心肌细胞和血管平滑肌细胞收缩调控机制的研究进展

心脏和血管构成体内的心血管系统,两者都具有收缩性。心脏收缩要求在很短的时间内升高室内压,因此要求细胞收缩快速和有力,这就需要细胞的收缩结构和钙调控过程能满足其要求。血管收缩缓慢而持久,其收缩结构及机制也正好与之功能相适应。本文从细胞水平讨论了心脏和血管的收缩结构和收缩机制,以及钙调控机制,并分别对两者之间的异同点作了介绍。     

Ex vivo generation of mature and functional human smooth muscle cells differentiated from skeletal myoblasts

We described the ex vivo production of mature and functional human smooth muscle cells (SMCs) derived from skeletal myoblasts. Initially, myoblasts expressed all myogenic cell-related markers such as Myf5, MyoD and Myogenin and differentiate into myotubes. After culture in a medium containing vascular endothelial growth factor (VEGF), these cells were shown to have adopted a differentiated SMC identity as demonstrated by alphaSMA, SM22alpha, calponin and smooth muscle-myosin heavy chain expression. Moreover, the cells cultured in the presence of VEGF did not express MyoD anymore and were unable to fuse in multinucleated myotubes. We demonstrated that myoblasts-derived SMCs (MDSMCs) interacted with endothelial cells to form, in vitro, a capillary-like network in three-dimensional collagen culture and, in vivo, a functional vascular structure in a Matrigel implant in nonobese diabetic-severe combined immunodeficient mice. Based on the easily available tissue source and their differentiation into functional SMCs, these data argue that skeletal myoblasts might represent an important tool for SMCs-based cell therapy.     

Incorporation of [3H]glucosamine into glycosaminoglycans in different cell culture conditions by rabbit aortic smooth muscle cells

This study sought to elucidate the optimal cell culture conditions for studies concerned with the incorporation of [³H]glucosamine into glycosaminoglycans by rabbit aortic smooth muscle cells. The incorporation of radioactivity into extracellular sulphated glycosaminoglycans was linear for at least 72 h and that into pericellular sulphated glycosaminoglycans for up to 24 h. The incorporation of radiolabel into hyaluronic acid was linear only up to 12 h. In the exponential growth phase the incorporation of [³H]glucosamine into sulphated glycosaminoglycans and hyaluronic acid proved to be less marked than in the stationary growth phase, but the highest values were nevertheless obtained immediately after trypsinisation. When studied in the stationary growth phase, cell density and incorporation of [³H]glucosamine were positively correlated in the case of hyaluronic acid, but in the case of sulphated glycosaminoglycans there was a negative correlation. The serum concentration of the incubation medium and the incorporation of radioactivity into hyaluronic acid were positively related. With sulphated glycosaminoglycans this was the case only after a 7-day preincubation in the different serum concentrations. when incorporation was studied without preincubation, the incorporation of radioactivity into sulphated glycosaminoglycans proved to be negatively associated with the serum concentration of the medium. The environmental pH of the cells was associated with the incorporation of radioactivity into hyaluronic acid and sulphated glycosaminoglycans in that between pH values 6.8 and 7.9 the incorporation of radioactivity increased when the pH of the medium was raised.     

Cell shape and arrangement of cultured aortic smooth muscle cells grown on collagen gels

Aortic smooth muscle cells (SMC) grown on conventional plastic culture dishes have morphological and functional properties of dedifferentiated cells in sub-culture. We examined the influence of collagen gels on the cell shape and arrangement. The cells grown on collagen gels showed a multilayered growth with formation of nodules. When the edge of the collagen gels was detached from the culture dish, the shape and arrangement of cells on the edge differed from that of the central, still attached region. The cells grown on floating collagen gels exhibited a spindle-like shape and were arranged in concentric circles. These findings suggest that the physical property of the substrate influences the cell shape and arrangement.     

