Coordinate regulation of matrix metalloproteinase-1 and tissue inhibitor of metalloproteinase-1 expression in human vascular smooth muscle cells

The expression of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) by human vascular smooth muscle cells (SMC) was monitored as a function of the phenotypic modulation in vitro. Cell phenotype was manipulated by varying serum concentration and cell density. Synthetic phenotype was characterized by a minimum expression of the contractile proteins and a maximal proliferation rate. Contractile phenotype was quiescent and expressed a maximal level of contractile proteins. Synthetic cells expressed the highest levels of both MMP-1 and TIMP-1 and displayed maximal collagenolytic activity. No significant change was detected in MMP-2 expression or catalytic activity. Enzyme immunoassays revealed that MMP-1 expression fell by 77+/-2.4-95+/-0.5%, and that of TIMP-1 by 34+/-0.5-59+/-1.9%, as the cells acquired a contractile phenotype. The level of the MMP-1/TIMP-1 complex was similarly reduced by 78+/-2.9-85+/-1.6%. These data demonstrate that the expression of MMP-1 and TIMP-1 are coordinately regulated with SMC phenotype.     

Coordinate Regulation of Matrix Metalloproteinase-1 and Tissue Inhibitor of Metalloproteinase-1 Expression in Human Vascular Smooth Muscle Cells

The expression of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) by human vascular smooth muscle cells (SMC) was monitored as a function of the phenotypic modulation in vitro. Cell phenotype was manipulated by varying serum concentration and cell density. Synthetic phenotype was characterized by a minimum expression of the contractile proteins and a maximal proliferation rate. Contractile phenotype was quiescent and expressed a maximal level of contractile proteins. Synthetic cells expressed the highest levels of both MMP-1 and TIMP-1 and displayed maximal collagenolytic activity. No significant change was detected in MMP-2 expression or catalytic activity. Enzyme immunoassays revealed that MMP-1 expression fell by 77 ± 2.4-95 ± 0.5%, and that of TIMP-1 by 34 ± 0.5-59 ± 1.9%, as the cells acquired a contractile phenotype. The level of the MMP-1/TIMP-1 complex was similarly reduced by 78 ± 2.9-85 ± 1.6%. These data demonstrate that the expression of MMP-1 and TIMP-1 are coordinately regulated with SMC phenotype.     

Decorin inhibition of PDGF-stimulated vascular smooth muscle cell function: potential mechanism for inhibition of intimal hyperplasia after balloon angioplasty

Decorin is a small proteoglycan that binds to transforming growth factor-beta (TGF-beta) and inhibits its activity. However, its interaction with platelet-derived growth factor (PDGF), involved in arterial repair after injury, is not well characterized. The objectives of this study were to assess decorin-PDGF and decorin-PDGF receptor (PDGFR) interactions, the in vitro effects of decorin on PDGF-stimulated smooth muscle cell (SMC) functions and the in vivo effects of decorin overexpression on arterial repair in a rabbit carotid balloon-injury model. Decorin binding to PDGF was demonstrated by solid-phase binding and affinity cross-linking assays. Decorin potently inhibited PDGF-stimulated PDGFR phosphorylation. Pretreatment of rabbit aortic SMC with decorin significantly inhibited PDGF-stimulated cell migration, proliferation, and collagen synthesis. Decorin overexpression by adenoviral-mediated gene transfection in balloon-injured carotid arteries significantly decreased intimal cross-sectional area and collagen content by approximately 50% at 10 weeks compared to beta-galactosidase-transfected or balloon-injured, non-transfected controls. This study shows that decorin binds to PDGF and inhibits its stimulatory activity on SMCs by preventing PDGFR phosphorylation. Decorin overexpression reduces intimal hyperplasia and collagen content after arterial injury. Decorin may be an effective therapy for the prevention of intimal hyperplasia after balloon angioplasty.     

The effect of interleukin-4 and amphiregulin on the proliferation of human airway smooth muscle cells and cytokine release

Airway smooth muscle (ASM) hyperplasia and angiogenesis are important features associated with airway remodeling. We investigated the effect of IL-4 and amphiregulin, an epidermal growth factor family member, on the proliferation of human ASM cells and on the release of vascular endothelial growth factor (VEGF) and monocyte chemotactic protein (MCP)-1 from human ASM cells. Human ASM cells were growth-arrested for 48 hr and incubated with platelet-derived growth factor (PDGF)- BB, interleukin (IL)-4, amphiregulin, and VEGF to evaluate cell proliferation. The cells were treated with PDGF, IL-4 and amphiregulin to evaluate the release of VEGF, MCP-1. IL-4 suppressed unstimulated and PDGF-stimulated ASM cell proliferation. Amphiregulin stimulated ASM cell proliferation in a dose-dependent manner. VEGF did not have any influence on ASM cell proliferation. IL-4 stimulated VEGF secretion by the ASM cells in a dose-dependent manner and showed added stimulatory effects when co-incubated with PDGF. Amphiregulin did not promote VEGF secretion. IL-4 and amphiregulin showed no stimulatory effects on MCP-1 secretion. The results of this study showed that IL-4 had bifunctional effects on airway remodeling, one was the suppression of the proliferation of the ASM cells and the other was the promotion of VEGF release by the ASM cells, and amphiregulin can promote human ASM cell proliferation.     

