Sox10 escalates vascular inflammation by mediating vascular smooth muscle cell transdifferentiation and pyroptosis in neointimal hyperplasia

1. Download : Download high-res image (187KB)
2. Download : Download full-size image

     

血管平滑肌细胞表型调节机制的研究进展

血管平滑肌细胞（VSMC）的增殖和迁移是动脉粥样硬化斑块形成、高血压和血管再狭窄的共同病理特征,而VSMC表型转化是VSMC增殖和迁移的基础,研究VSMC表型调节的分子机制,对上述疾病的防治具有重要意义。本文对VSMC表型转化的影响因素、信号转导途径和转录因子的研究进展作一综述。     

Regulation and characteristics of vascular smooth muscle cell phenotypic diversity

Vascular smooth muscle cells can perform both contractile and synthetic functions, which are associated with and characterised by changes in morphology, proliferation and migration rates, and the expression of different marker proteins. The resulting phenotypic diversity of smooth muscle cells appears to be a function of innate genetic programmes and environmental cues, which include biochemical factors, extracellular matrix components, and physical factors such as stretch and shear stress. Because of the diversity among smooth muscle cells, blood vessels attain the flexibility that is necessary to perform efficiently under different physiological and pathological conditions. In this review, we discuss recent literature demonstrating the extent and nature of smooth muscle cell diversity in the vascular wall and address the factors that affect smooth muscle cell phenotype. (Neth Heart J 2007;15:100-8.)     

Role of PCK2 in the proliferation of vascular smooth muscle cells in neointimal hyperplasia

Vascular smooth muscle cell (VSMC) proliferation is a hallmark of neointimal hyperplasia (NIH) in atherosclerosis and restenosis post-balloon angioplasty and stent insertion. Although numerous cytotoxic and cytostatic therapeutics have been developed to reduce NIH, it is improbable that a multifactorial disease can be successfully treated by focusing on a preconceived hypothesis. We, therefore, aimed to identify key molecules involved in NIH via a hypothesis-free approach. We analyzed four datasets ({"type":"entrez-geo","attrs":{"text":"GSE28829","term_id":"28829"}}GSE28829, {"type":"entrez-geo","attrs":{"text":"GSE43292","term_id":"43292"}}GSE43292, {"type":"entrez-geo","attrs":{"text":"GSE100927","term_id":"100927"}}GSE100927, and {"type":"entrez-geo","attrs":{"text":"GSE120521","term_id":"120521"}}GSE120521), evaluated differentially expressed genes (DEGs) in wire-injured femoral arteries of mice, and determined their association with VSMC proliferation in vitro. Moreover, we performed RNA sequencing on platelet-derived growth factor (PDGF)-stimulated human VSMCs (hVSMCs) post-phosphoenolpyruvate carboxykinase 2 (PCK2) knockdown and investigated pathways associated with PCK2. Finally, we assessed NIH formation in Pck2 knockout (KO) mice by wire injury and identified PCK2 expression in human femoral artery atheroma. Among six DEGs, only PCK2 and RGS1 showed identical expression patterns between wire-injured femoral arteries of mice and gene expression datasets. PDGF-induced VSMC proliferation was attenuated when hVSMCs were transfected with PCK2 siRNA. RNA sequencing of PCK2 siRNA-treated hVSMCs revealed the involvement of the Akt-FoxO-PCK2 pathway in VSMC proliferation via Akt2, Akt3, FoxO1, and FoxO3. Additionally, NIH was attenuated in the wire-injured femoral artery of Pck2-KO mice and PCK2 was expressed in human femoral atheroma. PCK2 regulates VSMC proliferation in response to vascular injury via the Akt-FoxO-PCK2 pathway. Targeting PCK2, a downstream signaling mediator of VSMC proliferation, may be a novel therapeutic approach to modulate VSMC proliferation in atherosclerosis.     

