The role of SIRT6 in the differentiation of vascular smooth muscle cells in response to cyclic strain

Vascular smooth muscle cells (VSMCs) may switch their phenotype between a quiescent contractile phenotype and a synthetic phenotype in response to cyclic strain, and this switch may contribute to hypertension, atherosclerosis, and restenosis. SIRT 6 is a member of the sirtuin family, and plays an important role in different cell processes, including differentiation. We hypothesized that cyclic strain modulates the differentiation of VSMCs via a transforming growth factor-β1 (TGF-β1)-Smad-SIRT6 pathway. VSMCs were subjected to cyclic strain using a Flexercell strain unit. It was demonstrated that the strain stimulated the secretion of TGF-β1 into the supernatant of VSMCs. After exposed to the strain, the expressions of contractile phenotype markers, including smooth muscle protein 22 alpha, alpha-actin, and calponin, and phosphorylated Smad2, phosphorylated Smad5, SIRT6 and c-fos were up-regulated in VSMCs by western blot and immunofluorescence. And the expression of intercellular-adhesion molecule-1 (ICAM-1) was also increased detected by flow cytometry. The strained-induced up-regulation of SIRT6 was blocked by a TGF-β1 neutralizing antibody. Furthermore, the effects of strain on VSMCs were abrogated by SIRT6-specific siRNA transfection via the suppression c-fos and ICAM-1. These results suggest that SIRT6 may play a critical role in the regulation of VSMC differentiation in response to the cyclic strain.     

Airway smooth muscle cell tone amplifies contractile function in the presence of chronic cyclic strain

Chronic contractile activation, or tone, in asthma coupled with continuous stretching due to breathing may be involved in altering the contractile function of airway smooth muscle (ASM). Previously, we (11) showed that cytoskeletal remodeling and stiffening responses to acute (2 h) localized stresses were modulated by the level of contractile activation of ASM. Here, we investigated if altered contractility in response to chronic mechanical strain was dependent on repeated modulation of contractile tone. Cultured human ASM cells received 5% cyclic (0.3 Hz), predominantly uniaxial strain for 5 days, with once-daily dosing of either sham, forskolin, carbachol, or histamine to alter tone. Stiffness, contractility (KCl), and "relaxability" (forskolin) were then measured as was cell alignment, myosin light-chain phosphorylation (pMLC), and myosin light-chain kinase (MLCK) content. Cells became aligned and baseline stiffness increased with strain, but repeated lowering of tone inhibited both effects (P < 0.05). Strain also reversed a negative tone-modulation dependence of MLCK, observed in static conditions in agreement with previous reports, with strain and tone together increasing both MLCK and pMLC. Furthermore, contractility increased 176% (SE 59) with repeated tone elevation. These findings indicate that with strain, and not without, repeated tone elevation promoted contractile function through changes in cytoskeletal organization and increased contractile protein. The ability of repeated contractile activation to increase contractility, but only with mechanical stretching, suggests a novel mechanism for increased ASM contractility in asthma and for the role of continuous bronchodilator and corticosteroid therapy in reversing airway hyperresponsiveness.     

Combined effects of microtopography and cyclic strain on vascular smooth muscle cell orientation

Cellular alignment studies have shown that cell orientation has a large effect on the expression and behavior of cells. Cyclic strain and substrate microtopography have each been shown to regulate cellular alignment. This study examined the combined effects of these two stimuli on the alignment of bovine vascular smooth muscle cells (VSMCs). Cells were cultured on substrates with microgrooves of varying widths oriented either parallel or perpendicular to the direction of an applied cyclic tensile strain. We found that microgrooves oriented parallel to the direction of the applied strain limited the orientation response of VSMCs to the mechanical stimulus, while grooves perpendicular to the applied strain enhanced cellular alignment. Further, the extent to which parallel grooves limited cell alignment was found to be dependent on the groove width. It was found that for both a small (15microm) and a large (70microm) groove width, cells were better able to reorient in response to the applied strain than for an intermediate groove width (40microm). This study indicates that microtopographical cues modulate the orientation response of VSMCs to cyclic strain. The results suggest that there is a range of microgroove dimensions that is most effective at maintaining the orientation of the cells in the presence of an opposing stimulus induced by cyclic strain.     

