Smooth muscle cell adhesion to tissue engineering scaffolds

Synthetic polyesters of lactic and glycolic acid, and the extracellular matrix molecule collagen are among the most widely-utilized scaffolding materials in tissue engineering. However, the mechanism of cell adhesion to these tissue engineering scaffolds has not been extensively studied. In this paper, the mechanism of adhesion of smooth muscle cells to these materials was investigated. Vitronectin was found to be the predominant matrix protein adsorbed from serum-containing medium onto polyglycolic acid, poly(lactic co-glycolic) acid, and collagen two-dimensional films and three-dimensional scaffolds. Fibronectin adsorbed to both materials as well, although to a much lower density. Smooth muscle cell adhesion was mediated through specific integrin receptors interacting with these adsorbed proteins, as evidenced by both immunostaining and blocking studies. The receptors involved in adhesion included the alpha(v)beta5 to vitronectin, the alpha5beta1 to fibronectin and the alpha2beta1 to collagen I. Identification of the specific receptors used to adhere to these polymers clarifies why smooth muscle tissue development differs on these scaffolds, and may allow one to design tissue formation by controlling the surface chemistry of tissue engineering scaffolds.     

Smooth muscle cell conditioned medium elevates angiotensin-converting enzyme of bovine pulmonary artery endothelial cells

Conditioned medium obtained from bovine pulmonary artery smooth muscle cells (SMC) in culture was found to elevate angiotensin-I-converting enzyme (ACE) of bovine pulmonary artery endothelial cells (EC) by 2- to 3-fold upon incubation for 24 to 48 h. The elevation in ACE was time dependent and inhibited by 10(-6) M cycloheximide, making it likely that the elevation was related to new protein synthesis by EC. Conditioned medium from EC failed to produce the same effect. The stimulatory effect of SMC conditioned medium on EC ACE was uninfluenced by exposures to anoxia as compared with room air, either during conditioning of medium or while assaying the effect of conditioned medium on EC ACE. Inhibitors of prostaglandin metabolism and calcium transport failed to influence the stimulatory effect of SMC conditioned medium on EC ACE. The stimulatory effect on EC ACE by conditioned medium was additive to that produced by Ca2+ ionophore A23187 and 3-isobutyl-1-methylxanthine and, similar to baseline ACE, was inhibited by 3 x 10(-8) M ouabain. Thus, SMC produce a factor that regulates the level of EC ACE.     

Expression of fibrinolytic and coagulation factors in cocultured human endothelial and smooth muscle cells

Interactions between endothelial cells and smooth muscle cells are interesting from a tissue-engineering point of view. We have developed a coculture system that allows direct contact between these two cell types. The fibrinolytic factors PAI-1, tPA, and uPA and the coagulation factor TF, were studied at the gene level by RT-PCR and at the protein level by ELISA. Significant changes of all studied factors were seen at the gene level in cocultured endothelial cells. tPA and TF were upregulated 4- and 7-fold, respectively, and PAI-1 and uPA were downregulated 4- and 1.5-fold, respectively, compared with single-cultured controls. In cocultured smooth muscle cells alterations of PAI-1 and TF were significant, with a 1.5-fold upregulation of PAI-1 and a 2.5-fold downregulation of TF. Results at the protein level mirrored the gene expression results. These findings indicate that cocultured endothelial cells are rendered both hypercoagulative and hyperfibrinolytic.     

Smooth muscle cells express Ia antigens during arterial response to injury

The atherosclerotic plaque is composed of vascular smooth muscle cells and of macrophages and lymphocytes derived from the blood. We have recently shown that smooth muscle cells express class II MHC (Ia) antigens in the atherosclerotic plaque but not in the normal vessel wall. In the present study, we have tested whether there is a relationship between arterial injury and smooth muscle Ia expression. Rat carotid arteries were deendothelialized with a balloon catheter, and the rat Ia antigens, I-A and I-E, were detected by immunocytochemistry. Leukocytes and smooth muscle cells were identified by monoclonal antibodies to cell type-specific antigens. Normal rat carotids were always I-A and I-E negative, but approximately 10% of the cells became I-A-positive and 5% I-E positive during the response to injury. Ia expression was limited to non-replicating smooth muscle cells, suggesting that Ia is expressed as part of an activation response in a nondividing subfraction of the cells. The frequency of Ia-positive smooth muscle cells was maximal at 2 weeks postinjury, which coincided with maximal leukocyte infiltration and with the appearance of T lymphocytes in the neointima. This suggests that T cells induce Ia expression in smooth muscle cells. This was confirmed in cell culture experiments, where conditioned media from activated T lymphocytes induced Ia antigens in arterial smooth muscle cells. A similar induction of Ia expression was induced by recombinant gamma interferon, suggesting that this lymphokine may be responsible for smooth muscle Ia expression also in vivo.     

