The influence of RGD-bearing hydrogels on the re-expression of contractile vascular smooth muscle cell phenotype

This study reports on the ability of poly(ethylene glycol) diacrylate (PEGDA) hydrogel scaffolds with pendant integrin-binding GRGDSP peptides (RGD-gels) to support the re-differentiation of cultured vascular smooth muscle cells (SMCs) toward a contractile phenotype. Human coronary artery SMCs were seeded on RGD-gels, hydrogels with other extracellular matrix derived peptides, fibronectin (FN) and laminin (LN). Differentiation was induced on RGD-gels with low serum medium containing soluble heparin, and the differentiation status was monitored by mRNA expression, protein expression, and intracellular protein organization of the contractile smooth muscle markers, smooth muscle α-actin, calponin, and SM-22α. RGD-gels supported a rapid induction (2.7- to 25-fold up-regulation) of SMC marker gene mRNA, with expression levels that were equivalent to FN and LN controls. Marker protein levels mirrored the changes in mRNA expression, with levels on RGD-gels indistinguishable from FN and LN controls. Furthermore, these markers co-localized in stress fibers within SMCs on RGD-gels suggesting the recapitulation of a contractile apparatus within the cells. These results indicate that SMCs cultured on RGD-bearing hydrogels can re-differentiate toward a contractile phenotype suggesting this material is an excellent candidate for further development as a bioactive scaffold that regulates SMC phenotype.     

The effects of stretch on vascular smooth muscle cell phenotype in vitro

Vascular smooth muscle cells (VSMC) situated in the tunica media of veins and arteries are central to maintaining conduit integrity in the face of mechanical forces inherent within the cardiovascular system. The predominant mechanical force influencing VSMC structural organisation and signalling is cyclic stretch. VSMC phenotype is manipulated by externally applied stretch which regulates the activity of their contractile apparatus. Stretch modulates cell shape, cytoplasmic organisation, and intracellular processes leading to migration, proliferation, or contraction. Drug therapy directed at the components of the signalling pathways influenced by stretch may ultimately prevent cardiovascular pathology such as myointimal hyperplasia.     

IGF-1 has plaque-stabilizing effects in atherosclerosis by altering vascular smooth muscle cell phenotype

Insulin-like growth factor-1 (IGF-1) signaling is important for the maintenance of plaque stability in atherosclerosis due to its effects on vascular smooth muscle cell (vSMC) phenotype. To investigate this hypothesis, we studied the effects of the highly inflammatory milieu of the atherosclerotic plaque on IGF-1 signaling and stability-related phenotypic parameters of murine vSMCs in vitro, and the effects of IGF-1 supplementation on plaque phenotype in an atherosclerotic mouse model. M1-polarized, macrophage-conditioned medium inhibited IGF-1 signaling by ablating IGF-1 and increasing IGF-binding protein 3, increased vSMC apoptosis, and decreased proliferation. Expression of α-actin and col3a1 genes was strongly attenuated by macrophage-conditioned medium, whereas expression of matrix-degrading enzymes was increased. Importantly, all of these effects could be corrected by supplementation with IGF-1. In vivo, treatment with the stable IGF-1 analog Long R3 IGF-1 in apolipoprotein E knockout mice reduced stenosis and core size, and doubled cap/core ratio in early atherosclerosis. In advanced plaques, Long R3 IGF-1 increased the vSMC content of the plaque by more than twofold and significantly reduced the rate of intraplaque hemorrhage. We believe that IGF-1 in atherosclerotic plaques may have a role in preventing plaque instability, not only by modulating smooth muscle cell turnover, but also by altering smooth muscle cell phenotype.     