Scaffolds for engineering smooth muscle under cyclic mechanical strain conditions

Cyclic mechanical strain has been demonstrated to enhance the development and function of engineered smooth muscle (SM) tissues, but appropriate scaffolds for engineering tissues under conditions of cyclic strain are currently lacking. These scaffolds must display elastic behavior, and be capable of inducing an appropriate smooth muscle cell (SMC) phenotype in response to mechanical signals. In this study, we have characterized several scaffold types commonly utilized in tissue engineering applications in order to select scaffolds that exhibit elastic properties under appropriate cyclic strain conditions. The ability of the scaffolds to promote an appropriate SMC phenotype in engineered SM tissues under cyclic strain conditions was subsequently analyzed. Poly(L-lactic acid)-bonded polyglycolide fiber-based scaffolds and type I collagen sponges exhibited partially elastic mechanical properties under cyclic strain conditions, although the synthetic polymer scaffolds demonstrated significant permanent deformation after extended times of cyclic strain application. SM tissues engineered with type I collagen sponges subjected to cyclic strain were found to contain more elastin than control tissues, and the SMCs in these tissues exhibited a contractile phenotype. In contrast, SMCs in control tissues exhibited a structure more consistent with the nondifferentiated, synthetic phenotype. These studies indicate the appropriate choice of a scaffold for engineering tissues in a mechanically dynamic environment is dependent on the time frame of the mechanical stimulation, and elastic scaffolds allow for mechanically directed control of cell phenotype in engineered tissues.     

STAT-3-dependent cytosolic phospholipase A2 expression is required for thrombin-induced vascular smooth muscle cell motility

Vascular smooth muscle cell (VSMC) migration from media to intima and its multiplication in intima is a contributing factor in the pathogenesis of atherosclerosis and restenosis after angioplasty. Previously, we have demonstrated that STAT-3-dependent cytosolic phospholipase A(2) (cPLA(2)) expression is needed for VSMC motility induced by platelet-derived growth factor-BB, a receptor tyrosine kinase agonist (Neeli et al. (2005) J. Biol. Chem. 279, 46122-46128). In order to learn more about the STAT-3-cPLA(2) axis in motogenic signaling, here we have studied its role in VSMC motility in response to a G protein-coupled receptor (GPCR) agonist, thrombin. Thrombin induced VSMC motility in a dose-dependent manner with a maximum effect at 0.5 units/ml. Thrombin activated STAT-3 as measured by its tyrosine phosphorylation and translocation from the cytoplasm to the nucleus. Forced expression of a dominant negative mutant of STAT-3 reduced thrombin-induced STAT-3 tyrosine phosphorylation and its translocation from the cytoplasm to the nucleus. Thrombin stimulated STAT-3-DNA binding and reporter gene activities in VSMC, and these responses were blocked by FS3DM, a dominant negative mutant of STAT-3. FS3DM also attenuated thrombin-induced VSMC motility. Thrombin induced the expression of cPLA(2) in a time- and STAT-3-dependent manner. In addition, pharmacological inhibition of cPLA(2) blocked thrombin-induced VSMC motility. Furthermore, exogenous addition of arachidonic acid rescued thrombin-induced VSMC motility from inhibition by blockade of STAT-3 activation. Forced expression of cPLA(2) also surpassed the inhibitory effect of dominant negative STAT-3 on thrombin-induced VSMC motility. Together, these results show that thrombin-induced VSMC motility requires STAT-3-dependent induction of expression of cPLA(2).     

Temporal phosphoproteomics to investigate the mechanotransduction of vascular smooth muscle cells in response to cyclic stretch

Vascular smooth muscle cells (VSMCs) are exposed to mechanical cyclic stretch in vivo, which play important roles in maintenance of vascular homeostasis and regulation of pathological vascular remodeling. Reversible protein phosphorylation is crucial for intracellular signaling transduction. However, the dynamic phosphorylated profile induced by cyclic stretch in VSMCs is still unclear. Using the stable isotope labeling by amino acid in cell culture, VSMCs were labeled and exposed to 10% physiological cyclic stretch in vitro at 1.25 Hz for 0 min, 15 min, 30 min, 1 h and 6 h, respectively. Using TiO₂ beads and liquid chromatography tandem mass spectrometry, the temporal phosphoproteomic profiles in response to cyclic stretch were then detected. Bioinformatics analysis including fuzzy c-means clustering, functional classifications, and Ingenuity Pathway Analysis were applied to further reveal the potential mechanotranduction networks. The results indicated that protein kinase C (PKCs) family, Rho-associated coiled-coil containing protein kinase 1 (ROCK1) and Akt may participate in cyclic-stretch induced VSMC functions. Cyclic stretch repressed the expression of ROCK1, while it had no significant effect on the phosphorylation of PKCα/βII, PKCζ/λ and PKCδ/θ. PKCθ was activated first at short time-phase (15 min and 30 min), and again at long time-phase (6 h, 12 h and 24 h). The activation of p-PKCμ was immediate and short-term, similar to p-Akt. Our present in vitro work hence revealed that cyclic stretch activates complex mechanotransduction networks, suggesting that novel mechanoresponsive molecules, i.e., PKCθ, PKCμ, and ROCK1, may participate in the mechanotransduction and modulation VSMC functions.     

