慢病毒载体构建小鼠CMKLR1基因缺陷性血管平滑肌细胞系

背景：趋化素在动脉粥样硬化时的表达水平上调,趋化素及其受体CMKLR1可能参与了动脉粥样硬化的病理过程。 目的：建立CMKLR1基因缺陷性小鼠血管平滑肌细胞株。 方法：以小鼠CMKLR1基因mRNA序列作为干扰靶点,设计3组靶向CMKLR1基因的shRNA序列,构建慢病毒载体,筛选出干扰最佳的shRNA,在293T细胞对慢病毒进行包装和滴度测定。体外培养小鼠血管平滑肌细胞,转染慢病毒形成CMKLR1基因缺陷性血管平滑肌细胞株,用real-time PCR检测感染慢病毒后细胞中CMKLR1 mRNA水平。 结果与结论：基因测序结果表明慢病毒载体构建成功,慢病毒的滴度约为8.7×10⁶ TU/mL,pLVX-shRNA3对CMKLR1基因的抑制效果最显著(P < 0.001)。将pLVX-shRNA3转染至血管平滑肌细胞中形成基因缺陷细胞株,用real time PCR血管平滑肌细胞中CMKLR1 mRNA水平,该细胞株中CMKLR1 mRNA水平显著下降(P < 0.001),表明慢病毒介导的siRNA可有效沉默小鼠血管平滑肌细胞中CMKLR1基因的表达。     

A novel monoclonal antibody identifies all avian embryonic myogenic cells and adult smooth muscle cells

A novel monoclonal antibody, designated GIIF3, recognized prospective and differentiated smooth muscle cells in avian species studied – guinea fowl, chicken and quail. The GIIF3 antigen appeared in the myocardial, and the myotomal cells of the embryos at Hamburger-Hamilton stages 10 and 14, respectively. The expression of the GIIF3 molecule in the vascular smooth muscle cells emerged in the ventral wall of the dorsal aorta at Hamburger-Hamilton stage 16. The visceral smooth muscle cells started to produce the GIIF3 molecule from Hamburger-Hamilton stage 28 onwards. In both cardiac and skeletal muscles the GIIF3 expression gradually diminished, and it was lost by the end of the embryonic period, unlike in the differentiated vascular and visceral smooth muscle cells. In the latter cells the GIIF3 immunoreactive product showed a fine granular pattern that accumulated in the central region of the cytoplasm; it also occurred in the nucleus. A heavily stained discontinuous layer was associated with the cell membrane. The immunoblotting of the GIIF3 antibody recognized protein bands at 50 and 42 kDa in lysates of adult avian gizzard. A detailed comparative immunohistochemical study was made by smooth muscle markers, which confirmed the results of the immunoblotting, namely, that the GIIF3 monoclonal antibody recognized an avian myogenic cell specific molecule. During smooth muscle cell differentiation the GIIF3 molecule appeared as early as the α-smooth muscle actin, and in adult birds continued to be expressed; therefore the GIIF3 molecule could be regarded as a novel avian smooth muscle specific marker. Accepted: 27 April 2001     

TSC-36/FRP inhibits vascular smooth muscle cell proliferation and migration

OBJECTIVE: In-stent restenosis is a vascular proliferation/migration disorder characterized by hyperplasia of vascular smooth muscle cells (VSMCs). Because mounting evidence suggests that the therapeutic potential of anti-proliferation and anti-migration therapy, we investigated possible inhibitory effects of the matricellular protein TGF-beta-stimulated clone 36 (TSC-36) on vascular smooth muscle cell proliferation and migration in vitro and in vivo. METHODS: Human umbilical artery smooth muscle cells (SMCs) were treated with inducting agents daidzein or estradiol. TSC-36 expression was detected by nested competitive PCR and in situ hybridization. TSC-36 was expressed in Origami (DE3) cells. The recombinant protein was used to immunize rabbits to produce polyclonal antibodies. VSMCs were treated with various concentrations of recombinant TSC-36 (rTSC-36) protein and daidzein. The MTT assay was used to analyze for cell proliferation. A transwell system was used to detect cell migration. Flow cytometry was used to detect cell phase. A rat carotid artery balloon injury model was duplicated. The rats were treated with daidzein or solvent control. Animals were sacrificed 5 weeks later, and injured arteries were taken for pathology and histology. RESULTS: TSC-36 mRNA and protein expression was induced in SMCs. Cell proliferation and migration were inhibited by rTSC-36. rTSC-36 caused accumulation of SMCs in G2 phase. The inducting agent daidzein decreased neo-intima proliferation. TSC-36 mRNA and protein expression was induced and expressed in the neo-intima. CONCLUSION: TSC-36 can be induced in VSMCs and inhibits VSMCs proliferation in vitro and in vivo.     