Vanin-1 pantetheinase drives smooth muscle cell activation in post-arterial injury neointimal hyperplasia

The pantetheinase vanin-1 generates cysteamine, which inhibits reduced glutathione (GSH) synthesis. Vanin-1 promotes inflammation and tissue injury partly by inducing oxidative stress, and partly by peroxisome proliferator-activated receptor gamma (PPARγ) expression. Vascular smooth muscle cells (SMCs) contribute to neointimal hyperplasia in response to injury, by multiple mechanisms including modulation of oxidative stress and PPARγ. Therefore, we tested the hypothesis that vanin-1 drives SMC activation and neointimal hyperplasia. We studied reactive oxygen species (ROS) generation and functional responses to platelet-derived growth factor (PDGF) and the pro-oxidant diamide in cultured mouse aortic SMCs, and also assessed neointima formation after carotid artery ligation in vanin-1 deficiency. Vnn1(-/-) SMCs demonstrated decreased oxidative stress, proliferation, migration, and matrix metalloproteinase 9 (MMP-9) activity in response to PDGF and/or diamide, with the effects on proliferation linked, in these studies, to both increased GSH levels and PPARγ expression. Vnn1(-/-) mice displayed markedly decreased neointima formation in response to carotid artery ligation, including decreased intima:media ratio and cross-sectional area of the neointima. We conclude that vanin-1, via dual modulation of GSH and PPARγ, critically regulates the activation of cultured SMCs and development of neointimal hyperplasia in response to carotid artery ligation. Vanin-1 is a novel potential therapeutic target for neointimal hyperplasia following revascularization.     

Exercise training attenuates coronary smooth muscle phenotypic modulation and nuclear Ca2+ signaling

Physical inactivity is an independent risk factor for coronary heart disease, yet the mechanism(s) of exercise-related cardioprotection remains unknown. We tested the hypothesis that coronary smooth muscle after exercise training would have decreased mitogen-induced phenotypic modulation and enhanced regulation of nuclear Ca(2+). Yucatan swine were endurance exercise trained (EX) on a treadmill for 16-20 wk. EX reduced endothelin-1-induced DNA content by 40% compared with sedentary (SED) swine (P < 0.01). EX decreased single cell peak endothelin-1-induced cytosolic Ca(2+) responses compared with SED by 16% and peak nuclear Ca(2+) responses by 33% (P < 0.05), as determined by confocal microscopy. On the basis of these results, we hypothesized that sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) and intracellular Ca(2+) stores in native smooth muscle are spatially localized to dissociate cytosolic Ca(2+) and nuclear Ca(2+). Subcellular localization of SERCA in living and fixed cells revealed a distribution of SERCA near the sarcolemma and on the nuclear envelope. These results show that EX enhances nuclear Ca(2+) regulation, possibly via SERCA, which may be one mechanism by which coronary smooth muscle cells from EX are less responsive to mitogen-induced phenotypic modulation.     

Bone marrow-derived mesenchymal stem cells inhibit vascular smooth muscle cell proliferation and neointimal hyperplasia after arterial injury in rats

We investigated whether mesenchymal stem cell (MSC)-based treatment could inhibit neointimal hyperplasia in a rat model of carotid arterial injury and explored potential mechanisms underlying the positive effects of MSC therapy on vascular remodeling/repair. Sprague-Dawley rats underwent balloon injury to their right carotid arteries. After 2 days, we administered cultured MSCs from bone marrow of GFP-transgenic rats (0.8 × 10⁶ cells, n = 10) or vehicle (controls, n = 10) to adventitial sites of the injured arteries. As an additional control, some rats received a higher dose of MSCs by systemic infusion (3 × 10⁶ cells, tail vein; n = 4). Local vascular MSC administration significantly prevented neointimal hyperplasia (intima/media ratio) and reduced the percentage of Ki67 + proliferating cells in arterial walls by 14 days after treatment, despite little evidence of long-term MSC engraftment. Notably, systemic MSC infusion did not alter neointimal formation. By immunohistochemistry, compared with neointimal cells of controls, cells in MSC-treated arteries expressed reduced levels of embryonic myosin heavy chain and RM-4, an inflammatory cell marker. In the presence of platelet-derived growth factor (PDGF-BB), conditioned medium from MSCs increased p27 protein levels and significantly attenuated VSMC proliferation in culture. Furthermore, MSC-conditioned medium suppressed the expression of inflammatory cytokines and RM-4 in PDGF-BB-treated VSMCs. Thus, perivascular administration of MSCs may improve restenosis after vascular injury through paracrine effects that modulate VSMC inflammatory phenotype.     