The effect of enzymatically degradable poly(ethylene glycol) hydrogels on smooth muscle cell phenotype

The formation of scar tissue due to dedifferentiation of smooth muscle cells (SMCs) is one of the major issues faced when engineering bladder tissue. Furthermore, cell sources for regenerating the SMC layer are also limiting. Here we explore if human mesenchymal stem cells (MCSs), cultured in enzymatically degradable poly(ethylene glycol) (PEG) hydrogel scaffolds can be differentiated into SMC-like cells. We explored the degree to which a less synthetic SMC phenotype can be achieved when primary human SMCs are cultured within these scaffolds, It was observed that when both MSCs and SMCs are cultured in the PEG hydrogel scaffolds, but not on traditional tissue culture plastic, they up-regulate markers associated with the less synthetic SMC phenotype, decreased expression of alpha(5) integrin and THY-1, and increased expression of alpha-smooth muscle actin (alphaSMA) and myosin. Furthermore, we show that MSCs and SMCs cultured in the PEG hydrogels are able to proliferate and express matrix metalloproteinases for up to 21d in culture, the duration of the study. This study addresses the importance of the cellular microenvironment on cell fate, and proposes synthetic instructive biomaterials as a means to direct cell differentiation and circumvent scar tissue formation during bladder reconstruction.     

成年合成型血管平滑肌细胞的研究进展

The intermediate phenotype of vascular smooth muscle cell in adult is the dedifferentiation state returned from the high differentiation state , appeared on the damaged blood vessels. It is regulated by many factors. Its distribution, the characteristics of morphology and structure, the regulated transform factors and the molecular biological mechanism are introduced, and its functional significance and the role in vascular diseases are also discussed in this article.     

去甲肾上腺素促进血管平滑肌增殖和细胞表型转化

目的:研究去甲肾上腺素(NE)对血管平滑肌细胞(VSMC)表型转化和增殖的影响及作用机制.方法:用NE分别作用体外培养和血清饥饿的血管平滑肌细胞,用5-BrdU标记VSMC的增殖、RT-PCR检测VSMC表型标志基因SM22a和增殖负性调控基因高血压相关基因-1(HRG-1)的表达.结果:NE和OX-LDL分别作用血清培养的VSMC后,SM22α和HRG-1表达下凋,5-BrdU标记的阳性增殖细胞增多;NE分别与α1-肾上腺素受体拮抗剂(α1-R-)和β1-肾上腺素受体拮抗剂(β1-R)共作用时,SM22α和HRG-1的表达明显上调.而当NE作用血清饥饿VSMC时,NE上调SM22α和HRG-1的表达.结论:NE对VSMC有促表型转化和增殖作用,且这种作用呈现像神经样的可逆性调节作用.其作用机制主要通过肾上腺索受体α1和β1来实现的.     

Differential effects of extracellular matrix proteins on human airway smooth muscle cell proliferation and phenotype

Mature airway smooth muscle cells are characterized by a low proliferative index and expression of contractile marker proteins such as smooth muscle alpha-actin (sm-alpha-actin), calponin, and smooth muscle myosin heavy chain (sm-MHC). In the present study, defined extracellular matrix (ECM) components were examined on the proliferative and phenotypic status of mitogen-stimulated, cultured human airway smooth muscle cells. The results demonstrate that although cells adhered and spread on plates precoated with (1 to 100 microg/ml) of fibronectin (FN), collagen I (Col I), laminin (LN), or Matrigel, their subsequent proliferative response varied qualitatively. FN and Col I enhanced proliferation in response to either platelet-derived growth factor (PDGF)-BB or alpha-thrombin, compared with cells on plastic. LN, however, reduced mitogen-stimulated proliferation. A similar reduction was found in cells cultured on Matrigel. The effect of ECM substrates on contractile phenotype was determined by examining cellular expression of sm-alpha-actin, sm-MHC, and calponin using immunocytochemical and flow cytometric methods. Approximately 75% of PDGF-BB-stimulated cells, cultured on LN or Matrigel, expressed sm-alpha-actin, calponin, and sm-MHC, but only 8 to 10% stained for the Ki67 nuclear antigen proliferation marker. In contrast, more than 75% of cells cultured on FN or Col I were positive for Ki67 antigen, but only 20% were positive for contractile proteins. Flow cytometric analysis of sm-alpha-actin and DNA content confirmed the immunocytochemical findings and showed that the observed reduction in sm-alpha-actin content after culture on FN or Col I, compared with LN and Matrigel, occurred in the majority of the cell population, supporting bidirectional phenotype modulation. Overall, the data suggest that ECM substrates modulate both proliferation and phenotype of human airway smooth muscle cells in culture.     