Oxygen- and glucose-deprived culture promotes cell proliferation and invasion of vascular smooth muscle cells

Excessive proliferation of vascular smooth muscle cells (VSMCs) is an important reason for the formation and development of many vascular remodeling diseases. In pathological conditions, necrosis of VSMCs may result in the release of inflammatory cytokines, which can lead to stimulation of other normal smooth muscle cells, and promote the proliferation of VSMCs. The purpose of this study was to investigate the effect of oxygen- and glucose-deprived (OGD) conditioned medium on VSMC cell proliferation and invasion. Following culture of VSMCs in OGD-conditioned medium, the cell cycle distributions were remarkably altered. The number of cells in the G0/G1 phase decreased, while the number of cells in G2/M and S phase increased. The expression of cell cycle proteins D1 (Cyclin D1) in VSMCs increased correspondingly. These results suggested that after being cultured in OGD medium, VSMCs can pass through the G0/G1 phase by up-regulation of Cyclin D1 expression, and promote cell proliferation. In addition, we found that the expression of matrix metalloproteinase (MMP)-2 and MMP-9 was increased in OGD medium cultured VSMCs. Using a Transwell invasion assay, we showed that the OGD medium enhanced VSMC cell invasion. These results suggest that MMP-2 and MMP-9 degraded the basement membrane and promoted VSMC invasion. Taken together, our data demonstrate that OGD-conditioned medium can promote VSMC proliferation and invasion by up-regulating Cyclin D1 and MMP-2 and MMP-9 expression, which may contribute to the formation and development of vascular remodeling diseases.     

AcSDKP对大鼠心成纤维细胞MMP-2、MMP-9活性和MMP-1表达的影响

目的探讨N-乙酰基-丝氨酰-天门冬酰-赖氨酰-脯氨酸(AcSDKP)对10%血清和血小板源性生长因子(PDGF)诱导的大鼠心成纤维细胞MMP-2、MMP-9活性和MMP-1表达的调节作用。方法明胶酶谱法检测心成纤维细胞MMP-2、MMP-9的活性。Western blot法检测心成纤维细胞MMP-1的表达。结果10%血清和PDGF使心成纤维细胞MMP-2、MMP-9活性增强,也促进MMP-1的表达;AcSDKP能够进一步增加由10%血清和PDGF诱导的心成纤维细胞MMP-2、MMP-9的活性,并促进MMP-1的表达。结论AcSDKP上调了由PDGF介导的心成纤维细胞MMPs活性或表达,这可能与AcSDKP抗心肌纤维化的作用相关。     

N-乙酰基-丝氨酰-天门冬酰-赖氨酰-脯氨酸对血小板洐生生长因子介导的NIH3T3细胞增殖的抑制调节作用

目的：探讨N-乙酰基-丝氨酰-天门冬酰-赖氨酰-脯氨酸（AcSDKP）对血小板洐生生长因子（PDGF）介导的NIH3T3细胞增殖的抑制调节作用。方法：采用MTT法和免疫组化法检测NIH3T3细胞增殖和增殖细胞核抗原（PCNA）的表达。结果：在10^-10～10^-8mol／L浓度范围内。AcSDKP对PDGF介导的NIH3T3细胞增殖均有抑制作用。并在10^-9mol／L浓度时其抑制作用最佳。PDGF对NIH3T3细胞PCNA的表达具有增强作用．而AcSDKP对PDGF介导的NIH3T3细胞PCNA的表达有显著抑制作用。结论：AcSDKP对PDGF介导的NIH3T3细胞增殖有明显抑制作用。     

Emodin inhibits tumor necrosis factor-α-induced migration and inflammatory responses in rat aortic smooth muscle cells