Functional muscarinic M2 and M3 receptors and beta-adrenoceptor in cultured rat bladder smooth muscle

A highly purified rat urinary bladder smooth muscle cell culture was obtained by a modified enzymic isolation method, and the presence of functional muscarinic as well as beta-adrenergic receptors were subsequently determined. At 7-10 days of culture, cells became elongated and spindle-shaped showing a typical "hills and valleys" form. They were stained with anti-alpha-actin and anti-myosin antibodies. Radiolabeled ligand binding using [3H]N-methylscopolamine and [3H]CGP12177 showed that these cells expressed muscarinic and beta-adrenergic receptors. Stimulation of cultured cells with carbachol inhibited the forskolin-stimulated cyclic AMP formation, caused an elevation of intracellular Ca2+ concentration measured by fura-2 fluorometry. The latter response was almost completely blocked by 4-DAMP, a selective muscarinic M3 antagonist. On the other hand, stimulation of cultured cells with isoproterenol enhanced the basal cyclic AMP formation, which was reversed by carbachol. Therefore, the presence of functional muscarinic (both M2 and M3) as well as beta-adrenergic receptors was confirmed in pure culture of the rat bladder smooth muscle cells obtained by using an enzymic isolation method.     

The inhibitory effect of neonatal rat smooth muscle cells and elastic extracellular matrix on development of the newly seeded cell population in culture

Neonatal smooth muscle cells were seeded in standard plastic Falcon flasks, on top of another 2-month-old culture of the same cell population or on top of an acellular matrix prepared by removal of these cells. The effect of both complete and acellular layers on the production of elastin, collagen and total extracellular matrix (EM) proteins as well as on cell division was measured. Compared with the standard population grown on plastic, the complete cell layer almost completely prevented the newly seeded cells from dividing. The acellular matrix did not affect cell doubling but caused a distinct decrease in the production of EM components.     

Secretion of a new growth factor, smooth muscle cell derived growth factor, distinct from platelet derived growth factor by cultured rabbit aortic smooth muscle cells

In attempts to determine the mechanism of proliferation of arterial smooth muscle cells (SMC) in intimal atheromatous lesions, autocrine secretion of growth factors by SMC has recently received much attention. Here we report a new growth factor named smooth muscle cell derived growth factor (SDGF). Cultured rabbit medial SMC secreted SDGF for 1 week during their incubation in serum-free media only after at least 4 passages. SDGF differed from platelet derived growth factor (PDGF) physicochemically, immunologically, and biologically. The properties of SDGF also seemed different from those of other known growth factors that stimulate the proliferation of mesenchymal cells.     

Application of Fourier linear spectral analyses to the characterization of smooth muscle contractile signals

In intestinal smooth muscle, peristaltic activity emerges from a depolarization wave generated by Cajal's interstitial cells that travels longitudinally and transversally towards adjacent smooth muscle. The electrophysiological diversity of cell populations involved in the generation and transmission of excitation between nerve and muscle cells, as well as the specialization in the excitation-contraction coupling of smooth muscle cells, makes it difficult to extrapolate individual cell responses to overall peristaltic activity. It is conceivable intestinal contractile activity as the macroscopic output from a multicellular system in time and frequency domains. Given that contractile signals are usually linear and stationary, application of frequency analyses using the discrete Fourier transform allows the accurate definition of amplitudes and phases of harmonic components in the frequency spectrum of contractile activity records, as well as the power spectrum of the signal. In addition, by using the short-time Fourier transform it is also possible to obtain the spectrogram of contractile signals, which allows the identification of non-stationary events. Often, the combined usage of these types of analyses together with specific pharmacological and molecular biology tools is sufficient to unveil the cellular and molecular locus of action of modulators of peristaltism, including hormones and different natural and synthetic compounds.     