苯丙氨酸对自发性高血压大鼠血管平滑肌细胞内钙的影响

目的 探索苯丙氨酸对自发性高血压大鼠血管平滑肌细胞内钙的影响，方法 将ＳＨＲ和ＷＫＹ的血管平滑肌细胞在不同浓度苯丙酸培养液中培养一定时间后，加入（＾４５ＣａＣｌ２）温育一定时间后，测定进入细胞内（＾４５Ｃａ＾２＋）的量；而后将在不同浓度苯丙氨酸培养液中培养一定时间后的ＳＨＲ和ＷＫＹ的血管平滑肌细胞同ＰＳＳ液温育，采用Ｆｕｒａ－２／ＡＭ荧光指示剂测定不同浓度苯丙氨酸对细胞内游离Ｃａ＾２＋的影响。结果     

Regulation of smooth muscle cell phenotype by glycosaminoglycan identity

The retention of lipoproteins in the arterial intima is an initial event in early atherosclerosis and occurs, in part, through interactions between negatively charged glycosaminoglycans (GAGs) and the positively charged residues of apolipoproteins. Smooth muscle cells (SMCs) which infiltrate into the lipoprotein-enriched intima have been observed to transform into lipid-laden foam cells. This phenotypic switch is associated with SMC acquisition of a macrophage-like capacity to phagocytose lipoproteins and/or of an adipocyte-like capacity to synthesize fatty acids de novo. The aim of the present work was to explore the impact of GAG identity on SMC foam cell formation using a scaffold environment intended to be mimetic of early atherosclerosis. In these studies, we focused on chondroitin sulfate C (CSC), dermatan sulfate (DS), and an intermediate molecular weight hyaluronan (HAIMW, ～400 kDa), the levels and/or distribution of each of which are significantly altered in atherosclerosis. DS hydrogels were associated with greater SMC phagocytosis of apolipoprotein B than HAIMW gels. Similarly, only SMCs in DS constructs maintained increased expression of the adipocyte marker A-FABP relative to HAIMW gels over 35 days of culture. The increased SMC foam cell phenotype in DS hydrogels was reflected in a corresponding decrease in SMC myosin heavy chain expression in these constructs relative to HAIMW gels at day 35. In addition, this DS-associated increase in foam cell formation was mirrored in an increased SMC synthetic phenotype, as evidenced by greater levels of collagen type I and glucose 6-phosphate dehydrogenase in DS gels than in HAIMW gels. Combined, these results support the increasing body of literature that suggests a critical role for DS-bearing proteoglycans in early atherosclerosis.     

血管平滑肌细胞凋亡在血管再狭窄中作用的研究进展

血管平滑肌细胞构成新生内膜增生的重要部分,且在血管腔内治疗术后再狭窄的心血管疾病的发生和发展中具有重要作用。血管平滑肌细胞凋亡能有效抑制血管球囊损伤和血管旁路移植术后新生内膜增生,从而可为血管术后再狭窄提供治疗手段。     

Emerging regulators of vascular smooth muscle cell migration

Journal of Muscle Research and Cell Motility - Vascular smooth muscle cells (VSMCs) are the predominant cell type in the blood vessel wall and normally adopt a quiescent, contractile phenotype....     

Molecular control of vascular smooth muscle cell differentiation

Changes in the differentiated state of the vascular smooth muscle cell (SMC) including enhanced growth responsiveness, altered lipid metabolism, and increased matrix production are known to play a key role in development of atherosclerotic disease. As such, there has been extensive interest in understanding the molecular mechanisms and factors that regulate differentiation of vascular SMC, and how this regulation might be disrupted in vascular disease. Key questions include determination of mechanisms that control the coordinate expression of genes required for the differentiated function of the smooth muscle cell, and determination as to how these regulatory processes are influenced by local environmental cues known to be important in control of smooth muscle differentiation. Of particular interest, a number of common cis regulatory elements including highly conserved CArG [CC(A/T)₆GG] motifs or CArG-like motifs and a TGF_β control element have been identified in the promoters of virtually all smooth muscle differentiation marker genes characterized to date including smooth muscle α-actin, smooth muscle myosin heavy chain, telokin, and SM22α and shown to be required for expression of these genes both in vivo and in vitro. In addition, studies have identified a number of trans factors that interact with these cis elements, and shown how the expression or activity of these factors is modified by local environmental cues such as contractile agonists that are known to influence differentiation of smooth muscle.     

