Inhibitory role of reactive oxygen species in the differentiation of multipotent vascular stem cells into vascular smooth muscle cells in rats: a novel aspect of traditional culture of rat aortic smooth muscle cells

Proliferative or synthetic vascular smooth muscle cells (VSMCs) are widely accepted to be mainly derived from the dedifferentiation or phenotypic modulation of mature contractile VSMCs, i.e., a phenotype switch from a normally quiescent and contractile type into a proliferative or synthetic form. However, this theory has been challenged by recent evidence that synthetic VSMCs predominantly originate instead from media-derived multipotent vascular stem cells (MVSCs). To test these hypotheses further, we re-examine whether the conventional rat aortic SMC (RASMC) culture involves the VSMC differentiation of MVSCs or the dedifferentiation of mature VSMCs and the potential mechanism for controlling the synthetic phenotype of RASMCs. We enzymatically isolated RASMCs and cultured the cells in both a regular growth medium (RGM) and a stem cell growth medium (SCGM). Regardless of culture conditions, only a small portion of freshly isolated RASMCs attaches, survives and grows slowly during the first 7 days of primary culture, while expressing both SMC- and MVSC-specific markers. RGM-cultured cells undergo a process of synthetic SMC differentiation, whereas SCGM-cultured cells can be differentiated into not only synthetic SMCs but also other somatic cells. Notably, compared with the RGM-cultured differentiated RASMCs, the SCGM-cultured undifferentiated cells exhibit the phenotype of MVSCs and generate greater amounts of reactive oxygen species (ROS) that act as a negative regulator of differentiation into synthetic VSMCs. Knockdown of phospholipase A2, group 7 (Pla2g7) suppresses ROS formation in the MVSCs while enhancing SMC differentiation of MVSCs. These results suggest that cultured synthetic VSMCs can be derived from the SMC differentiation of MVSCs with ROS as a negative regulator.     

Close relation of arterial ICC-like cells to the contractile phenotype of vascular smooth muscle cell

This work aimed to establish the lineage of cells similar to the interstitial cells of Cajal (ICC), the arterial ICC-like (AIL) cells, which have recently been described in resistance arteries, and to study their location in the artery wall. Segments of guinea-pig mesenteric arteries and single AIL cells freshly isolated from them were used. Confocal imaging of immunostained cells or segments and electron microscopy of artery segments were used to test for the presence and cellular localization of selected markers, and to localize AIL cells in intact artery segments. AIL cells were negative for PGP9.5, a neural marker, and for von Willebrand factor (vWF), an endothelial cell marker. They were positive for smooth muscle alpha-actin and smooth muscle myosin heavy chain (SM-MHC), but expressed only a small amount of smoothelin, a marker of contractile smooth muscle cells (SMC), and of myosin light chain kinase (MLCK), a critical enzyme in the regulation of smooth muscle contraction. Cell isolation in the presence of latrunculin B, an actin polymerization inhibitor, did not cause the disappearance of AIL cells from cell suspension. The fluorescence of basal lamina protein collagen IV was comparable between the AIL cells and the vascular SMCs and the fluorescence of laminin was higher in AIL cells compared to vascular SMCs. Moreover, cells with thin processes were found in the tunica media of small resistance arteries using transmission electron microscopy. The results suggest that AIL cells are immature or phenotypically modulated vascular SMCs constitutively present in resistance arteries.     