Multinucleated giant cell phenotype in response to stimulation

Macrophages fuse into multinucleated giant cells (MGC) in many pathological conditions. Despite MGC correlations with granulomas, their functional contribution to inflammation is relatively unknown. An in vitro mouse model of IL-4-induced bone marrow-derived macrophage fusion and microfiltration were used to generate enriched MGC and macrophage populations. Phenotypes were compared in response to well-known inflammatory stimuli, including lipopolysaccharide and crocidolite asbestos. Surface markers were assessed by flow cytometry: CD11b, CD11c, F4/80, and MHC II. Secreted cytokines were assessed by multiplex immunoassay: IFN-γ, IL-1β, IL-6, TNF-α, IL-10, IL-13, and IL-33. Results show that MGC maintained macrophage surface protein expression but lost the ability to produce a cytokine response. This suggests a potentially beneficial role of MGC in isolating the host from a foreign body without contributing to excessive inflammation. This study and future research using other stimulants and environments are important to gaining a fundamental MGC cell biology understanding. This will inform approaches to controlling the foreign body response to particle exposure, medical implants, and many diseases associated with granulomas.     

Vascular apolipoprotein e expression and recruitment from circulation to modulate smooth muscle cell response to endothelial denudation

Apolipoprotein E (apoE) has been shown previously to have anti-proliferative and anti-migratory effects on smooth muscle cells in culture. In addition, overexpression of the apoE gene also reduces neointimal hyperplasia in mice after endothelial denudation. In this investigation, immunohistochemical techniques were used to demonstrate that apoE was present in the medial smooth muscle layers of the carotid artery between 1 and 28 days after endothelial cell denudation. Analysis of transgenic mice overexpressing human apoE in the liver revealed that apoE was recruited from the circulation to the injured site of the vessel wall. In situ hybridization using a mouse-specific apoE mRNA probe confirmed that apoE was also synthesized in the carotid artery after endothelial denudation. Interestingly, apoE accumulation in apoE transgenic mice followed a layer-specific pattern, and was inversely associated with smooth muscle alpha-actin expression. The vascular accumulation of apoE after endothelial denudation, and its association with alpha-actin-depleted smooth muscle cells, suggest that apoE inhibition of injury-induced neointimal hyperplasia is not due to the inhibition of injury-induced smooth muscle cell de-differentiation, but is likely a direct effect of apoE on smooth muscle cell migration and proliferation.     

Smooth muscle cell adhesion on crosslinked hyaluronan gels

Hyaluronic acid (HA)-based polymers (hylans) are highly biocompatible and can be structurally modified to obtain desired mechanical properties. This study evaluated divinyl sulfone-crosslinked solid and particulate hylans as cellular scaffolds. These two hylan types differ in surface characteristics, mode of preparation, HA content, and extent of crosslinking. Neonatal rat aortic smooth muscle cells were cultured on hylan gels coated with matrix factors including collagen I, ECM gel, laminin, and fibronectin and on uncoated controls for < or =4 weeks. Cell attachment was sparse on uncoated controls but significantly enhanced on coated gels. Cell morphology was influenced by the identity of the matrix factors coated and the surface topography of the hylan gels. Cells attached to coated particulate gels appeared either highly spread (collagen, fibronectin) or irregularly shaped (ECM gel, laminin). Cells on laminin and fibronectin-coated solid gels were rounded and nonproliferative. Cells proliferated most rapidly on ECM gel-coated gels. The uneven surface of particulate gels induced more protein deposition and the subsequent attachment and active proliferation of cells. This study shows that surface texturizing and subsequent surface treatment with matrix factors enhances cell attachment and proliferation of hylans. These results are useful toward developing bioengineered materials based on cell-hylan composites.     