Smoothelin expression during chicken embryogenesis: detection of an embryonic isoform.

Two isoforms of a novel smooth muscle cell (SMC) -specific cytoskeletal protein, smoothelin, have been described. In the adult chick, the 55-kDa smoothelin-A is expressed in visceral SMC, whereas the 120-kDa smoothelin-B is the major product in vascular SMC. Chicken was chosen to study smoothelin expression during embryogenesis and neonatally. Smoothelin-B was found in vascular SMC from stage 20 onward. In visceral SMC, smoothelin-B was present from stage 29 until hatching. Perinatally, a strong up-regulation of smoothelin synthesis was observed in visceral tissues, coinciding with a switch to the A-isoform. Transient smoothelin synthesis was observed in the somites and the developing heart. Western blotting revealed in these tissues a 62-kDa smoothelin isoform, designated smoothelin-C. Expression of the smoothelin isoforms seems to be strictly controlled with respect to cell type and developmental stage and may be related to the mode of contraction of the different cells.     

组织工程平滑肌构建的研究进展

平滑肌在人和动物体内分布广泛,普遍存在于消化道、呼吸道、血管、泌尿、生殖等诸多内脏器官中,通过收缩蠕动来协助器官的功能发挥,是重要的功能组织。平滑肌组织工程的研究目的就是要为这些组织和器官的功能化重建提供支撑。目前关于单纯平滑肌组织工程研究的报道并不太多,大多的研究主要结合在诸如血管、胃肠等组织/器官的构建中。对于平滑肌的构建仍然存在诸如生物材料的性能不可控、支架的制备技术较单一、种子细胞的培养技术和生物功能较欠缺、以及动物体内实验不完善等问题,实验室研究与实际应用还存在较大距离。本文主要从种子细胞的获取及支架构建两大方面阐述平滑肌组织工程的研究进展。从干细胞诱导分化为平滑肌细胞的研究示例中展望干细胞作为种子细胞的前景。     

Isolation of vascular smooth muscle cells from a single murine aorta

The vascular smooth muscle cell plays a significant role in many important cardiovascular disorders, and smooth muscle biology is therefore important to cardiovascular research. The mouse is critical to basic cardiovascular research, largely because techniques for genetic manipulation are more fully developed in the mouse than in any othermammalian species. We describe here a technique for isolating smooth muscle cells from a single mouse aorta. This technique is particularly useful when material is limiting, as is frequently the case when genetically modified animals are being characterized.     

大鼠提睾肌缺血再灌注损伤时血管平滑肌细胞和内皮细胞的增殖与凋亡

目的研究大鼠提睾肌缺血再灌注后血管平滑肌细胞和内皮细胞的增殖和凋亡。方法以免疫组织化学SP法和末端脱氧核苷酸转移酶介导生物素标记法 (TUNEL)对大鼠提睾肌缺血再灌注后的血管平滑肌细胞和内皮细胞的Bcl-2、Bax、P53和增殖细胞核抗原 (PCNA)的表达进行观察研究。结果缺血再灌注后 ,血管平滑肌细胞和内皮细胞均有Bcl-2、Bax、P53和PCNA的表达 ,在平滑肌细胞 ,Bcl-2阳性细胞数量明显高于Bax和P53。PCNA阳性细胞明显增多。在内皮细胞 ,Bax和P53的表达最强 ,TUNEL阳性细胞率最高。结论大鼠提睾肌缺血再灌注可造成平滑细胞的增殖和内皮细胞的凋亡 ,其结果可能与微循环障碍有关。     