Stem cell marker expression,proliferation and apoptosis of vascular smooth muscle cells

Vascular endothelial Flt-1 and other stem cell markers are variably expressed in vascular smooth muscle cells (SMCs) during normal and pathological conditions, but their biological role remains uncertain. In normal rat aorta, rare flt-1+ and c-kit+ SMCs were detected. Fifteen days after injury, 61.8+3.8, 45.7+3% of the intimal cells resulted flt-1+ and c-kit+ and expressed low level of alpha-smooth muscle actin; CD133+ cells were 5.6+0.7%. BrDU+/flt-1+ largely predominated in the neointima, whereas BrDU+/CD133+ cells were rare. Forty-five and sixty days after injury, intimal proliferation such as BrDU+ cells was greatly reduced. After sixty days, intimal stem marker expression had almost disappeared whereas alpha-smooth muscle actin was restored. Flk-1 and Oct-4 SMC immunodection was consistently negative. In vitro, intimal cells obtained fifteen days after injury exhibited an epithelioid phenotype and increased flt-1 and c-kit protein and mRNA and low smooth muscle markers compared to spindle-shaped medial and intimal SMCs obtained after sixty days. Epithelioid clones, independently from layer of origin, were similar in stem cell marker expression. The anti-flt-1 blocking antibody added to epithelioid SMC cultures reduced serum-deprived apoptosis and migration but not PDGF-BB-induced proliferation, and increased cell-populated collagen lattice contraction. In conclusion, stem marker expression in vascular SMCs was variable, chronologically regulated and prevailed in epithelioid populations and clones; among stem markers, flt-1 expression critically regulates intimal SMC response to microenviromental changes.     

Modulation of pulmonary vascular smooth muscle cell phenotype in hypoxia: role of cGMP-dependent protein kinase

Chronic hypoxia triggers pulmonary vascular remodeling, which is associated with a modulation of the vascular smooth muscle cell (SMC) phenotype from a contractile, differentiated to a synthetic, dedifferentiated state. We previously reported that acute hypoxia represses cGMP-dependent protein kinase (PKG) expression in ovine fetal pulmonary venous SMCs (FPVSMCs). Therefore, we tested if altered expression of PKG could explain SMC phenotype modulation after exposure to hypoxia. Hypoxia-induced reduction in PKG protein expression strongly correlated with the repressed expression of SMC phenotype markers, myosin heavy chain (MHC), calponin, vimentin, alpha-smooth muscle actin (alphaSMA), and thrombospondin (TSP), indicating that hypoxic exposure of SMC induced phenotype modulation to dedifferentiated state, and PKG may be involved in SMC phenotype modulation. PKG-specific small interfering RNA (siRNA) transfection in FPVSMCs significantly attenuated calponin, vimentin, and MHC expression, with no effect on alphaSMA and TSP. Treatment with 30 microM Drosophila Antennapedia (DT-3), a membrane-permeable peptide inhibitor of PKG, attenuated the expression of TSP, MHC, alphaSMA, vimentin, and calponin. The results from PKG siRNA and DT-3 studies indicate that hypoxia-induced reduction in protein expression was also similarly impacted by PKG inhibition. Overexpression of PKG in FPVSMCs by transfection with a full-length PKG construct tagged with green fluorescent fusion protein (PKG-GFP) reversed the effect of hypoxia on the expression of SMC phenotype marker proteins. These results suggest that PKG could be one of the determinants for the expression of SMC phenotype marker proteins and may be involved in the maintenance of the differentiated phenotype in pulmonary vascular SMCs in hypoxia.     

Differentiation of Human Induced-Pluripotent Stem Cells into Smooth-Muscle Cells: Two Novel Protocols

Conventional protocols for differentiating human induced-pluripotent stem cells (hiPSCs) into smooth-muscle cells (SMCs) can be inefficient and generally fail to yield cells with a specific SMC phenotype (i.e., contractile or synthetic SMCs). Here, we present two novel hiPSC-SMC differentiation protocols that yield SMCs with predominantly contractile or synthetic phenotypes. Flow cytometry analyses of smooth-muscle actin (SMA) expression indicated that ~45% of the cells obtained with each protocol assumed an SMC phenotype, and that the populations could be purified to ~95% via metabolic selection. Assessments of cellular mRNA and/or protein levels indicated that SMA, myosin heavy chain II, collagen 1, calponin, transgelin, connexin 43, and vimentin expression in the SMCs obtained via the Contractile SMC protocol and in SMCs differentiated via a traditional protocol were similar, while SMCs produced via the Sythetic SMC protocol expressed less calponin, more collagen 1, and more connexin 43. Differences were also observed in functional assessments of the two SMC populations: the two-dimensional surface area of Contractile SMCs declined more extensively (to 12% versus 44% of original size) in response to carbachol treatment, while quantification of cell migration and proliferation were greater in Synthetic SMCs. Collectively, these data demonstrate that our novel differentiation protocols can efficiently generate SMCs from hiPSCs.     