Smooth muscle cell markers in developing rat lung

We employed a panel of antibodies directed against cytoskeletal and contractile proteins in a developmental study to follow the differentiation and distribution of smooth muscle-like cells in the rat lung. We observed that, in the mesenchyme around developing airways and vessels, desmin replaces vimentin as the predominant intermediate filament as specialization toward smooth muscle occurs. Normally, desmin and smooth muscle myosin were expressed together in the cells and their acquisition appeared indicative of terminal differentiation of smooth muscle. In this regard, the maturation of vascular smooth muscle is delayed in the lung relative to that surrounding the developing air passages. alpha-smooth muscle actin-containing cells form a thicker coat around the primitive airway tubes and extend farther down the tree than desmin or smooth muscle myosin-positive cells. This suggests that the alpha-actin is a marker for initial differentiation of smooth muscle cells and that these cells arise from the enveloping mesenchyme. In the pseudoglandular and canalicular lung, alpha-actin-containing cells were also found in regions of epithelial tube cleft formation, suggesting an association with the process of branching morphogenesis. In addition, a large complement of alpha-actin-positive but smooth muscle myosin-negative cells were observed in the saccular interstitium during the period of secondary saccule formation and capillary reorganization that leads to final alveolarization. In summary, we note an association of smooth muscle-like, alpha-actin-containing cells with areas and periods of remodeling during normal pulmonary development. This observation may have relevance to the repair process in the adult lung.     

血管平滑肌细胞表型转变与糖尿病血管病变

血管平滑肌细胞按其形态、功能及细胞标志蛋白的不同可分为收缩型和合成型。血管平滑肌细胞受生化因子、细胞外基质成分、机械性刺激等多种因素的调节,其表型转变的分子机制主要与KLF4/Myo D/SRF轴相关。糖尿病的高血糖、胰岛素抵抗、脂类代谢紊乱、炎症反应可诱导血管平滑肌细胞表型转变,使其迁移、增殖能力增强,从而导致糖尿病血管病变。     

Myostatin knockout mice increase oxidative muscle phenotype as an adaptive response to exercise

Myostatin-deficient mice (MSTN ^−/−) display excessive muscle mass and this is associated with a profound loss of oxidative metabolic properties. In this study we analysed the effect of two endurance-based exercise regimes, either a forced high-impact swim training or moderate intensity voluntary wheel running on the adaptive properties of the tibialis anterior and plantaris muscle from MSTN ^−/− mice. MSTN ^−/− and wild type (MSTN ^+/+) animals had comparable performances in the wheel running regime in terms of distance, average speed and time, but MSTN ^−/− mice showed a reduced ability to sustain a high-impact activity via swimming. Swim training elicited muscle specific adaptations on fibre type distribution in MSTN ^−/−; the tibialis anterior displaying a partial transformation in contrast to the plantaris which showed no change. Conversely, wheel running induced similar changes in fibre type composition of both muscles, favouring transitions from IIB-to-IIA. Succinate dehydrogenase activity, an indicator of mitochondrial oxidative potential was increased in response to either exercise regime, with wheel running eliciting more robust changes in the MSTN ^−/− muscles. Examination of the cross sectional area of individual fibre types showed genotype-specific responses with MSTN ^−/− mice exhibiting an incapability of fibre enlargement following the wheel running regime, as opposed to MSTN ^+/+ mice and a greater susceptibility to muscle fibre area loss following swimming. In conclusion, the muscle fibre hypertrophy, oxidative capacity and glycolytic phenotype of myostatin deficient muscle can be altered with endurance exercise regimes.     

B prolymphocytic leukemia cell lines: phenotype and response to B cell differentiation factor

Three recently-derived cell lines of prolymphocytic leukemia origin were studied. The phenotype of the cells, analysed in terms of expression of immunoglobulin and other B cell maturation markers, indicates that the cells are mature B lymphocytes close to the plasma cell stage of differentiation. All three cell lines secrete IgM, and two of the three lines respond to B cell differentiation factor with an increased secretion of IgM.     

Mast cell tryptase may modulate endothelial cell phenotype in healing myocardial infarcts

Mast cells and macrophages infiltrate healing myocardial infarcts and may play an important role in regulating fibrous tissue deposition and extracellular matrix remodelling. This study examined the time-course of macrophage and mast cell accumulation in healing infarcts and studied the histological characteristics and protease expression profile of mast cells in a canine model of experimental infarction. Although macrophages were more numerous than mast cells in infarct granulation tissue, macrophage density decreased during maturation of the scar, whereas mast cell numbers remained persistently elevated. During the inflammatory phase of infarction, newly recruited leucocytes infiltrated the injured myocardium and appeared to be clustered in close proximity to degranulating cardiac mast cells. During the proliferative phase of healing, mast cells had decreased granular content and were localized close to infarct neovessels. In contrast, macrophages showed no selective localization. Mast cells in healing canine infarcts were alcian blue/safranin-positive cells that expressed both tryptase and chymase. In order to explain the pro-inflammatory and angiogenic actions of tryptase--the major secretory protein of mast cells--its effects on endothelial chemokine expression were examined. Chemokines are chemotactic cytokines that play an important role in leucocyte trafficking and angiogenesis and are highly induced in infarcts. Tryptase, a proteinase-activated receptor (PAR)-2 agonist, induced endothelial expression of the angiogenic chemokines CCL2/MCP-1 and CXCL8/IL-8, but not the angiostatic chemokine CXCL10/IP-10. Endothelial PAR-2 stimulation with the agonist peptide SLIGKV induced a similar chemokine expression profile. Mast cell tryptase may exert its angiogenic effects in part through selective stimulation of angiogenic chemokines.     