The role of Exo70 in vascular smooth muscle cell migration

Background

As a key subunit of the exocyst complex, Exo70 has highly conserved sequence and is widely found in yeast, mammals, and plants. In yeast, Exo70 mediates the process of exocytosis and promotes anchoring and integration of vesicles with the plasma membrane. In mammalian cells, Exo70 is involved in maintaining cell morphology, cell migration, cell connection, mRNA splicing, and other physiological processes, as well as participating in exocytosis. However, Exo70’s function in mammalian cells has yet to be fully recognized. In this paper, the expression of Exo70 and its role in cell migration were studied in a rat vascular smooth muscle cell line A7r5.

Methods

Immunofluorescent analysis the expression of Exo70, α-actin, and tubulin in A7r5 cells showed a co-localization of Exo70 and α-actin, we treated the cells with cytochalasin B to depolymerize α-actin, in order to further confirm the co-localization of Exo70 and α-actin. We analyzed Exo70 co-localization with actin at the edge of migrating cells by wound-healing assay to establish whether Exo70 might play a role in cell migration. Next, we analyzed the migration and invasion ability of A7r5 cells before and after RNAi silencing through the wound healing assay and transwell assay.

Results

The mechanism of interaction between Exo70 and cytoskeleton can be clarified by the immunoprecipitation techniques and wound-healing assay. The results showed that Exo70 and α-actin were co-localized at the leading edge of migrating cells. The ability of A7r5 to undergo cell migration was decreased when Exo70 expression was silenced by RNAi. Reducing Exo70 expression in RNAi treated A7r5 cells significantly lowered the invasion and migration ability of these cells compared to the normal cells. These results indicate that Exo70 participates in the process of A7r5 cell migration.

Conclusions

This research is importance for the study on the pathological process of vascular intimal hyperplasia, since it provides a new research direction for the treatment of cardiovascular diseases such as atherosclerosis and restenosis after balloon angioplasty.

     

Role of cyclic strain frequency in regulating the alignment of vascular smooth muscle cells in vitro

The arterial system is subjected to cyclic strain because of periodic alterations in blood pressure, but the effects of frequency of cyclic strain on arterial smooth muscle cells (SMCs) remain unclear. Here, we investigated the potential role of the cyclic strain frequency in regulating SMC alignment using an in vitro model. Aortic SMCs were subject to cyclic strain at one elongation but at various frequencies using a Flexercell Tension Plus system. It was found that the angle information entropy, the activation of integrin-β1, p38 MAPK, and F/G actin ratio of filaments were all changed in a frequency-dependent manner, which was consistent with SMC alignment under cyclic strain with various frequencies. A treatment with anti-integrin-β1 antibody, SB202190, or cytochalasin D inhibited the cyclic strain frequency-dependent SMC alignment. These observations suggested that the frequency of cyclic strain plays a role in regulating the alignment of vascular SMCs in an intact actin filament-dependent manner, and cyclic strain at 1.25 Hz was the most effective frequency influencing SMC alignment. Furthermore, integrin-β1 and p38 MAPK possibly mediated cyclic strain frequency-dependent SMC alignment.     