Effects of cyclic stretch on proliferation of mesenchymal stem cells and their differentiation to smooth muscle cells

Bone marrow mesenchymal stem cells (MSCs) are capable of differentiating into a variety of cell types such as vascular smooth muscle cells (SMCs). In this study, we investigated influence of cyclic stretch on proliferation of hMSCs for different loading conditions, alignment of actin filaments, and consequent differentiation to SMCs. Isolated cells from bone marrow were exposed to cyclic stretch utilizing a customized device. Cell proliferation was examined by MTT assay, alignment of actin fibers by a designed image processing code, and cell differentiation by fluorescence staining. Results indicated promoted proliferation of hMSCs by cyclic strain, enhanced by elevated strain amplitude and number of cycles. Such loading regulated smooth muscle α-actin, and reoriented actin fibers. Cyclic stretch led to differentiation of hMSCs to SMCs without addition of growth factor. It was concluded that applying appropriate loading treatment on hMSCs could enhance proliferation capability, and produce functional SMCs for engineered tissues.     

Impact of glafenine hydrochloride on human endothelial cells and human vascular smooth muscle cells: a substance reducing proliferation, migration and extracellular matrix synthesis

The aim of this study was to examine the effects of glafenine hydrochloride (a nonsteroidal anti-inflammatory drug) on proliferation, clonogenic activity, cell-cycle, migration, and the extracellular matrix protein tenascin of human aortic smooth muscle cells (haSMCs) and human endothelial cells (ECs) in vitro.HaSMCs and ECs were seeded in tissue culture flasks. The cells were treated for 4 days with glafenine hydrochloride (10 microM, 50 microM, 100 microM). Half of the treated groups were incubated again with glafenine hydrochloride, the other half received medium free of glafenine hydrochloride every 4 days until day 20. The growth kinetics and clonogenic activity were assessed. Cell cycle distribution was investigated by FACS, migratory ability was evaluated, and effects on extracellular matrix synthesis were assessed by immunofluorescence.Glafenine hydrochloride inhibited the proliferation and clonogenic activity of haSMCs and ECs in a dose-dependent manner. A block in the G2/M phase and a reduction in the G1 phase occurred. The migratory ability of haSMCs was impaired in a dose-dependent manner and the extracellular matrix protein tenascin was reduced. As glafenine hydrochloride has the ability to fully inhibit proliferation and to partially inhibit migration in haSMCs, it could be an interesting substance for further research in the field of restenosis therapy.     

Development of the enteric nervous system,smooth muscle and interstitial cells of Cajal in the human gastrointestinal tract

The generation of functional neuromuscular activity within the pre-natal gastrointestinal tract requires the coordinated development of enteric neurons and glial cells, concentric layers of smooth muscle and interstitial cells of Cajal (ICC). We investigated the genesis of these different cell types in human embryonic and fetal gut material ranging from weeks 4–14. Neural crest cells (NCC), labelled with antibodies against the neurotrophin receptor p75^NTR, entered the foregut at week 4, and migrated rostrocaudally to reach the terminal hindgut by week 7. Initially, these cells were loosely distributed throughout the gut mesenchyme but later coalesced to form ganglia along a rostrocaudal gradient of maturation; the myenteric plexus developed primarily in the foregut, then in the midgut, and finally in the hindgut. The submucosal plexus formed approximately 2–3 weeks after the myenteric plexus, arising from cells that migrated centripetally through the circular muscle layer from the myenteric region. Smooth muscle differentiation, as evidenced by the expression of -smooth muscle actin, followed NCC colonization of the gut within a few weeks. Gut smooth muscle also matured in a rostrocaudal direction, with a large band of -smooth muscle actin being present in the oesophagus at week 8 and in the hindgut by week 11. Circular muscle developed prior to longitudinal muscle in the intestine and colon. ICC emerged from the developing gut mesenchyme at week 9 to surround and closely appose the myenteric ganglia by week 11. By week 14, the intestine was invested with neural cells, longitudinal, circular and muscularis mucosae muscle layers, and an ICC network, giving the fetal gut a mature appearance.A.S.W. is funded by a PhD studentship awarded to A.J.B. by the Child Health Research Appeal Trust.     