Reduction of perifusate magnesium alters inotropic response of papillary muscle to ion channel modulators.

Magnesium has a significant role in the regulation of ion transport. Marginal deficiency of Mg can therefore affect myocardial excitability and contractility. This study was taken up with the objective of examining the inotropic response of the myocardium to variation in extracellular [Mg]o and identifying the ion channels and pumps mediating the inotropic changes. Electrically stimulated rat papillary muscle was used as the experimental model and mechanical changes were recorded using a physiograph. Channel specific antagonists were used to identify the channels mediating the functional changes. Diastolic Ca2+ levels were determined in isolated myocytes by the ratiometric method using the fluorescent indicator Fura2-AM. A negative association was observed between the level of [Mg]o and force of contraction, with a peak at 0.48 mM Mg. The force of contraction in Mg deficient medium (0.48 mM) was 158% of control (1.2 mM Mg) (p < 0.001). Inotropic response to the L-type channel antagonist (verapamil-1 microm) and NaK ATPase inhibitor (Ouabain-0.3 mM) was augmented in Mg deficiency (p < 0.005), indicating activation of the channel and the pump. The response to T-type channel inhibitor (NiCl2-40 microM) was attenuated in Mg deficiency (p < 0.05). The response to the sarcoplasmic reticular Ca pump inhibitor (caffeine-10 mM) and the SR Ca2+ release channel inhibitor (ryanodine-1 microM) were not significantly affected by Mg deficiency. Diastolic level of Ca2+ increased with a decrease in Mg (p < 0.05). The observations of the study lead to the conclusion that the positive inotropic response in Mg deficiency is mediated by an increase in basal Ca2+ combined with Ca-induced-Ca release consequent to Ca2+ influx through L-type Ca channel. Variation in sensitivity to Ca channel blockers and NaK ATPase inhibitor in Mg deficiency can have pharmacological implications.     

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.     

Proteoglycans on endothelial cells present adhesion-inducing cytokines to leukocytes

Leukocyte recruitment from the blood circulation into tissue is essential for effective immune responses, and is, consequently, carefully regulated. In this article Yoshiya Tanaka and co-workers describe a model in which proteoglycans on the luminal surface of endothelium capture pro-adhesive cytokines. These cytokines provide the adhesion-inducing signal to particular leukocyte subsets which initiates their transmigration.     

Smooth muscle and other cell sources for human blood vessel engineering

Despite substantial progress in the field of vascular tissue engineering over the past decades, transition to human models has been rather challenging. The limited replicative life spans of human adult vascular cells, and their slow rate of collagenous matrix production in vitro, have posed important hurdles in the development of mechanically robust and biologically functional engineered grafts. With the more recent advances in the field of stem cells, investigators now have access to a plethora of new cell source alternatives for vascular engineering. In this paper, we review various alternative cell sources made available more recently for blood vessel engineering and also present some recent data on the derivation of smooth muscle cells from human induced pluripotent stem cells.     

Smooth muscle cell mineralocorticoid receptors: role in vascular function and contribution to cardiovascular disease

The mineralocorticoid receptor (MR), a member of the steroid receptor family, regulates blood pressure by mediating the effects of the hormone aldosterone on renal sodium handling. In recent years, it has become clear that MR is expressed in vascular smooth muscle cells (SMCs), and interest has grown in understanding the direct role of SMC MR in regulating vascular function. This interest stems from multiple clinical studies where MR inhibitor treatment reduced the incidence of cardiovascular events and mortality. This review summarizes the most recent advances in our understanding of SMC MR in regulating normal vascular function and in promoting vascular disease. Many new studies suggest a role for SMC MR activation in stimulating vascular contraction and contributing to vessel inflammation, fibrosis, and remodeling. These detrimental vascular effects of MR activation appear to be independent of changes in blood pressure and are synergistic with the presence of endothelial dysfunction or damage. Thus, in humans with underlying cardiovascular disease or cardiovascular risk factors, SMC MR activation may promote hypertension, atherosclerosis, and vascular aging. Further exploration of the molecular mechanisms for the effects of SMC MR activation has the potential to identify novel therapeutic targets to prevent or treat common cardiovascular disorders.