27nt-miRNA对血管平滑肌细胞SM22α表达的调节及其对细胞活力、迁移和表型改变的影响

目的:探讨27nt-微小RNA(27nt-miRNA)对血管平滑肌细胞(VSMCs)中平滑肌22α蛋白(SM22α表达的调节及其对细胞活力、迁移和表型改变的影响。方法:构建27nt-miRNA高表达、反义序列(anti-27nt-miRNA)以及阴性对照的表达质粒,经慢病毒包装后分别转染大鼠原代VSMCs,加入血小板源性生长因子BB(PDGF-BB)诱导VSMCs表型转换。MTT实验检测细胞活力,划痕实验检测细胞迁移能力,RT-PCR、Western blot和免疫细胞化学染色法检测细胞中SM22α的mRNA和蛋白表达情况。结果:与正常组相比,PDGF-BB组的细胞活力上升(P 0.05)、迁移能力上升(P 0.05),SM22α的mRNA及蛋白表达量下降(P 0.05);与阴性对照慢病毒组相比,27nt-miRNA高表达组的细胞活力下降(P 0.05),迁移能力下降(P 0.05),SM22α的mRNA及蛋白表达量明显升高(P 0.05);而anti-27nt-miRNA组细胞的细胞活力上升(P 0.05),迁移能力上升(P 0.05),SM22α的mRNA及蛋白表达量下降(P 0.05)。结论:27nt-miRNA促进SM22α表达,同时抑制VSMCs的活力及迁移,并有可能抑制VSMCs从收缩型转变为合成型。     

Paladin (X99384) is expressed in the vasculature and shifts from endothelial to vascular smooth muscle cells during mouse development

Background: Angiogenesis is implicated in many pathological conditions. The role of the proteins involved remains largely unknown, and few vascular‐specific drug targets have been discovered. Previously, in a screen for angiogenesis regulators, we identified Paladin (mouse: X99384, human: KIAA1274), a protein containing predicted S/T/Y phosphatase domains. Results: We present a mouse knockout allele for Paladin with a β‐galactosidase reporter, which in combination with Paladin antibodies demonstrate that Paladin is expressed in the vasculature. During mouse embryogenesis, Paladin is primarily expressed in capillary and venous endothelial cells. In adult mice Paladin is predominantly expressed in arterial pericytes and vascular smooth muscle cells. Paladin also displays vascular‐restricted expression in human brain, astrocytomas, and glioblastomas. Conclusions: Paladin, a novel putative phosphatase, displays a dynamic expression pattern in the vasculature. During embryonic stages it is broadly expressed in endothelial cells, while in the adult it is selectively expressed in arterial smooth muscle cells. Developmental Dynamics 241:770–786, 2012. © 2012 Wiley Periodicals, Inc.     

Circumferential alignment of vascular smooth muscle cells in a circular microfluidic channel

The circumferential alignment of human aortic smooth muscle cells (HASMCs) in an orthogonally micropatterned circular microfluidic channel is reported to form an in vivo-like smooth muscle cell layer. To construct a biomimetic smooth muscle cell layer which is aligned perpendicular to the axis of blood vessel, a half-circular polydimethylsiloxane (PDMS) microchannel is first fabricated by soft lithography using a convex PDMS mold. Then, the orthogonally microwrinkle patterns are generated inside the half-circular microchannel by a strain responsive wrinkling method. During the UV treatment on a PDMS substrate with uniaxial 40% stretch and a subsequent strain releasing step, the microwrinkle patterns perpendicular to the axial direction of the circular microchannel are generated, which can guide the circumferential alignment of HASMCs during cultivation. The analysis of orientation angle, shape index, and contractile protein marker expression indicates that the cultured HASMCs reveal the in vivo-like cell phenotype. Finally, a fully circular microchannel is produced by bonding two half-circular microchannels, and the HASMCs are cultured circumferentially inside the channels with high alignment and viability for 5 days. These results demonstrated the creation of an in vivo-like 3D smooth muscle cell layer in the circular microfluidic channel which can provide a bioassay platforms for in-depth study of HASMC biology and vascular function.     