Coexpression of Notch3 and Rgs5 in the pericyte-vascular smooth muscle cell axis in response to pulp injury

Recent studies have shown that the pulp of human teeth contains a population of cells with stem cell properties and it has been suggested that these cells originate from pericytes. Molecules of the Notch signaling pathway regulate stem cell fate specification, while Rgs5 represents an excellent marker for pericytes. Pathological conditions such as dental trauma and carious lesion stimulate pulp stem cells to elaborate reparative dentin. Previous studies have shown that genes involved in the Notch pathway are activated in response to pulp injury in rodent and humans. To demonstrate the importance of pericytes as a source of stem cells during dental repair, we have studied Rgs5 and Notch3 mRNA expression by in situ hybridization in developing, adult intact and injured rodent teeth. Furthermore, we have examined the distribution of Notch3 protein in carious and injured human teeth using immunohistochemistry. Overlapping expression patterns of Rgs5 and Notch3 were observed during rodent tooth development as well as immediately after injury. Both genes were expressed in vascular structures during development and in perivascular and single capillary cells of injured teeth. However, the expression patterns of Rgs5 and Notch3 were different during tooth repair, with relatively extensive Rgs5 expression along the pericyte-vascular smooth muscle cell axis in central pulp arterioles. These results show co-expression of Rgs5 and Notch3 in pericytes of developing and injured teeth and furthermore indicate the importance of vascular-derived stem cells during pulp healing.     

The role of cyclic GMP in cells with the properties of smooth muscle cultured from the rat myometrium

Cells growing in culture with previously described properties of rat uterine smooth muscle accumulated 45Ca2+ from the medium. Ca2+ uptake by these cells was stimulated by the addition to the medium of 8-bromo-cGMP but not by 8-bromo-cAMP. Ca2+ uptake was also stimulated by carbachol and by the nitro-vasodilator nitroprusside. Although cholinergic agonists have been shown previously to stimulate contraction but not cGMP synthesis in the rat myometrium, both carbachol and nitroprusside stimulated cGMP production by the cultured cells. These results suggested the cells had cholinergic receptor-mediated functions that reflected some neurotransmitter-sensitive properties of uterine smooth muscle in situ. When determined by a specific radioligand binding assay, subcellular fractions of the cultured cells bound muscarinic cholinergic agonists and antagonists with affinities expected of the muscarinic receptor. The cells were also sensitive to the beta-adrenergic catecholamine agonist isoproterenol, which stimulated cAMP production but not Ca2+ uptake. Carbachol failed to inhibit isoproterenol-dependent cAMP production, which is an important property of the cholinergic receptor in uterine smooth muscle in situ. These results suggest some but not all acetylcholine-sensitive properties of uterine smooth muscle may be retained in cell culture.     

The role of cytoplasmic nanospaces in smooth muscle cell Ca2+ signalling

We address the importance of cytoplasmic nanospaces in Ca(2+) transport and signalling in smooth muscle cells and how quantitative modelling can shed significant light on the understanding of signalling mechanisms. Increasingly more convincing evidence supports the view that these nanospaces--nanometre-scale spaces between organellar membranes, hosting cell signalling machinery--are key to Ca(2+) signalling as much as Ca(2+) transporters and Ca(2+) storing organelles. Our research suggests that the origin of certain diseases is to be sought in the disruption of the proper functioning of cytoplasmic nanospaces. We begin with a historical perspective on the study of smooth muscle cell plasma membrane-sarcoplasmic reticulum nanospaces, including experimental evidence of their role in the generation of asynchronous Ca(2+) waves. We then summarize how stochastic modelling approaches have aided and guided our understanding of two basic functional steps leading to healthy smooth muscle cell contraction. We furthermore outline how more sophisticated and realistic quantitative stochastic modelling is now being employed not only to deepen our understanding but also to aid in the hypothesis generation for further experimental investigation.     

The role of arterial smooth muscle in vasorelaxation

The concept of endothelium-derived relaxing factor (EDRF) implies that nitric oxide (NO) produced by NO synthase (NOS) in the endothelium in response to vasorelaxants such as acetylcholine (ACh) acts on the underlying vascular smooth muscle cells (VSMC) inducing vascular relaxation. The EDRF concept was derived from experiments on denuded blood vessel strips and, in frames of this concept, VSMC were regarded as passive recipients of NO from endothelial cells. However, it was later found that VSMC express NOS by themselves, but the principal question remained unanswered, is the NO generation by VSMC physiologically relevant? We hypothesized that the destruction of the vascular wall anatomical integrity by rubbing off the endothelial layer might increase vascular superoxides that, in turn, reduced the NO bioactivity as a relaxing factor. To test our hypothesis, we examined ACh-induced vasorelaxation under protection against oxidative stress and found that superoxide scavengers restored vasodilatory responses to ACh in endothelium-deprived blood vessels. These findings imply that VSMC can release NO in amounts sufficient to account for the vasorelaxatory response and challenge the concept of the obligatory role of endothelial cells in the relaxation of arterial smooth muscle.     