Ablation of connexin43 in smooth muscle cells of the mouse intestine: functional insights into physiology and morphology

Connexin43 (Cx43) gap-junction channels are highly abundant in intestinal smooth muscle but their functional impact has not been studied so far. Here, we have aimed to elucidate the functional role of Cx43 in the tunica muscularis of the mouse intestine in vivo. Transgenic mice with conditional deletion of Cx43 in smooth muscle cells (SMC) were generated. Histological investigations by immunofluorescence analyses and organ-bath recordings to assess the contractility of intestinal tissue strips were carried out. Measurements of gastrointestinal transit and of the visceromotor response by utilizing a standardized colorectal distension model to quantify alterations of visceral sensory function were also performed in SMC-specific Cx43 null mice and control littermates. Histologically, we found thickening of the tunica muscularis and a 13-fold increase of neutrophil infiltration of the gastrointestinal wall of SMC-specific Cx43 null mice. These animals also exhibited a decrease of 29% in gastrointestinal transit time. In contrast, the visceromotor response to a standardized colorectal distension was elevated, as was the contractility in SMC-specific Cx43 null mice, compared with controls. Thus, SMC-specific ablation of Cx43 in mice leads to morphological and functional alterations of the intestinal tunica muscularis, to gastrointestinal motor dysfunction and to altered visceral sensory function. This study was supported by a grant from the German Research Association (Wi 270/25-1,2) to K.W. and in part by the IFORES program of the University Hospital, Essen, Germany.     

Inhibitory effect of phorbol ester on carbachol-induced signal transduction in cultured canine tracheal smooth muscle cells

Regulation of the increases in inositol 1,4,5-trisphosphate (IP₃) production and intracellular Ca²⁺ concentration ([Ca²⁺]_i) by activation of protein kinase C (PKC) was investigated in cultured canine tracheal smooth muscle cells (TSMCs). Stimulation of TSMCs by carbachol led to IP₃ formation and caused an initial transient peak of [Ca²⁺]_i followed by a sustained elevation in a concentration-dependent manner. Pretreatment of TSMCs with phorbol 12-myristate 13-acetate (PMA, 1 µM) for 30 min blocked the carbachol-induced IP₃ formation and Ca²⁺ mobilization. Following preincubation, carbachol-induced Ca²⁺ mobilization recovered within 24 h. The concentrations of PMA that gave half-maximal inhibition of carbachol-induced IP₃ formation and increase in [Ca²⁺]_i were 7 and 4 nM, respectively. Prior treatment of TSMCs with staurosporine (1 µM), a PKC inhibitor, inhibited the ability of PMA to attenuate carbachol-induced responses. Inactive phorbol ester, 4-phorbol 12,13-didecanoate at 1 µM, did not inhibit these responses to carbachol. The K_d and B_max of the muscarinic receptor for [³H]N-methylscopolamine binding were not significantly changed by PMA treatment. PMA also decreased PKC activity in the cytosol of TSMCs, while increasing it transiently in the membranes within 30 min. Thereafter, the membrane-associated PKC activity decreased and persisted for at least 24 h of PMA treatment. Taken together, these results suggest that activation of PKC may inhibit phosphoinositide hydrolysis and consequently attenuate the [Ca²⁺]_i increase or inhibit both responses independently. The inhibition by PMA of carbachol-induced responses was inversely correlated with membranous PKC activity.     

Featured Article: Temporal responses of human endothelial and smooth muscle cells exposed to uniaxial cyclic tensile strain

The physiology of vascular cells depends on stimulating mechanical forces caused by pulsatile flow. Thus, mechano-transduction processes and responses of primary human endothelial cells (ECs) and smooth muscle cells (SMCs) have been studied to reveal cell-type specific differences which may contribute to vascular tissue integrity. Here, we investigate the dynamic reorientation response of ECs and SMCs cultured on elastic membranes over a range of stretch frequencies from 0.01 to 1 Hz. ECs and SMCs show different cell shape adaptation responses (reorientation) dependent on the frequency. ECs reveal a specific threshold frequency (0.01 Hz) below which no responses is detectable while the threshold frequency for SMCs could not be determined and is speculated to be above 1 Hz. Interestingly, the reorganization of the actin cytoskeleton and focal adhesions system, as well as changes in the focal adhesion area, can be observed for both cell types and is dependent on the frequency. RhoA and Rac1 activities are increased for ECs but not for SMCs upon application of a uniaxial cyclic tensile strain. Analysis of membrane protrusions revealed that the spatial protrusion activity of ECs and SMCs is independent of the application of a uniaxial cyclic tensile strain of 1 Hz while the total number of protrusions is increased for ECs only. Our study indicates differences in the reorientation response and the reaction times of the two cell types in dependence of the stretching frequency, with matching data for actin cytoskeleton, focal adhesion realignment, RhoA/Rac1 activities, and membrane protrusion activity. These are promising results which may allow cell-type specific activation of vascular cells by frequency-selective mechanical stretching. This specific activation of different vascular cell types might be helpful in improving strategies in regenerative medicine.     