Tenascin‐C in Development and Disease of Blood Vessels

Tenascin‐C (TNC) is an extracellular glycoprotein categorized as a matricellular protein. It is highly expressed during embryonic development, wound healing, inflammation, and cancer invasion, and has a wide range of effects on cell response in tissue morphogenesis and remodeling including the cardiovascular system. In the heart, TNC is sparsely detected in normal adults but transiently expressed at restricted sites during embryonic development and in response to injury, playing an important role in myocardial remodeling. Although TNC in the vascular system appears more complex than in the heart, the expression of TNC in normal adult blood vessels is generally low. During embryonic development, vascular smooth muscle cells highly express TNC on maturation of the vascular wall, which is controlled in a way that depends on the embryonic site of cell origin. Strong expression of TNC is also linked with several pathological conditions such as cerebral vasospasm, intimal hyperplasia, pulmonary artery hypertension, and aortic aneurysm/ dissection. TNC synthesized by smooth muscle cells in response to developmental and environmental cues regulates cell responses such as proliferation, migration, differentiation, and survival in an autocrine/paracrine fashion and in a context‐dependent manner. Thus, TNC can be a key molecule in controlling cellular activity in adaptation during normal vascular development as well as tissue remodeling in pathological conditions. Anat Rec, 297:1747–1757, 2014. © 2014 Wiley Periodicals, Inc.     

The expression of adiponectin receptors and the effects of adiponectin and leptin on airway smooth muscle cells

Purpose

Obesity is a major risk factor for asthma and it influences airway smooth muscle function and responsiveness. Adiponectin is inversely associated with obesity and its action is mediated through at least 2 cell membrane receptors (AdipoR1 and AdipoR2). Leptin is positively associated with obesity. We investigated whether human airway smooth muscle (ASM) cells express adiponectin receptors and whether adiponectin and leptin regulate human ASM cell proliferation and vascular endothelial growth factor (VEGF) release.

Materials and Methods

Human ASM cells were growth-arrested in serum-deprived medium for 48 hours and then stimulated with PDGF, adiponectin and leptin. After 48 hours of stimulation, proliferation was determined using a cell proliferation ELISA kit. Human AdipoR1 and -R2 mRNA expressions were determined by RT-PCR using human-specific AdipoR1 and -R2 primers. Concentrations of VEGF, monocyte chemotactic protein (MCP)-1 and macrophage inflammatory protein (MIP)-1α in cell culture supernatant were determined by ELISA.

Results

Both AdipoR1 and AdipoR2 mRNA were expressed in the cultured human ASM cells. However, adiponectin did not suppress PDGF-enhanced ASM cell proliferation, nor did leptin promote ASM cell proliferation. Leptin promoted VEGF release by human ASM cells, while adiponectin did not influence VEGF release. Neither leptin nor adiponectin influenced MCP-1 secretion from human ASM cells. Adiponectin and MIP-1α were not secreted by human ASM cells.

Conclusion

Human ASM cells expressed adiponectin receptors. However, adiponectin did not regulate human ASM cell proliferation or VEGF release, while leptin stimulated VEGF release by human ASM cells.     

Analysis of human muscle stem cells reveals a differentiation‐resistant progenitor cell population expressing Pax7 capable of self‐renewal

Studies using mouse models have established a critical role for resident satellite stem cells in skeletal muscle development and regeneration, but little is known about this paradigm in human muscle. Here, using human muscle stem cells, we address their lineage progression, differentiation, migration, and self‐renewal. Isolated human satellite cells expressed α7‐integrin and other definitive muscle markers, were highly motile on laminin substrates and could undergo efficient myotube differentiation and myofibrillogenesis. However, only a subpopulation of the myoblasts expressed Pax7 and displayed a variable lineage progression as measured by desmin and MyoD expression. Analysis identified a differentiation‐resistant progenitor cell population that was Pax7⁺/desmin^? and capable of self‐renewal. This study extends our understanding of the role of Pax7 in regulating human satellite stem cell differentiation and self‐renewal. Developmental Dynamics 238:138–149, 2009. © 2008 Wiley‐Liss, Inc.     