Emodin, a naturally occurring anthraquinone derivative in oriental herbal medicine, has been shown to exert a variety of pharmacological activities. The goal of this study was to determine the effects of emodin on the modulation of cell proliferation, migration, inflammatory responses, and matrix metalloproteinase (MMP)-2 and MMP-9 expression in tumor necrosis factor (TNF)-α-induced rat aortic smooth muscle cells (RASMCs). Cell proliferation and migration were measured using the MTT assay and the transwell chamber assay, respectively. Quantitative real-time PCR and western blot analysis were used to detect MMP expression. Gel shift was used for analysis of nuclear factor (NF)-κB activation. In addition, the expression of several inflammatory genes was also analyzed. Treatment of RASMCs with emodin significantly and dose-dependently attenuated TNF-α-induced proliferation, migration, mRNA and protein expression of MMP-2 and MMP-9, and NF-κB activation. Furthermore, emodin significantly inhibited TNF-α-evoked inflammatory responses, as demonstrated by the reduction in the expression of inflammatory genes. These results suggest that emodin inhibits TNF-α-induced proliferation, migration, MMP-2 and MMP-9 expression as well as inflammatory responses in cultured RASMCs, supporting the notion that emodin may have potential application in clinical atherosclerosis disease.     

Regulation and function of an activation-dependent epitope of the beta 1 integrins in vascular cells after balloon injury in baboon arteries and in vitro.

Migration and proliferation of endothelial cells (ECs) and smooth muscle cells (SMCs) contribute to the response to injury in damaged and atherosclerotic vessels. These events might be regulated by cellular interactions with extracellular matrix through the expression and activation of integrins. To study the functions of beta 1 integrins in the vessel wall, we used monoclonal antibody (MAb) 15/7, which recognizes an activation epitope of beta 1 integrin subunits, and MAb 8A2, which induces a high affinity form of beta 1 integrins recognized by MAb 15/7. Immunohistochemical analyses were done on samples of normal baboon saphenous arteries and from arteries subjected to balloon injury. EC and SMC expressed the activation epitope of beta 1 integrin in uninjured arteries. By contrast, in balloon-injured arteries 6 weeks after injury, regenerating EC did not express the activation epitope, and there was no decrease in the expression of total beta 1 integrin, whereas SMC migrating into the intima exhibited decreased expression of the total and activated beta 1 integrin. Flow cytometer analysis of cultured cells indicated that baboon EC and SMC weakly express the activation epitope of beta 1 integrin. Next, we determined by utilizing MAb 8A2 the effects of increased expression of activation epitope of beta 1 integrin on the functions of SMC and EC. The activation of beta 1 integrins on SMC induced by MAb 8A2 enhanced SMC adhesion and suppressed SMC migration in a Boyden chamber assay. SMC proliferation was inhibited by MAb 8A2 dose-dependently. Similarly, MAb 8A2-induced activation of beta 1 integrins on EC suppressed EC migration into a wound. However, MAb 8A2 did not affect the basic fibroblast growth factor-induced proliferation of EC, although it blocked the decrease in EC number caused by the removal of basic fibroblast growth factor. These results suggest that activation of beta 1 integrins in vascular cells is regulated in a cell-type dependent manner and plays an important role in modulating vascular cell functions.     

胡椒碱抑制AngⅡ诱导大鼠气道平滑肌细胞增殖和迁移

目的探究胡椒碱对血管紧张素Ⅱ(AngⅡ)对大鼠气道平滑肌细胞(ASMCs)增殖和迁移的影响。方法用改良组织块和胰蛋白酶消化联合培养原代细胞ASMCs。MTT检测AngⅡ及其受体拮抗剂losartan对细胞增殖活性的影响。AngⅡ和不同浓度胡椒碱作用ASMCs后,MTT、流式细胞术和Transwell分别检测细胞增殖、细胞周期以及细胞迁移;ERK1/2抑制剂PD98059和losartan干预后,Western blot检测cyclin D1、MMP-9、p-ERK1/2、ERK1/2和β-actin等蛋白表达。结果 AngⅡ(10~(-8)、10~(-7)、10~(-6)和10~(-5)mol/L)作用24 h后,可浓度依赖性地促进ASMCs增殖(P0.05),其中10~(-7)mol/L AngⅡ促进效果最为显著;losartan处理则抑制AngⅡ诱导的ASMCs增殖(P0.05)。10~(-7)mol/L AngⅡ处理组ASMCs增殖活性、S期细胞分布比例、细胞迁移数目和蛋白质(cyclin D1、MMP-9和p-ERK1/2)表达均明显增加(P0.05);而胡椒碱(10、25、50和100μmol/L)处理可浓度依赖性地减轻AngⅡ诱导的上述效应。并且,PD98059和losartan亦能阻断AngⅡ对ASMCs p-ERK1/2、cyclin D1和MMP-9的上调作用。结论胡椒碱能通过ERK1/2通路抑制AngⅡ诱导的大鼠ASMCs增殖和迁移。     