Dual effects of estrogen on vascular smooth muscle cells: receptor-mediated proliferative vs. metabolite-induced pro-senescent actions

Objective

To investigate the mechanism for the dual effects of estrogen on vascular smooth muscle cells (VSMCs).

Methods

Cultured rat VSMCs were exposed to gradient concentrations (10⁻⁹-10⁻⁵ M) of 17β-estradiol (E₂) with or without pre-administration of a broad-spectrum CYP450 inhibitor 1-aminobenzotriazole (ABT) (10 × 10⁻⁶ M) and an estrogen receptor (ER) antagonist ICI 182,780 (10⁻⁶ M), respectively. The growth, cell cycle progression, premature senescence, estrogen metabolites, reactive oxygen species (ROS) and DNA damage of the cells were analyzed with cell counting assay, flow cytometry, Western blot, liquid chromatography-mass spectrometry and comet assay, respectively.

Results

E₂ in its physiological levels from 10⁻⁹ M to 10⁻⁸ M had a concentration-dependent promoting effect on growth of VSMCs. However, when the concentration increased over 10⁻⁸ M, the growth-promoting effect gradually reversed to a growth-inhibiting action. When the activity of CYP450s was blocked by ABT, the growth-promoting effect of E₂ increased and did not reverse at high concentrations. Whereas when the ERs were blocked by ICI 182,780, E₂ showed a pure growth-inhibiting effect. The E₂ metabolites 2- and 4-hydroxyestradiols accumulated with the increase of E₂ over 10⁻⁸ M, which accompanied by increased ROS, DNA damage and cellular senescence. All of these changes were eliminated by block of CYP450s, indicating that the VSMC growth inhibition by E₂ is due to an increased production of ROS from accumulated E₂ metabolites which induces DNA damage, leading to VSMC premature senescence.

Conclusion

The complex effect of E₂ is due to two opposite actions: one ER-mediated and proliferative, and the other estrogen metabolite-induced and pro-senescent.     

Research of smooth muscle cells response to fluid flow shear stress by hyaluronic acid micro-pattern on a titanium surface

The morphology of vascular smooth muscle cells (SMCs) in the normal physiological state depends on cytoskeletal distribution and topology beneath, and presents vertical to the direction of blood flow shear stress (FFSS) although SMCs physiologically are not directly exposed to the shear conditions of blood flow. However, this condition is relevant for arteriosclerotic plaques and the sites of a vascular stent, and little of this condition in vitro has been studied and reported till now. It is unclear what will happen to SMC morphology, phenotype and function when the direction of the blood flow changed. In this paper, the distribution of SMCs in a specific area on Ti surface was regulated by micro-strips of hyaluronic acid (HA). Cell morphology depended on the distribution of the cytoskeleton extending along the micrographic direction. Simulated vascular FFSS was perpendicular or parallel to the direction of the cytoskeleton distribution. Based on investigating the morphology, apoptotic number, phenotypes and functional factors of SMCs, it was obtained that SMCs of vertical groups showed more apoptosis, expressed more contractile types and secreted less TGF-β1 factor compared with SMCs of parallel groups, the number of ECs cultured by medium from SMCs of parallel groups was larger than vertical groups. This study could help to understand the effect of direction change of FFSS on patterned SMC morphology, phenotype and function.