Roles of Hemodynamic Forces in Vascular Cell Differentiation

The pulsatile nature of blood flow is a key stimulus for the modulation of vascular cell differentiation. Within the vascular media, physiologic stress is manifested as cyclic strain, while in the lumen, cells are subjected to shear stress. These two respective biomechanical forces influence the phenotype and degree of differentiation or proliferation of smooth muscle cells and endothelial cells within the human vasculature. Elucidation of the effect of these mechanical forces on cellular differentiation has led to a surge of research into this area because of the implications for both the treatment of atherosclerotic disease and the future of vascular tissue engineering. The use of mechanical force to directly control vascular cell differentiation may be utilized as an invaluable engineering tool in the future. However, an understanding of the role of hemodynamics in vascular cell differentiation and proliferation is critical before application can be realized. Thus, this review will provide a current perspective on the latest research and controversy behind the role of hemodynamic forces for vascular cell differentiation and phenotype modulation. Furthermore, this review will illustrate the application of hemodynamic force for vascular tissue engineering and explicate future directions for research.     

Collagen production by human smooth muscle cells isolated during intestinal organogenesis

Summary The extracellular matrix influences organogenesis by modulating cell behavior. In humans, collagen is the major matrix constituent of the adult intestinal wall and is synthesized by smooth muscle cells. The objective of the current study was to examine collagen production by fetal human intestinal smooth muscle cells isolated during intestinal morphogenesis. Techniques were developed for the isolation and culture of human fetal intestinal smooth muscle cells. The cultured cells were confirmed as muscle by immunohistochemical stains for cytoskeletal filaments and documentation of contractile behavior. In culture, these cells stained for mesenchymal and muscle cytoskeletal proteins: vimentin, actin, and desmin, and did not stain for neural or epithelial markers. The muscle cells contracted in response to acetylcholine, in contrast to human fetal dermal fibroblasts which did not contract appreciably. Collagen production was assayed by the uptake of [³H]-proline into collagenase-digestible protein. Collagen production was greatest at 11 weeks gestation, the youngest age studied. By 20 weeks gestation, collagen production had decreased to adult levels. However, when compared to another matrix-producing fetal mesenchymal cell, the dermal fibroblast, intestinal smooth muscle cells produced twice as much collagen. Collagen types were determined by polyacrylamide slab gel electrophoresis. Smooth muscle cells predominantly produced types I and III collagen chains. Therefore, collagen production is a significant function of human fetal intestinal smooth muscle cells, and probably plays a major role in the development of intestinal structure. The in vitro model presented here provides a means of studying the regulation of this collagen production throughout intestinal organogenesis.     

Insulin enhances angiotensin II induced DNA synthesis in vascular smooth muscle cells of the rat

Summary Hypertension has a high prevalence among subjects with decreased insulin sensitivity and/or hyperinsulinemia. Furthermore, angiotensin II plays a pivotal role in the regulation of vascular tone and is known to induce hypertrophy and/or hyperplasia in vascular smooth muscle cells. In the present study, the effect of insulin on angiotensin II induced smooth muscle cell growth (Wistar-Kyoto rat) was investigated. Cell growth was assessed by the measurement of [³H]thymidine incorporation into cell DNA. Insulin in a concentration range of 1.7 × 10^–10–1.7 × 10^–6 M lacked any effect on cell DNA synthesis. However, insulin enhanced the angiotensin 11 induced DNA synthesis in a concentration-dependent manner. This effect was similar in cells with a weak and in cells with a marked response in DNA synthesis to stimulation with 100 nM angiotensin 11. In conclusion, insulin is able to enhance angiotensin 11 induced DNA synthesis and may therefore function as a growth cofactor in vascular smooth muscle cells.Abbreviations AngII angiotensin II - EGF epidermal growth factor - bFGF basic fibroblast growth factor - PDGF-BB platelet-derived growth factor-BB - VSMC vascular smooth muscle cells Dedicated to Prof. Dr. N. Zöllner on the occasion of his 70th birthday     