MicroRNA-145-based differentiation of human mesenchymal stem cells to smooth muscle cells

Objectives

To investigate the role of microRNA-145, that regulates gene expression of genes related to differentiation, proliferation and the phenotype of smooth muscle cells (SMCs), in the differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs) to SMCs.

Results

Real-time PCR analysis indicated significant upregulation of SMC markers, including SM-α-actin, calponin, caldesmon and SMMHC, in SMCs compared to hBM-MSCs. Conversely, Krüppel-like factor 4, the direct target of microRNA-145 and the suppressor of smooth muscle differentiation, was suppressed in hBM-MSC-derived SMCs. Western blot analysis and immunocytochemistry also confirmed that the introduction of microRNA-145 into hBM-MSCs induced mature contractile SMCs. The functionality of hBM-MSC-derived SMCs was assessed by proliferation assay using PDGF-BB and contractility assay using carbachol. The results showed that the produced SMCs contracted in response to carbachol stimulation.

Conclusion

Overexpression of microRNA-145 in undifferentiated hBM-MSCs results in functionally mature contractile SMCs that can be used in drug discovery and cell therapy in SMC disorders such as vascular disease.

     

Phenotypic changes of human smooth muscle cells during development: late expression of heavy caldesmon and calponin.

Expression of the regulatory contractile proteins, heavy caldesmon (h-caldesmon) and calponin was studied in human aortic smooth muscle cells (SMCs) during development and compared with the expression of alpha-SM-actin and smooth muscle-myosin heavy chain (SM-MHCs). For this study, novel monoclonal antibodies specific to SM-MHCs, h-caldesmon, and calponin were developed and characterized. Aortic SMCs from fetuses of 8-10 and 20-22 weeks of gestation express alpha-SM-actin and SM-MHCs, but neither h-caldesmon nor calponin were expressed as demonstrated by immunoblotting and immunofluorescence techniques. In the adult aortic tunica media, SMCs contain all four markers. Thus, the expression of calponin, similar to the expression of alpha-SM-actin, SM-MHCs, and h-caldesmon, is developmentally regulated in aortic SMCs. In the adult aortic subendothelial (preluminal) part of tunica intima, numerous cells containing SM-MHCs, but lacking h-caldesmon and calponin, were found. These results illustrate the similarity of SMCs from intimal thickenings and immature (fetal) SMCs. Expression of contractile proteins in the developing SMCs is coordinately regulated; however, distinct groups of proteins appear to exist whose expression is regulated differently. Actin and myosin, being major contractile proteins, also play a structural role and appear rather early in development, whereas caldesmon and calponin, being involved in regulation of contraction, can serve as markers of higher SMC differentiation steps. In contrast, h-caldesmon and calponin were already present in visceral SMCs (trachea, esophagus) of the 10-week-old fetus. These results demonstrate that the time course of maturation of visceral SMCs is different from that of vascular SMCs.     

