, closely associated with reducing its phosphorylation. Serine383 (S383) was identified as the crucial phosphorylation site for stabilizing protein expression and nuclear location of c-Ski, which was responsible for its transcriptional suppression of autophagy-related genes. Decreased S383 phosphorylation facilitated nuclear export and degradation of c-Ski, thereby lessened its inhibitory effect on induction of autophagy genes. These findings provide a novel view of c-Ski modification and function modulation under some vascular injury factors, which point to a new potential therapeutic strategy by targeting c-Ski.

Circadian misalignment promotes vascular smooth muscle cell apoptosis via defective autophagy.

Defective autophagy in vascular smooth muscle cells (VSMCs) in response to oxidative stress can lead to cellular apoptosis and plaque instability. Previous studies have revealed that the circadian clock system is involved in autophagic regulation and plaque progression. However, the mechanism by which circadian rhythmicity influences VSMC autophagy and plaque stability remains unclear. Our study described the circadian profiles in atheromatous plaques and verified the role of circadian misalignment in VSMC autophagy and apoptosis. We found that the mRNA expression levels of circadian locomotor output cycles protein kaput (CLOCK) and Beclin 1 were significantly decreased in unstable plaques compared with stable plaques. No significant differences were observed in other circadian rhythm genes. VSMCs treated with oxidized low-density lipoprotein (ox-LDL, 80μg/ml) exhibited abnormal circadian rhythmicity and impaired autophagy, as evidenced by consistent decreases in CLOCK and Beclin 1 expression, suggesting a correlation between CLOCK and autophagy. CLOCK protein expression was inhibited by ox-LDL, accompanied by defective autophagy and an increased apoptosis rates (P < 0.05). Administration of rapamycin (10nM) reversed the effect of ox-LDL on VSMC autophagy and apoptosis. Finally, CLOCK silencing led to a considerable decrease in autophagy. VSMCs with stable CLOCK silencing also showed an increased apoptosis rate. In addition, gene silencing of CLOCK in VSMCs counteracted the effects of moderate rapamycin concentrations on autophagy and apoptosis. In conclusion, these findings suggested that the CLOCK-dependent rapamycin signaling pathway is a critical mediator in ox-LDL-induced VSMCs with defective autophagy that exacerbates plaque destabilization.

Nkx2-3 induces autophagy inhibiting proliferation and migration of vascular smooth muscle cells via AMPK/mTOR signaling pathway.

Vascular remodeling and restenosis are common complications after percutaneous coronary intervention. Excessive proliferation and migration of vascular smooth muscle cells (VSMCs) play important roles in intimal hyperplasia-induced vascular restenosis. NK2 Homeobox 3 (Nkx2-3), a critical member of Nkx family, is involved in tissue differentiation and organ development. However, the role of Nkx2-3 in VSMCs proliferation and migration remains unknown. In this study, we used carotid balloon injury model and platelet-derived growth factor-BB (PDGF)-treated VSMCs as in vivo and in vitro experimental models. EdU assay and CCK-8 assay were used to detect cell proliferation. Migration was measured by scratch test. Hematoxylin and eosin staining and immunohistochemistry staining were used to evaluate the intimal hyperplasia. The autophagy level was detected by fluorescent mRFP-GFP-LC3 in vitro and by transmission electron microscopy in vivo. It was shown that Nkx2-3 was upregulated both in balloon injured carotid arteries and PDGF-stimulated VSMCs. Adenovirus-mediated Nkx2-3 overexpression inhibited intimal hyperplasia after balloon injury, and suppressed VSMCs proliferation and migration induced by PDGF. Conversely, silencing of Nkx2-3 by small interfering RNA exaggerated proliferation and migration of VSMCs. Furthermore, we found that Nkx2-3 enhanced autophagy level, while the autophagy inhibitor 3-MA eliminated the inhibitory effect of Nkx2-3 on VSMCs proliferation and migration both in vivo and in vitro. Moreover, Nkx2-3 promoted autophagy in VSMCs by activating the AMP-activated protein kinase/mammalian target of rapamycin (AMPK/mTOR) signaling pathway. These results demonstrated for the first time that Nkx2-3 inhibited VSMCs proliferation and migration through AMPK/mTOR-mediated autophagy.