SSAO/VAP-1 protein expression during mouse embryonic development

SSAO/VAP-1 is a multifunctional enzyme depending on in which tissue it is expressed. SSAO/VAP-1 is present in almost all adult mammalian tissues, especially in highly vascularised ones and in adipocytes. SSAO/VAP-1 is an amine oxidase able to metabolise various endogenous or exogenous primary amines. Its catalytic activity can lead to cellular oxidative stress, which has been implicated in several pathologies (atherosclerosis, diabetes, and Alzheimer's disease). The aim of this work is to achieve a study of SSAO/VAP-1 protein expression during mouse embryogenesis. Our results show that SSAO/VAP-1 appears early in the development of the vascular system, adipose tissue, and smooth muscle cells. Moreover, its expression is strong in several epithelia of the sensory organs, as well as in the development of cartilage sites. Altogether, this suggests that SSAO/VAP-1 enzyme could be involved in the differentiation processes that take place during embryonic development, concretely in tissue vascularisation. Developmental Dynamics 237:2585-2593, 2008. (c) 2008 Wiley-Liss, Inc.     

Temporal and spatial characterization of cellular constituents during neointimal hyperplasia after vascular injury: Potential contribution of bone-marrow-derived progenitors to arterial remodeling

BACKGROUND: Exuberant smooth muscle cells (SMCs) hyperplasia is the major cause of postangioplasty restenosis. We suggested that circulating smooth muscle progenitor cells might contribute to lesion formation after vascular injury. METHODS: We extensively investigated the cellular constituents during neointimal formation after mechanical vascular injury. RESULTS: A large wire was inserted into the mouse femoral artery, causing complete endothelial denudation and marked enlargement of the lumen with massive apoptosis of medial SMCs. At 2 h, the injured artery remained dilated with a thin media containing very few cells. A scanning electron microscopy showed fibrin and platelet deposition at the luminal side. One week after the injury, CD45-positive hematopoietic cells accumulated at the luminal side. Those CD45-positive cells gradually disappeared, whereas neointimal hyperplasia was formed with alpha-smooth muscle actin (SMA) positive cells. Bone marrow cells and peripheral mononuclear cells differentiated into alpha-SMA-positive cells in the presence of PDGF and basic FGF. Moreover, in bone marrow chimeric mice, bone-marrow-derived cells substantially contributed to neointimal hyperplasia after wire injury. CONCLUSION: These results suggest that early accumulation of hematopoietic cells may play a role in the pathogenesis of SMC hyperplasia under certain circumstances.     

Spatial and temporal pattern of smooth muscle cell differentiation during development of the vascular system in the mouse embryo

The initial phase of smooth muscle differentiation in the vascular system of the mouse embryo was observed immunohistochemically with monoclonal antibody against -smooth muscle actin. Few smooth muscle cells were detected in the vascular system of the 9.5-day embryo, where only the dorsal aorta and umbilical artery showed signs of smooth muscle differentiation. In the 10.5-day embryo, smooth muscle cells were observed in the dorsal aorta, ventral aorta, omphalomesenteric artery and vein, umbilical artery and vein, internal carotid artery, aortic arches III and IV, and subclavian artery. The extent of smooth muscle differentiation varied among these vessels and among regions of a vessel. At 11.5 days of gestation, smooth muscle cells appeared in the basilar artery, vertebral artery, aortic arches VI, intersomitic artery, ductus venosus, and caudal artery. Smooth muscle cells were absent from the venous system characteristic of the embryo at the stages examined. Alpha-smooth muscle actin-positive cells were also observed in allantoic mesoderm in the placenta at 9.5 days, when the umbilical vessels were not surrounded by smooth muscle cells. Vascular smooth muscle cells appear to arise independently from mesenchyme at multiple sites in the vascular system.