Myricetin suppresses the proliferation and migration of vascular smooth muscle cells and inhibits neointimal hyperplasia via suppressing TGFBR1 signaling pathways

BackgroundNeointimal formation, mediated by the proliferation and migration of vascular smooth muscle cells (VSMCs), is a common pathological basis for atherosclerosis and restenosis. Myricetin, a natural flavonoid, reportedly exerts anti-atherosclerotic effects. However, the effect and mechanism of myricetin on VSMCs proliferation and migration and neointimal hyperplasia (NIH) remain unknown.PurposeWe investigated myricetin's effect on NIH, as well as the potential involvement of transforming growth factor-beta receptor 1 (TGFBR1) signaling in mediating myricetin's anti-atherosclerotic and anti-restenotic actions.MethodsMyricetin's effects on the proliferation and migration of HASMCs and A7R5 cells were determined by CCK-8, EdU assays, wound healing, Transwell assays, and western blotting (WB).Molecular docking, molecular dynamics (MD) simulation, surface plasmon resonance (SPR) and TGFBR1 kinase activity assays were employed to investigate the interaction between myricetin and TGFBR1. An adenovirus vector encoding TGFBR1 was used to verify the effects of myricetin. In vivo, the left common carotid artery (LCCA) ligation mouse model was adopted to determine the impacts of myricetin on neointimal formation and TGFBR1 activation.ResultsMyricetin dose-dependently inhibited the migration and proliferation in VSMCs, suppressed the expression of CDK4, cyclin D3, MMP2, and MMP9. Molecular docking revealed that myricetin binds to key regions for TGFBR1 antagonist binding, and the binding energy was -9.61 kcal/mol. MD simulation indicated stable binding between TGFBR1 and myricetin. Additionally, SPR revealed an equilibrium dissociation constant of 4.35 × 10⁻⁵ M between myricetin and TGFBR1. According to the TGFBR1 kinase activity assay, myricetin directly inhibited TGFBR1 kinase activity (IC₅₀ = 8.551 μM). Furthermore, myricetin suppressed the phosphorylation level of TGFBR1, Smad2, and Smad3 in a dose-dependent pattern, which was partially inhibited by TGFBR1 overexpression. Consistently, TGFBR1 overexpression partially rescued the suppressive roles of myricetin on VSMCs migration and proliferation. Moreover, myricetin dramatically inhibited NIH and reduced TGFBR1, Smad2, and Smad3 phosphorylation in the LCCA.ConclusionThis is the first study to demonstrate that myricetin suppresses NIH and VSMC proliferation and migration via inhibiting TGFBR1 signaling. Myricetin can be developed as a potential therapeutic candidate for treating atherosclerosis and vascular restenosis.     

The role of lysophosphatidic acid receptors in phenotypic modulation of vascular smooth muscle cells