Matrix stiffness modulates the differentiation of neural crest stem cells in vivo

Stem cells are often transplanted with scaffolds for tissue regeneration; however, how the mechanical property of a scaffold modulates stem cell fate in vivo is not well understood. Here we investigated how matrix stiffness modulates stem cell differentiation in a model of vascular graft transplantation. Multipotent neural crest stem cells (NCSCs) were differentiated from induced pluripotent stem cells, embedded in the hydrogel on the outer surface of nanofibrous polymer grafts, and implanted into rat carotid arteries by anastomosis. After 3 months, NCSCs differentiated into smooth muscle cells (SMCs) near the outer surface of the polymer grafts; in contrast, NCSCs differentiated into glial cells in the most part of the hydrogel. Atomic force microscopy demonstrated a stiffer matrix near the polymer surface but much lower stiffness away from the polymer graft. Consistently, in vitro studies confirmed that stiff surface induced SMC genes whereas soft surface induced glial genes. These results suggest that the scaffold’s mechanical properties play an important role in directing stem cell differentiation in vivo, which has important implications in biomaterials design for stem cell delivery and tissue engineering.     

Derivation of cranial neural crest-like cells from human embryonic stem cells

The neural crest is a transient population of multipotent progenitors contributing to a diverse array of tissues throughout the vertebrate embryo. Embryonic stem (ES) cells are able to form embryoid body and spontaneously differentiate to various lineages, following a reproducible temporal pattern of development that recapitulates early embryogenesis. Embryoid bodies were triturated and the dissociated cells were processed for fluorescence-activated cell sorting (FACS), and more than 1% of cells were identified as frizzled-3⁺/cadherin-11⁺. Expression of marker genes associated with various terminal fates was detected for chondrocytes, glia, neurons, osteoblasts and smooth muscles, indicating that the FACS-sorted frizzled-3⁺/cadherin-11⁺ cells were multipotent progenitor cells capable of differentiating to fates associated with cranial neural crest. Moreover, the sorted cells were able to self-renew and maintain multipotent differentiation potential. The derivation of cranial neural crest-like multipotent progenitor cells from ES cells provides a new tool for cell lineage analysis of neural crest in vitro.     

Shear Stress Modulation of Smooth Muscle Cell Marker Genes in 2-D and 3-D Depends on Mechanotransduction by Heparan Sulfate Proteoglycans and ERK1/2

Background

During vascular injury, vascular smooth muscle cells (SMCs) and fibroblasts/myofibroblasts (FBs/MFBs) are exposed to altered luminal blood flow or transmural interstitial flow. We investigate the effects of these two types of fluid flows on the phenotypes of SMCs and MFBs and the underlying mechanotransduction mechanisms.

Methodology/Principal Findings

Exposure to 8 dyn/cm² laminar flow shear stress (2-dimensional, 2-D) for 15 h significantly reduced expression of α-smooth muscle actin (α-SMA), smooth muscle protein 22 (SM22), SM myosin heavy chain (SM-MHC), smoothelin, and calponin. Cells suspended in collagen gels were exposed to interstitial flow (1 cmH₂O, ∼0.05 dyn/cm², 3-D), and after 6 h of exposure, expression of SM-MHC, smoothelin, and calponin were significantly reduced, while expression of α-SMA and SM22 were markedly enhanced. PD98059 (an ERK1/2 inhibitor) and heparinase III (an enzyme to cleave heparan sulfate) significantly blocked the effects of laminar flow on gene expression, and also reversed the effects of interstitial flow on SM-MHC, smoothelin, and calponin, but enhanced interstitial flow-induced expression of α-SMA and SM22. SMCs and MFBs have similar responses to fluid flow. Silencing ERK1/2 completely blocked the effects of both laminar flow and interstitial flow on SMC marker gene expression. Western blotting showed that both types of flows induced ERK1/2 activation that was inhibited by disruption of heparan sulfate proteoglycans (HSPGs).

Conclusions/Significance

The results suggest that HSPG-mediated ERK1/2 activation is an important mechanotransduction pathway modulating SMC marker gene expression when SMCs and MFBs are exposed to flow. Fluid flow may be involved in vascular remodeling and lesion formation by affecting phenotypes of vascular wall cells. This study has implications in understanding the flow-related mechanobiology in vascular lesion formation, tumor cell invasion, and stem cell differentiation.     