Antiproliferative effects of novel, nonanticoagulant heparin derivatives on vascular smooth muscle cells in vitro and in vivo.

The proliferation of vascular smooth muscle cells (VSMC) is strongly inhibited by whole heparin both in vitro and in vivo. To identify and characterize antiproliferative, but nonanticoagulant heparin derivatives, heparin fragments made by periodate treatment were produced and acylated with 2-, 4-, or 6-carbon chain lengths. In culture, the 4- and 6-carbon acylated compounds were more effective than whole heparin in inhibiting serum stimulated VSMC growth at equal mass or approximately equal mean molar concentrations. Further testing was performed in the rat carotid balloon injury model. Myointimal VSMC proliferation produced by balloon catheterization of rat carotid arteries was inhibited by the 4-carbon acylated compound as effectively as heparin at the same mass dose. Importantly, unlike heparin, the 4-carbon acylated compound had no anticoagulant effect in vivo. These experiments suggest nonanticoagulant, acylated heparin derivatives may have a pharmacologic role in preventing myointimal proliferative lesions that are responsible for failures of vascular surgeries and angioplasties.     

In vitro effects of desoxycorticosterone on vascular smooth muscle.

We present evidence in accord with the observations of S. Kalsner (Br. J. Pharmacol. 36: 582-593, 1969) that in the rabbit aorta, desoxycorticosterone (DOC) potentiates the contractile response to certain catecholamines by inhibiting their degradation by catechol-O-methyltransferase. In contrast, DOC depresses the contractile responses in rat aorta and tail arteries. To elucidate the mechanism of this depression the effect of DOC was evaluated under various conditions. DOC depressed the contractile response to epinephrine, phenylephrine, KCl, and angiotensin II. The depression was unaltered by ouabain or by a potassium-free solution, indicating that DOC did not produce its depression by altering Na-K-ATPase activity. The depression is unaltered in a chloride-free solution, demonstrating that the DOC effect is not caused by a change in membrane permeability to chloride. Radioisotope studies demonstrate that DOC does not alter membrane permeability to potassium. Removal of extracellular calcium with EGTA (ethylene glycol-bis (beta-aminoethyl ether) N, N'-tetraacetic acid) significantly reduced the magnitude of the DOC depression. Indirect evidence is presented suggesting that DOC might increase calcium binding to the plasma membrane, resulting in its stabilization and hence in a depression of the contractile response.     

雌激素对血管平滑肌的作用

血管平滑肌细胞是保持血管功能完整性的重要结构和功能单位,在动脉粥样硬化、高血压、冠心病及损伤后再狭窄的发病机制中具有关键性作用。本文对雌激素通过功能性的雌激素受体调节血管平滑肌细胞的结构和功能进行综述。     

Directing phenotype of vascular smooth muscle cells using electrically stimulated conducting polymer

Vascular smooth muscle cells (VSMCs) isolated from rabbit aorta and immortalised A7r5 cells were cultured on conducting polypyrrole (PPy) substrates and were subjected to a 50muA sinusoidal electrical stimulation at 0.05, 5 and 500 Hz. These substrates were doped with hyaluronic acid and coated with collagen IV followed by Matrigel in order to mimic the basement membrane and encourage cell attachment. Increased proliferation and expression of smooth muscle phenotype markers (smooth muscle alpha-actin and smooth muscle myosin heavy chain) were observed in cultures stimulated at 5 and 500 Hz. This increased proliferation and expression of contractile proteins were found to be significantly decreased when L-type voltage-gated calcium channels (VGCC) were blocked with the drug nifedipine. To the best of our knowledge, this is the first work that demonstrates that VSMCs cultured on a conducting polymer substrate and subject to electrical stimulation not only exhibit enhanced proliferation but can be simultaneously encouraged to increase contractile protein expression. This behaviour is somewhat contrary to the classical definition of smooth muscle contractile and synthetic phenotypes that, in general, requires a modulation in phenotype as a prerequisite for smooth muscle proliferation. This interesting result highlights both the inherent plasticity of vascular smooth muscle cells and the potential of electrical stimulation via conducting polymer substrates to manipulate their behaviour.     

