Migration of human vascular endothelial and smooth muscle cells.

Migration of endothelial and smooth muscle cells was studied in vitro by measuring the increase in surface area at specific time intervals of confluent cell colonies advancing under agarose gels that contained both Morgan's medium 199 and variuos types of sera. First passage cultures of endothelial cells or 3 to 6 passage smooth muscle cells were plated into wells punched in agarose gels, at a seeding density of 50,000 cells per well. At zero time the size of the cell colonies was 35.4 sq. mm. +/- standard error 0.1. Irradiation (1500 rads) did not affect the expansion of the cell colonies although 3H-thymidine uptake was inhibited. Endothelial cells migrated under the agarose gels concentrically as contiguous sheets. When exposed to either 20 per cent platelet-poor plasma serum, platelet-rich plasma serum, or whole blood serum, the average increase in surface area was approximately 9 sq. mm. per day. In contrast, arterial smooth muscle cell colonies expanded with an increment of approximately 9 sq. mm. per day when exposed to 10 per cent platelet-poor plasma serum but 12 sq. mm. per day when exposed to 10 per cent platelet-rich plasma serum (p less than 0.001). Platelet factors also had stimulatory effects on the migration of venous smooth muscle cells. Cytochalasin B, dibutyryl cyclic AMP, and theophylline inhibited the migration of both endothelial and smooth muscle cells, but the latter responded more to the inhibitory effects of all three agents. It is concluded that in contrast to vascular smooth muscle, endothelial cells do not require platelet factors for migration and are less responsive to specific inhibitors affecting cell movement.     

The role of ERK signaling in protein hydrogel remodeling by vascular smooth muscle cells

Collagen type I and fibrin hydrogels have been used for cell-based therapies and tissue engineering. These matrices can be broken down and remodeled by cells, but the effects that these proteins have on cell function are not completely understood. We examined activation of the extracellular signal-regulated kinase (ERK) signaling pathway by vascular smooth muscle cells (VSMC) in response to 2D and 3D matrices of type I collagen, fibrin, or a 1:1 composite mixture of these proteins. After 3 days of culture, ERK phosphorylation, osteopontin secretion, and MMP-2 activation were all markedly increased in 3D matrices, compared with 2D substrates. A strong positive correlation existed between these protein markers of the synthetic phenotype and phosphorylated ERK levels, and this relationship persisted across matrix geometries and compositions. Cell proliferation in 3D matrices was inversely correlated to ERK activation, while on 2D substrates a modest positive correlation was observed. Pharmacologic inhibition of ERK signaling confirmed that this pathway was involved in the observed phenotype shifts. This study suggests that contextual activation of the ERK pathway results in different effects on cell phenotype, depending on the geometry and composition of the ECM. These findings add to our understanding of cell function and remodeling in protein-based hydrogel biomaterials.     

Molecular responses of vascular smooth muscle cells and phagocytes to curcumin-eluting bioresorbable stent materials

A major complication of coronary stenting is restenosis, often accompanied by inflammatory reactions and smooth muscle cell proliferation. Curcumin has been shown to possess anti-inflammatory and anti-proliferative properties, thus we hypothesize that locally released curcumin by coronary stent would diminish in-stent restenosis. As a first test of this hypothesis, curcumin-eluting PLLA films (C-PLLA) were produced and the anti-inflammatory and anti-proliferative properties were then tested using peritoneal phagocytes and human coronary artery smooth muscle cell (hCASMCs) culture systems. We find that the addition of curcumin reduced phagocyte accumulation and activation on C-PLLA films. On the other hand, C-PLLA significantly reduced the proliferation, but not the adhesion, of hCASMCs. The molecular responses of hCASMCs to C-PLLA were further assessed by cDNA microarray analysis. Curcumin up-regulated genes related to apoptosis and enhanced the expression of anti-proliferative and anti-inflammatory factors, and of antioxidants. Equally important, C-PLLA inhibited the cell cycle progression of adherent hCASMCs. The results suggest that curcumin regulates gene expression and cell function through the protein kinase (PK) and mitogen-activated protein kinase (MAPK) pathways. These results support the use of curcumin to inhibit in-stent restenosis.     