Lysophosphatidic acid (LPA) is a bioactive lipid with diverse physiological effects via activation of G protein-coupled receptors (GPCRs). It has been implicated as a specific dedifferentiation factors that can promote phenotypic modulation of cultured vascular smooth muscle cells (VSMCs) which is critically involved in various vascular disease. However, the role of LPA receptors and details of their signaling in LPA induced phenotypic modulation are largely unexplored. In this study we detect the expression of LPA1 and LPA3 in rat aortic smooth muscle cells (RASMCs). LPA promoted RASMCs phenotypic modulation in a dose-dependent manner and coordinated induced the phosphorylation of p38 mitogen-activated protein kinase (p38MAPK) and extracellular signal-regulated kinase (ERK). LPA-induced cell phenotypic modulation was significantly inhibited by specific LPA1/LPA3-receptor antagonist dioctyl-glycerol pyrophosphate (DGPP8:0) at concentration, but this inhibitive effect was lost when the antagonist was coadministered with a highly selective LPA3 agonist,1-oleoyl-2-Omethyl-rac-glycero-phosphothionate (OMPT). In addition, pertussis toxin (PTX), a Gi protein inhibitor had little affect on the LPA-induced phenotypic modulation in RASMC. These data suggest that LPA-induced phenotypic modulation is mediated through the PTX-insensitive G-protein(s), possibly Gq-coupled LPA3 receptor.     

Dual role of PKA in phenotypic modulation of vascular smooth muscle cells by extracellular ATP

Extracellular ATP is released from activated platelets and endothelial cells and stimulates proliferation of vascular smooth muscle cells (VSMC). We found that ATP stimulates a profound but transient activation of protein kinase A (PKA) via purinergic P2Y receptors. The specific inhibition of PKA by adenovirus-mediated transduction of the PKA inhibitor (PKI) attenuates VSMC proliferation in response to ATP, suggesting a positive role for transient PKA activation in VSMC proliferation. By contrast, isoproterenol and forskolin, which stimulate a more sustained PKA activation, inhibit VSMC growth as expected. On the other hand, the activity of serum response factor (SRF) and the SRF-dependent expression of smooth muscle (SM) genes, such as SM--actin and SM22, are extremely sensitive to regulation by PKA, and even transient PKA activation by ATP is sufficient for their downregulation. Analysis of the dose responses of PKA activation, VSMC proliferation, SRF activity, and SM gene expression to ATP, with or without PKI overexpression, suggests the following model for the phenotypic modulation of VSMC by ATP, in which the transient PKA activation plays a critical role. At low micromolar doses, ATP elicits a negligible effect on DNA synthesis but induces profound SRF activity and SM gene expression, thus promoting the contractile VSMC phenotype. At high micromolar doses, ATP inhibits SRF activity and SM gene expression and promotes VSMC growth in a manner dependent on transient PKA activation. Transformation of VSMC by high doses of ATP can be prevented and even reversed by inhibition of PKA activity. adenosine triphosphate; purinergic receptors; protein kinase A; serum response factor; proliferation; -actin; SM22     

Changes in three-dimensional architecture of microfilaments in cultured vascular smooth muscle cells during phenotypic modulation

To investigate changes in the three-dimensional microfilament architecture of vascular smooth muscle cells (SMC) during the process of phenotypic modulation, rabbit aortic SMCs cultured under different conditions and at different time points were either labelled with fluorescein-conjugated probes to cytoskeletal and contractile proteins for observation by confocal laser scanning microscopy, or extracted with Triton X-100 for scanning electron microscopy. Densely seeded SMCs in primary culture, which maintain a contractile phenotype, display prominent linear myofilament bundles (stress fibres) that are present throughout the cytoplasm with alpha-actin filaments predominant in the central part and beta-actin filaments in the periphery of the cell. Intermediate filaments form a meshed network interconnecting the stress fibres and linking directly to the nucleus. Moderately and sparsely seeded SMCs, which modulate toward the synthetic phenotype during the first 5 days of culture, undergo a gradual redistribution of intermediate filaments from the perinuclear region toward the peripheral cytoplasm and a partial disassembly of stress fibres in the central part of the upper cortex of the cytoplasm, with an obvious decrease in alpha-actin and myosin staining. These changes are reversed in moderately seeded SMCs by day 8 of culture when they have reached confluence. The results reveal two changes in microfilament architecture in SMCs as they undergo a change in phenotype: the redistribution of intermediate filaments probably due to an increase in synthetic organelles in the perinuclear area, and the partial disassembly of stress fibres which may reflect a degradation of contractile components.