Regulation of the cell magnesium in vascular smooth muscle

1. The relation between [Mg](o) and the Mg content of the rat tail artery incubated in Ca-free solutions was studied.2. Variation of [Mg](o) in Ca-free Krebs solution between 0.6 and 2.4 mm did not affect the cell Mg. In ATP-depleted arteries, a sizeable fraction of the cell Mg was found to be proportional to [Mg](o).3. An even larger fraction of the cell Mg became proportional to [Mg](o) in metabolically active arteries in which the transmembrane gradient of Na had been dissipated as a result of inhibition of the Na pump. In contrast to the extracellular Mg, the fraction responded to a change in [Mg](o) at a very slow rate. The size of the fraction was reflected in net uptake of Mg if [Mg](o) was higher than 1.2 mm. The rate of the uptake was lowered markedly by external Ca. All the Mg taken up by the cells was extruded again after restoration of the Na gradient.4. The uptake of Mg in Ca-free, ouabain-containing solution took place even if the concomitant swelling of cells was prevented by substitution of isethionate for external Cl.5. Under steady-state conditions and at const. [Mg](o) and [Na](o), [Mg](i) increased with increasing [Na](i).6. The results are consistent with the hypothesis that the outwardly directed Mg pump in rat vascular smooth muscle utilizes the energy released in the course of spontaneous influx of Na.     

Elastogenic effects of exogenous hyaluronan oligosaccharides on vascular smooth muscle cells

Prior studies suggest that hyaluronan (HA), a glycosaminoglycan, may upregulate innately poor elastin matrix synthesis by adult vascular smooth muscle cells (SMCs). HA scaffolds could thus be useful to regenerate damaged vascular elastin. In an earlier study, we established that the elastogenic effects of non-oligomeric HA are fragment size- and/or dose-specific. We currently investigate the pro-elastogenic effects of exogenous HA oligomers on rat aortic smooth muscle cells (RASMCs). RASMCs were cultured with pure HA oligomers (4-mers) and mixtures (4-8mers) obtained by enzymatic digestion of long-chain HA (MW approximately 2000kDa). Polyacrylamide gel electrophoresis (PAGE)/Matrix Assisted Laser Desorption/Ionization Spectroscopy Time-Of-Flight Analysis (MALDI-TOF) showed HA digestates to contain a mixture of 4-8mers with a predominance of 4-mers (75+/-0.4% w/w). Cell layers supplemented with both pure HA 4-mers or oligomer mixtures showed proliferation levels similar to non-HA controls over 21 days of culture. Pure 4-mers and oligomer mixtures enhanced DNA-normalized output of tropoelastin by 1.6 and 1.8 times, respectively, and that of matrix elastin by approximately 2.7 times relative to controls. Sodium dodecyl sulfate (SDS)-PAGE/Western Blot and a desmosine assay semi-quantitatively confirmed the observed biochemical trends for tropoelastin and matrix elastin, respectively. HA oligomers induced enhanced synthesis of the elastin crosslinker, desmosine, and appeared to stabilize the elastin matrix by suppression of elastin-laminin receptor (ELR) activity relative to controls. Transmission electron micrographs (TEMs) showed elastin deposits within oligomer-supplemented cultures to be distinct, longitudinally oriented, aggregating fibrils, and clumps, and to be less abundant and mostly amorphous in controls. HA oligomers preserved normal fibrillin-mediated elastin matrix deposition. Results suggest that HA oligos are highly pro-elastogenic, promote elastin fibril formation, and stabilize elastin matrix and may thus be usefully incorporated into scaffolds for guided elastin regeneration.     

高糖、高脂血症大鼠血管内皮细胞及平滑肌细胞的形态和功能改变

复制高糖、是动物模型,利用血管功能测定,组织培养和放射免疫分析等方法观察长期高糖,高脂血症对血管内皮细胞和平骨肌细胞功能的影响及相互作用,结果表明,高糖、高脂对血和内皮细胞均有影响,两者有协同作用;对血管平滑肌细胞两者均可促进其增强,加速了糖尿病血管并发症的发生发展。