剪切力条件下血管内皮细胞与平滑肌细胞的相互作用

血管内皮细胞(endothelial cells,ECs)与平滑肌细胞(smooth muscle cells,SMCs)是血管壁的主要细胞,它们之间的相互作用在维持血管正常的生理功能以及心血管疾病的发生发展过程中至关重要。为真实模拟ECs与SMCs体内条件下的位置关系与生长状态,人们建立了多种共培养系统。介绍当前几种常用的能够加载流动剪切力的共培养系统,并分别比较其优势与不足;简要总结剪切力条件下ECs与SMCs相互作用对ECs与SMCs表型与分布、SMCs生长与迁移、ECs表面相关黏附分子表达的影响。研究表明,一氧化氮(NO)、细胞因子、microRNA等可以作为信号分子介导ECs与SMCs之间的相互作用。     

Aortic smooth muscle cells express and secrete vascular endothelial growth factor.

We examined whether cultured bovine aortic smooth muscle (ASM) cells express VEGF. RNA blot analysis of total cellular RNA derived from ASM cells demonstrates the expression of the VEGF gene. ASM cells release in the medium a VEGF-like endothelial cell mitogen which binds to heparin-sepharose and has an apparent molecular weight of 40-45 kDa as assessed by an HPLC gel filtration column. Consistent with VEGF, this mitogen does not stimulate the proliferation of ASM cells. Immunoblot analysis of the bioactive material with an antibody specific for VEGF demonstrates the presence of a major immunoreactive band with an apparent molecular mass of 23 kDa and a minor band with a molecular mass of approximately 18 kDa, in reducing conditions. The major band has very similar apparent molecular weight as the 165 amino-acid species of human recombinant VEGF of folliculo-stellate cells derived VEGF. These data demonstrate the expression and synthesis of VEGF by cultured ASM cells and suggest that the 164 amino-acid species is the predominant molecular form of the growth factor secreted by such cells. VEGF released by ASM cells may play a paracrine role in the maintenance of the integrity of the endothelial lining or in the abnormal proliferation of the vasa vasorum which takes place in atherosclerosis.     

Differential effects of substrate modulus on human vascular endothelial, smooth muscle, and fibroblastic cells

Regenerative medicine approaches offer attractive alternatives to standard vascular reconstruction; however, the biomaterials to be used must have optimal biochemical and mechanical properties. To evaluate the effects of biomaterial properties on vascular cells, heparinized poly(ethylene glycol) (PEG)-based hydrogels of three different moduli, 13.7, 5.2, and 0.3 kPa, containing fibronectin and growth factor were utilized to support the growth of three human vascular cell types. The cell types exhibited differences in attachment, proliferation, and gene expression profiles associated with the hydrogel modulus. Human vascular smooth muscle cells demonstrated preferential attachment on the highest-modulus hydrogel, adventitial fibroblasts demonstrated preferential growth on the highest-modulus hydrogel, and human umbilical vein endothelial cells demonstrated preferential growth on the lowest-modulus hydrogel investigated. Our studies suggest that the growth of multiple vascular cell types can be supported by PEG hydrogels and that different populations can be controlled by altering the mechanical properties of biomaterials.     

Heme oxygenase activity modulates vascular endothelial growth factor synthesis in vascular smooth muscle cells