Role of NEAT1/MiR-9-5p/SLC26A2 Pathway on Human Airway Smooth Muscle Cell

PurposeAsthma is a serious inflammatory disease of the respiratory system in which airway smooth muscle cells (ASMCs) play a key role. This study aimed to investigate the expression of SLC26A2 in human ASMCs (HASMCs) and the regulatory mechanism of SLC26A2 in the proliferation and inflammatory factor production of HASMCs.Materials and MethodsWe obtained the asthma-associated differential mRNA SLC26A2 by bioinformatics analysis in childhood acute asthma samples. To investigate its role in airway inflammation and airway remodeling, we treated HASMCs with platelet-derived growth factor (PDGF) in an in vitro model and determined SLC26A2 expression in cells using western blotting. Cell proliferation was detected by MTT and EdU assays, and cell contractile phenotype marker proteins were measured. Cell migration and production of inflammatory factors were determined by Transwell and ELISA assays. Additionally, the upstream regulatory miRNA and LncRNA of SLC26A2 were identified by bioinformatics, luciferase reporter gene, and RIP analyses.ResultsSLC26A2 was significantly upregulated in bioinformatics analysis of pediatric asthma-related sample. PDGF treatment up-regulated SLC26A2 expression in HASMCs, whereas the knockdown of SLC26A2 inhibited PDGF-stimulated proliferation, migration, and production of inflammatory factors, and enhanced the expression of cell contractile phenotype marker proteins in HASMCs. Luciferase reporter and RIP experiments validated that NEAT1 targeted miR-9-5p to regulate SLC26A2, thereby influencing the biological function of PDGF-induced HASMCs.ConclusionThese findings indicate that NEAT1-mediated miR-9-5p targeting of SLC26A2 inhibits the PDGF-induced proliferation and production of inflammatory factors in HASMCs. These findings highlight potential therapeutic targets for asthma and airway inflammation.     

血小板源生长因子对大鼠肝层星状细胞增殖和胶原及血小板源 …

目的 证实血小板源生长因子（ＰＤＧＦ）对体外培养大鼠肝星状细胞增殖和胶原基因表达及ＰＤＧＦ自分泌的作用。方法 ⑴采用完全无血清培养,用＾３Ｈ－ＴｄＲ掺入检测了ＰＤＧＦ、转化生长因子β１（ＴＧＦ－β１）和表皮生长因子（ＥＧＦ）对肝星状细胞的ＤＮＡ合成作用;⑵应用Ｎｏｒｔｈｅｒｎ分子杂交检测了ＰＤＧＦ对肝星状细胞的Ⅰ、Ⅲ型前胶原及ＰＤＧＦ－Ｂ等ｍＲＮＡ表达水平的影响。结果 ＰＤＧＦ、ＥＧＦ均可剂量依赖     

供体年龄影响融合生长内皮祖细胞对平滑肌细胞表型转换及增殖迁移的作用

目的:探讨老龄和年轻个体来源融合生长状态内皮祖细胞(EPCs)对血管平滑肌细胞(SMCs)表型转换以及增殖和迁移的调节作用。方法:脱臼处死1~2月龄、19~26月龄SD大鼠,应用含15%FBS的DMEM/F12培养基(含内皮细胞生长添加剂100 mg/L、肝素100 mg/L、青霉素、链霉素各1×105U/L)培养EPCs,取1~2月龄大鼠腹主动脉,组织块法培养血管SMCs,应用Di I-Ac-LDL与FITC-UEA-1荧光双染以及α-SM-actin免疫荧光分别对EPCs和SMCs进行鉴定。建立细胞共培养体系,上室为融合生长状态的EPCs,下室为SMCs,实验分4组:(1)第3代SMCs(P3)组;(2)第4代SMCs(P4)组;(3)第4代SMCs与年轻大鼠来源EPCs共培养(P4YE)组;(4)第4代SMCs与老龄大鼠来源EPCs共培养(P4AE)组。Western blotting检测α-SM-actin和osteopontin蛋白的表达;[3H]-TdR掺入法检测SMCs增殖;细胞划痕实验检测SMCs的迁移能力。结果:与P3组相比,P4组的SMCsα-SM-actin表达显著下调,而osteopontin表达显著增强;P4YE组SMCs的α-SM-actin及osteopontin表达与P3组比较未见有显著差别;与P4组相比,年轻和老龄大鼠来源的EPCs均显著促进第4代SMCs的α-SM-actin和下调osteopontin的表达,抑制第4代SMCs的增殖和迁移;与老龄大鼠来源的EPCs相比,年轻大鼠来源的EPCs更能够显著延迟SMCs表型由收缩型向合成型转换,抑制SMCs增殖和迁移。结论:共培养融合生长状态的EPCs使血管SMCs表型转换延迟、抑制SMCs增殖和迁移,年轻大鼠来源的EPCs较老龄大鼠来源的EPC更显著延迟血管SMCs表型由收缩型向合成型转换,并具有更强的抑制血管SMCs增殖和迁移的能力。     