The smooth muscle cell. III. Elastin synthesis in arterial smooth muscle cell culture

Primate arterial smooth muscle cells and skin fibroblasts were examined for their ability to synthesize elastin in culture. In the presence of the lathyrogen beta-aminopropionitrile, the smooth muscle cells incorporate [3H]lysine into a lysyl oxidase substrate that was present in the medium and associated with the cell layer. A component having a mol wt of 72,000 and an electrophoretic mobility similar to that of authentic tropoelastin was isolated from the labeled smooth muscle cells by coacervation and fractionation with organic solvents. In the absence of beta-aminopropionitrile, long-term cultures of smooth muscle cells incorporated [14C]lysine into desmosine and isodesmosine, the cross-link amino acids unique to elastin. In contrast, no desmosine formation occurred in the fibroblast cultures. These characteristics demonstrate that arterial smooth muscle cells are capable of synthesizing both soluble and cross-lined elastin in culture.     

A new in vitro system for studying cell response to mechanical stimulation : Different effects of cyclic stretching and agitation on smooth muscle cell biosynthesis

An experimental model has been devised to permit morphologic and metabolic characterization of cells subjected to a range of cyclic mechanical stimuli similar to those which may prevail in blood vessel walls. A unique feature is the use of purified elastin membranes prepared from bovine aortas as extensible substrates for cell growth. Cells attached firmly to such membranes which could then be subjected to continuous stretching and relaxation or displacement without stretching by a motor coupled to a movable supporting frame. Various combinations of frequencies, amplitudes and rates of deformation have been used over extended periods with minimal fatigue or disruption of the elastin substrate. The effects of cyclic stretching on [¹⁴C]proline incorporation into protein and collagen and [³H]thymidine incorporation into DNA by rabbit aortic smooth muscle cells were distinct from those attributable to agitation without stretching, indicating that cells responded differently to these modes of stimulation. Increases in rate of protein or DNA synthesis induced by stretching were just as marked after 48 h of stimulation as they were at the outset of an experimental period. Since the system permits observations of cell response to independently variable components of pulsatile stress over extended periods and under a variety of culture conditions, it may be expected to provide new data concerning the interaction of mechanical with hormonal and genetic factors in the elaboration of connective tissue components.     

The effect of ions upon the response of smooth muscle to cooling

The slow tonic responses of the anterior byssus retractor of Mytilus edulis to rapid cooling were investigated by simultaneously recording tension and resting potential changes after soaking the muscle in banthine, a powerful neuromuscular blocking agent. The quantitative relations between the amount of cooling and the amount of associated depolarization necessary for contraction at various concentrations of potentiating potassium can be expressed in a family of curves. The plateaus of the curves for sea water and for potassium-free sea water were beneath the depolarization value necessary for contraction, so that it is clear that no amount of cooling with sea water alone or with potassium-free sea water would ever be effective. When the muscle is treated with subthreshold amounts of potassium and rapidly cooled in various concentrations of sodium ion and calcium ion, respectively, the sodium and calcium do not affect the amount of depolarization. Acetylcholine, in subthreshold amounts, has a potentiating effect, but, unlike potassium and cooling, acts through the nervous apparatus. Mytilus muscle will respond to cooling with tonic contraction whenever a critical threshold amount of depolarization is achieved. Cooling alone cannot trigger the contraction since it cannot bring about sufficient depolarization. Cooling can result in contraction, however, if used in conjunction with some other subthreshold depolarizing agent. Cooling affects the contractile mechanism by first causing membrane breakdown and depolarization.     

Molecular pathways of cyclic nucleotide-induced inhibition of arterial smooth muscle cell proliferation

Cyclic adenosine 3',5'-monophosphate (cAMP) and cyclic guanosine 3',5'-monophosphate (cGMP) are second messengers involved in the intracellular signal transduction of a wide variety of extracellular stimuli. These signals regulate many biological processes including cell proliferation, differentiation, migration, and apoptosis. Recently, significant progress has been achieved in the molecular basis underlying cyclic nucleotide regulation of cell proliferation. This review summarizes our knowledge of the signaling pathways regulated by cyclic nucleotides in arterial smooth muscle cells.