Hypoxia, cytokines, and nitric oxide (NO) stimulate the generation of vascular endothelial growth factor (VEGF) and induce heme oxygenase-1 (HO-1) expression in vascular tissue. HO-1 degrades heme to carbon monoxide (CO), iron, and biliverdin, the latter being reduced to bilirubin by biliverdin reductase. In the present study, we investigated the role of HO-1 in the modulation of VEGF synthesis in rat vascular smooth muscle cells (VSMC). In VSMC stimulated with cytokines, inhibition of NO production significantly, but not completely, reduced VEGF release. In contrast, inhibition of HO activity by tin protoporphyrin IX (SnPPIX) totally prevented cytokine-induced increase in VEGF, despite an augmented synthesis of intracellular NO. Stimulation of HO-1 activity by hemin enhanced VEGF production; this effect was abrogated by blockade of the HO pathway. Similarly, VEGF synthesis induced by hypoxia was down-regulated by SnPPIX, but not by inhibitors of NO synthase. To elucidate further a direct involvement of HO-1 in the observed effects, we generated transfected cells that overexpressed the HO-1 gene. Notably, these cells synthesized significantly more VEGF protein than cells transfected with a control gene. Among the products of HO-1, biliverdin and bilirubin showed no effect, whereas iron ions inhibited VEGF synthesis. Exposure of cells to 1% CO resulted in a marked accumulation of VEGF (20-fold increase) over the basal level. Our data indicate that HO-1 activity influences the generation of VEGF in VSMC in both normoxic and hypoxic conditions. As CO and iron, respectively the inducer and the inhibitor of VEGF synthesis, are concomitantly produced during the degradation of heme, these data indicate that HO by-products may differentially modulate VEGF production.     

Studies on the mechanism of swelling-induced lysosomal alkalinization in vascular smooth muscle cells

Previous studies in renal cells and hepatocytes have shown that cell swelling leads to a rapid and reversible increase in pH in acidic cellular compartments, including lysosomes. Among the consequences are an inhibition of proteolysis. The present study shows that a similar lysosomal alkalinization occurs upon osmotic swelling of vascular smooth muscle cells, as evidenced by acridine orange and fluorescein isothiocyanate fluorescence. Furthermore, we have studied the mechanism underlying lysosomal alkalinization, which had remained unclear. The lysosomal alkalinization was not abolished by inhibition of vacuolar H⁺-ATPases (100 nM bafilomycin), Cl^– channels [100 M] 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), carbonic anhydrase (100 M acetazolamide) or Na⁺/H⁺ exchange (10 M HOE 694). The Ca²⁺ ionophore A23187 (10 M) led to a slight increase in lysosomal pH, but removal of extracellular Ca²⁺ and depletion of cellular Ca²⁺ stores (100 nM thapsigargin) did not appreciably blunt the swelling-induced lysosomal alkalinization. In the presence of bafilomycin the alkalinizing effect of osmotic cell swelling was not reversible, in contrast to that of NH₄Cl. In conclusion, osmotic swelling of vascular smooth muscle cells leads to lysosomal alkalinization, presumably in large part through activation of a hydrogen ion leak.     

Influence of endothelial cells on vascular smooth muscle cells phenotype after irradiation: implication in radiation-induced vascular damages

Damage to vessels is one of the most common effects of therapeutic irradiation on normal tissues. We undertook a study in patients treated with preoperative radiotherapy and demonstrated in vivo the importance of proliferation, migration, and fibrogenic phenotype of vascular smooth muscle cells (VSMCs) in radiation-induced vascular damage. These lesions may result from imbalance in the cross talk between endothelial cells (ECs) and VSMCs. Using co-culture models, we examined whether ECs influence proliferation, migration, and fibrogenic phenotype of VSMCs. In the presence of irradiated ECs, proliferation and migration of VSMCs were increased. Moreover, expressions of alpha-smooth muscle actin, connective tissue growth factor, plasminogen activator inhibitor type 1, heat shock protein 27, and collagen type III, alpha 1 were up-regulated in VSMCs exposed to irradiated ECs. Secretion of transforming growth factor (TGF)-beta1 was increased after irradiation of ECs, and irradiated ECs activated the Smad pathway in VSMCs by inducing Smad3/4 nuclear translocation and Smad-dependent promoter activation. Using small interferring RNA targeting Smad3 and a TGFbeta-RII neutralizing antibody, we demonstrate that a TGF-beta1/TGF-beta-RII/Smad3 pathway is involved in the fibrogenic phenotype of VSMCs induced by irradiated ECs. In conclusion, we show the importance of proliferation, migration, and fibrogenic phenotype of VSMCs in patients. Moreover, we demonstrate in vitro that ECs influence these fundamental mechanisms involved in radiation-induced vascular damages.     