低氧对肺血管周细胞的影响

目的 研究低氧直接或通过内皮细胞的介导对体外培养的肺血管周细胞的能量代谢。细胞周期和血小板源性生长因子及其受体ｍＲＮＡ表达的影响。方法 应用 ＭＴＴ比色分析法,流式细胞术,核酸原位分子杂交及图像分析等方法进行定量分析。结果 低氧量直接或通过内皮细胞介导,促进肺血管周期由静止期进入ＤＮＡ合成期及有丝分裂期,且能促进周细胞ＰＤＧＦ及其受体ｍＲＮＡ的表达。     

Retinoic acid inhibits airway smooth muscle cell migration

Airway remodeling in chronic asthma is characterized by increased smooth muscle mass that is associated with the reduction of the bronchial lumen as well as airway hyperresponsiveness. The development of agents that inhibit smooth muscle growth is therefore of interest for therapy to prevent asthma-associated airway remodeling. All-trans retinoic acid (ATRA) suppresses growth of vascular smooth muscle cells (SMCs) from the systemic and pulmonary circulation. The present study investigated the effects of ATRA on human bronchial (airway) SMCs. Human bronchial SMCs were found to express mRNAs for retinoic acid receptor (RAR)-alpha, -beta, -gamma, and retinoid X receptor (RXR)-alpha, -beta, but not RXR-gamma. Although ATRA was not effective in inhibiting proliferation or in inducing apoptosis in airway SMCs, we found that ATRA (0.2-2 microM) inhibited the SMC migration in response to platelet-derived growth factor (PDGF), as determined in a modified Boyden chamber assay. Both RAR and RXR agonists also blocked PDGF-induced airway SMC migration. ATRA also inhibited PDGF-induced actin reorganization associated with migration. PDGF-induced actin reorganization and migration were blocked by inhibitors of phosphatidylinositol 3 kinase (PI3K) and Akt. However, migration was blocked by inhibitors of the MEK/ERK pathway, with no effect on cytoskeletal reorganization. ATRA suppressed PDGF-induced Akt activation without influencing ERK activation. RAR was found to form protein-protein interactions with the p85 PI3K subunit. These results suggest that retinoic acid inhibits airway SMC migration through the modulation of the PI3K/Akt pathway.     

Inflammatory regulation of extracellular matrix remodeling in calcific aortic valve stenosis.

BACKGROUND: Calcific aortic stenosis (AS), the most frequent heart valve disorder in developed countries, leads to the calcification and fibrous thickening of the valve. While several studies have addressed the process of valvular calcification, the molecular pathomechanisms of the extensive matrix remodeling remain unclear. Because inflammation is present in stenotic valves, we hypothesized that the proinflammatory cytokine tumor necrosis factor alpha (TNFalpha) might influence cell proliferation and regulate the expression and activation of matrix metalloproteinases (MMPs)--enzymes that are thought to be involved in calcific AS. METHODS: Immunohistochemistry for leukocytes, TNFalpha, MMP-1, and the endogenous MMP inhibitor tissue inhibitor of metalloproteinase (TIMP)-1 was performed on human stenotic (n = 19) and control (n = 8) valves. Primary cultures of human aortic valve myofibroblasts were incubated with and without TNFalpha, and cell proliferation was assessed. The expression and activation of MMP-1 were detected by Western blotting and a specific MMP-1 activity assay. RESULTS: Control valves showed scattered macrophages and low expression of TNFalpha, MMP-1, and TIMP-1. In stenotic valves, leukocyte infiltration and a strong, colocalized expression of TNFalpha and MMP-1 were present, while TIMP-1 remained unchanged. Double-label immunofluorescence localized TNFalpha mainly to macrophages. In cultured human aortic valve myofibroblasts, TNFalpha stimulated proliferation and induced a time-dependent increase in MMP-1 expression and activation, while TIMP-1 remained unchanged. CONCLUSION: The results indicate that matrix remodeling in calcific AS involves the expression and activation of MMPs. Activated leukocytes, by the secretion of TNFalpha, may stimulate valvular myofibroblasts to proliferate and express MMPs, thus regulating actively the matrix remodeling in calcific AS.