Human vascular smooth muscle cells and endothelial cells lack catalase activity and are susceptible to hydrogen peroxide

⁵Cr release as lytic and cell detachment as nonlytic injury were employed to estimate neutrophil-mediated injury of cultured human vascular smooth muscle cells and endothelial cells. The reagents hydrogen peroxide or hypoxanthine-xanthine oxidase produced dose-dependent killing and nonlytic cell detachment, which were specifically inhibited by catalase but not by superoxide dismutase. The concentration of hydrogen peroxide or xanthine oxidase to induce cell detachment was less than lytic dose, suggesting that cell detachment was a much more sensitive assay of injury. Neutrophil-mediated cell lysis averaged 15% at most and was mostly dependent on hydrogen peroxide, while neutrophil-mediated cell detachment was nearly 100% and its dependency on hydrogen peroxide varied from 46% to 60%. These results suggest that vascular smooth muscle cells and endothelial cells in neutrophil-mediated events are destroyed by a hydrogen peroxide-dependent process, mainly via a nonlytic cell detachment mechanism. There was no striking difference of sensitivity to hydrogen peroxide between vascular smooth muscle cells and endothelial cells. Vascular smooth muscle ceils and endothelial cells contained fairly high concentrations of superoxide dismutase, but not catalase, activity. The sensitivity of these cells to hydrogen peroxide but not to superoxide may arise from the fact that these cells lack intracellular catalase activity. The injury of vascular cells, which constitute important components of blood vessels, may lead to vascular injury and subsequent tissue damage.     

血管平滑肌细胞对联合培养内皮祖细胞黏附、增殖与形态的影响

目的：探讨血管平滑肌细胞对联合培养的内皮祖细胞黏附、增殖与分化的影响。方法：从人脐血中分离纯化内皮祖细胞并鉴定；建立内皮祖细胞和平滑肌细胞的联合培养模型；细胞粘附实验测定内皮祖细胞的粘附能力；形态学观察内皮祖细胞的分化与增殖。结果：与血管平滑肌细胞联合培养的内皮祖细胞的粘附能力比单独培养时提高了63％，长梭形细胞数量增加，同时克隆形成单位的数量下降了约60％。结论：血管平滑肌细胞促进了内皮祖细胞的粘附与分化，同时抑制了内皮祖细胞的增殖能力。     

缝隙连接在血管内皮和平滑肌细胞中的作用及其与高血压的关系

细胞间的直接通讯--缝隙连接在控制和协调血管功能方面起重要作用。缝隙连接是沟通相邻细胞质膜的唯一一类通道,为离子、小分子代谢物质、第二信使等细胞间信号分子的直接交换提供一个通道。血管内皮细胞和平滑肌细胞表达多种类型缝隙连接蛋白(connexin,Cx),多种Cx 在心血管系统中的共同表达并协调一致地工作,对心血管系统的发育、平滑肌和内皮细胞功能的整合、以及对血管壁细胞功能协调等起着重要作用。在血管发生疾病时Cx表达相应发生改变,以与缝隙连接调节血管紧张度和动脉血压的作用相一致。本综述讨论了在血管内皮和平滑肌细胞在生理和病理情况下(以高血压为例)缝隙连接对血管结构和功能的影响。     

Human aortic smooth muscle cells promote arteriole formation by coengrafted endothelial cells

Collagen-fibronectin gels containing Bcl-2-transduced human umbilical vein endothelial cells (Bcl-2-HUVEC) implanted in the abdominal walls of immunodeficient mice form mature microvessels invested by host-derived smooth muscle cells (SMC) by 8 weeks. We tested the hypothesis that coengraftment of human aortic SMC (HASMC) could accelerate vessel maturation. To prevent SMC-mediated gel contraction, we polymerized the gel within a nonwoven poly(glycolic acid) (PGA) scaffold. Implanted grafts were evaluated at 15, 30, and 60 days. Acellular PGA-supported protein gels elicited a macrophage-rich foreign body reaction and transient host angiogenic response. When transplanted alone, HASMC tightly associated with the fibers of the scaffold and incorporated into the walls of angiogenic mouse microvessels, preventing their regression. When transplanted alone in PGA-supported gels, Bcl-2-HUVEC retained the ability to form microvessels invested by mouse SMC. Interestingly, grafts containing both Bcl-2-HUVEC and HASMC displayed greater numbers of smooth muscle alpha-actin-expressing cells associated with human EC-lined arteriole-like microvessels at all times examined and showed a significant increase in the number of larger caliber microvessels at 60 days. We conclude that SMC coengraftment can accelerate vessel development by EC and promote arteriolization. This strategy of EC-SMC coengraftment in PGA-supported protein gels may have broader application for perfusing bioengineered tissues.     

The phosphatidylserine receptor mediates phagocytosis by vascular smooth muscle cells

Apoptosis participates in every step of atherogenesis, but the process of clearance of apoptotic cells by phagocytosis has been underestimated. Rapid removal of apoptotic cells is critical for tissue homeostasis, in order to avoid accumulation of necrotic material and subsequent inflammation in the pathological vascular wall. We have demonstrated by RT-PCR, western blot and immunocytofluorescence that vascular smooth muscle cells (VSMCs) express the phosphatidylserine receptor (PSR). We then tested the involvement of PSR in the ability of VSMCs to bind and engulf apoptotic cells. We used a model of senescent erythrocytes, which expose PS after 4 days of culture (85% of cells relative to 8% in freshly isolated erythrocytes). The pseudo-peroxidase activity of haemoglobin contained within erythrocytes allowed us to quantify per se both binding and phagocytosis by VSMCs. We have also shown by light and confocal microscopy that VSMCs were able to ingest aged erythrocytes. Addition of a blocking antibody or transfection of VSMCs by a siRNA directed against PSR reduced the binding and engulfment of aged erythrocytes by more than 90%. These results suggest that PSR is involved in phagocytosis of PS-presenting cells. Incubation of aged erythrocytes with VSMCs also significantly increased the expression of PSR, suggesting that the tethering/ingestion of apoptotic cells triggers this process. Immunostaining for PSR in complicated atherosclerotic plaques shows positivity in the media and macrophage-rich areas. The mechanisms underlying phagocytosis and involving PSR in vivo, within the pathological arterial wall, deserve further investigation.     

RNA干扰内皮细胞分泌白细胞介素6影响平滑肌细胞的迁移

背景：血管支架置入后靶血管部位易发生炎症反应。 目的：利用siRNA技术抑制内皮细胞白细胞介素6的生成,观察其对平滑肌细胞迁移的影响。 方法：采用RT-PCR测定脂多糖刺激EA.HY926细胞表达白细胞介素6 mRNA的时间梯度与浓度梯度,针对白细胞介素6构建短发卡状siRNA真核表达载体pGensil-1.1-白细胞介素6,通过lipofectamine 2000转染EA.HY926,抑制其白细胞介素6的产生。 结果与结论：pGensil-1.1-白细胞介素6转染EA.HY926细胞后,脂多糖刺激下EA.HY926细胞表达的白细胞介素6 mRNA及蛋白明显减少。共培养模型中,转染pGensil-1.1-白细胞介素6的EA.HY926细胞作用下,人脐静脉平滑肌细胞表达的基质金属蛋白酶9 mRNA及蛋白明显降低,结晶紫染色显示人脐静脉平滑肌细胞迁移数量减少。说明siRNA技术可抑制内皮细胞白细胞介素6的生成,并通过降低平滑肌细胞基质金属蛋白酶9的表达减弱平滑肌细胞的迁移能力。     

丹参对血管平滑肌细胞抑制内皮细胞分泌的纤溶酶原激活物活性的影响

培养的猪内皮细胞(EC)产生和分泌纤溶酶原激活物(PA),血管平滑肌细胞(SMC)的无血清条件培养液(SMC-CM)与EC的无血清条件培养液(EC-CM)混合后,后者的PA活力明显减弱。EC与SMC复合培养时,其产生的PA活力亦降低(降低70.7%)。以上结果提示SMC产生纤溶酶原激活物抑制剂PAI抑制了EC分泌的PA活性。SMC-CM经SDS-PAGE后作反向纤维蛋白自显影显示PAI的分子量范围在49,000—62,000,与Laug(1985)报道的相似(55,000)。我们研究了中药丹参(Radix Salviae Miltiorrhizae,RSM)对SMC分泌PAI活性的影响。结果表明,培养的SMC经丹参作用后,其CM对尿激酶及内皮细胞产生的PA活性的中和作用明显减弱(P<0.05、P<0.01或P<0.001)。     

E1A激活基因阻遏子蛋白过表达可抑制人血管平滑肌细胞的迁移

背景：成熟动脉中膜血管平滑肌细胞(vascular smooth muscle cells, VSMCs)分化稳态的丧失是血管老化的一个重要原因。 目的：观察E1A激活基因阻遏子(cellular repressor of E1A-stimulated genes,CREG)抑制人血管平滑肌细胞迁移的分子机制。 方法：应用已建立的稳定转染CREG过表达细胞hVSMCs-CREG和CREG表达沉默的hVSMCs-siCREG细胞株通过刮伤实验和Transwell小室细胞移行实验检测细胞迁移能力,通过Western blot检测转染前后细胞中CREG蛋白、基质金属蛋白酶和JNK表达及活化情况,应用JNK和基质金属蛋白酶9抑制剂阻断研究分析上述信号分子表达变化在细胞迁移中的作用。 结果与结论：Western blot结果证实,CREG蛋白表达在hVSMCs-CREG组中增加(P < 0.05),而在hVSMCs-siCREG组中减少(P < 0.05)。刮伤实验和Transwell实验分析结果证实hVSMCs-CREG组细胞较正常对照组细胞迁移能力受抑。相反,hVSMCs-siCREG组细胞迁移能力显著增加(P< 0.05)。Western blot和明胶酶谱分析证实hVSMCs-siCREG组细胞中MMP9活性明显增加(P < 0.05),同时JNK蛋白活化。进一步应用JNK抑制剂阻断研究证实,CREG蛋白表达抑制引起的血管平滑肌细胞迁移能力增加与细胞中基质金属蛋白酶9活性均受到剂量依赖性抑制。结果证实,CREG蛋白表达可抑制JNK-基质金属蛋白酶9信号通路的活化,以此抑制体外培养的人血管平滑肌细胞迁移。     

microRNA-133b在低切应力诱导血管内皮细胞影响血管平滑肌细胞增殖中的作用

目的研究血管内皮细胞(endothelial cells,ECs)直接感受低切应力刺激后分泌类胰岛素生长因子-1(insulinlike growth factor-1,IGF-1)影响血管平滑肌细胞(vascular smooth muscle cells,VSMCs)增殖这一过程中microRNAs(miRs)的作用。方法用平行平板流动腔系统对ECs施加1.5 Pa正常切应力(normal shear stress,NSS)和0.5 Pa低切应力(low shear stress,Low SS),Real-time PCR检测VSMCs的miRs变化。用miRs预测网站预测miR-133b靶基因并验证。Western blotting检测核糖核酸结合蛋白1(polypyrimidine tract binding protein 1,Ptbp1)和N-myc下游调节基因1(N-myc downstream regulated 1,Ndrg1)的蛋白水平变化。Ed U流式检测miR-133b对VSMCs增殖的影响。结果 IGF-1静态刺激后,VSMCs的miR-133b和miR-378a表达上升。Low SS条件下,VSMCs的miR-133b表达显著上升,miR-378a表达无明显变化。下调VSMCs的miR-133b表达,Ptbp1、Ndrg1的mRNA水平均显著升高,上调VSMCs的miR-133b的表达,Ptbp1、Ndrg1的mRNA和蛋白水平显著降低,并且显著促进VSMCs增殖。结论在Low SS条件下ECs分泌IGF-1可能通过调控联合培养VSMCs的miR-133b和靶基因Ptbp1和Ndrg1促进VSMCs增殖。研究结果为心血管疾病治疗提供了一个新的潜在靶标。