Myocardin overexpression is sufficient for promoting the development of a mature smooth muscle cell-like phenotype from human embryonic stem cells

Background

Myocardin is thought to have a key role in smooth muscle cell (SMC) development by acting on CArG-dependent genes. However, it is unclear whether myocardin-induced SMC maturation and increases in agonist-induced calcium signalling are also associated with increases in the expression of non-CArG-dependent SMC-specific genes. Moreover, it is unknown whether myocardin promotes SMC development from human embryonic stem cells.

Methodology/Principal

Findings The effects of adenoviral-mediated myocardin overexpression on SMC development in human ESC-derived embryoid bodies were investigated using immunofluorescence, flow cytometry and real time RT-PCR. Myocardin overexpression from day 10 to day 28 of embryoid body differentiation increased the number of smooth muscle α-actin⁺ and smooth muscle myosin heavy chain⁺ SMC-like cells and increased carbachol-induced contractile function. However, myocardin was found to selectively regulate only CArG-dependent SMC-specific genes. Nevertheless, myocardin expression appeared to be sufficient to specify the SMC lineage.

Conclusions/Significance

Myocardin increases the development and maturation of SMC-like cells from human embryonic stem cells despite not activating the full repertoire of SMC genes. These findings have implications for vascular tissue engineering and other applications requiring large numbers of functional SMCs.     

Differential effects of equiaxial and uniaxial strain on mesenchymal stem cells

Bone marrow mesenchymal stem cells (MSCs) can differentiate into a variety of cell types, including vascular smooth muscle cells (SMCs), and have tremendous potential as a cell source for cardiovascular regeneration. We postulate that specific vascular environmental factors will promote MSC differentiation into SMCs. However, the effects of the vascular mechanical environment on MSCs have not been characterized. Here we show that mechanical strain regulated the expression of SMC markers in MSCs. Cyclic equiaxial strain downregulated SM alpha-actin and SM-22alpha in MSCs on collagen- or elastin-coated membranes after 1 day, and decreased alpha-actin in stress fibers. In contrast, cyclic uniaxial strain transiently increased the expression of SM alpha-actin and SM-22alpha after 1 day, which subsequently returned to basal levels after the cells aligned in the direction perpendicular to the strain direction. In addition, uniaxial but not equiaxial strain induced a transient increase of collagen I expression. DNA microarray experiments showed that uniaxial strain increased SMC markers and regulated the expression of matrix molecules without significantly changing the expression of the differentiation markers (e.g., alkaline phosphatase and collagen II) of other cell types. Our results suggest that uniaxial strain, which better mimics the type of mechanical strain experienced by SMCs, may promote MSC differentiation into SMCs if cell orientation can be controlled. This study demonstrates the differential effects of equiaxial and uniaxial strain, advances our understanding of the mechanical regulation of stem cells, and provides a rational basis for engineering MSCs for vascular tissue engineering and regeneration.     

Construction of vascular tissues with macro-porous nano-fibrous scaffolds and smooth muscle cells enriched from differentiated embryonic stem cells

Vascular smooth muscle cells (SMCs) have been broadly used for constructing tissue-engineered blood vessels. However, the availability of mature SMCs from donors or patients is very limited. Derivation of SMCs by differentiating embryonic stem cells (ESCs) has been reported, but not widely utilized in vascular tissue engineering due to low induction efficiency and, hence, low SMC purity. To address these problems, SMCs were enriched from retinoic acid induced mouse ESCs with LacZ genetic labeling under the control of SM22α promoter as the positive sorting marker in the present study. The sorted SMCs were characterized and then cultured on three-dimensional macro-porous nano-fibrous scaffolds in vitro or implanted subcutaneously into nude mice after being seeded on the scaffolds. Our data showed that the LacZ staining, which reflected the corresponding SMC marker SM22α expression level, was efficient as a positive selection marker to dramatically enrich SMCs and eliminate other cell types. After the sorted cells were seeded into the three-dimensional nano-fibrous scaffolds, continuous retinoic acid treatment further enhanced the SMC marker gene expression level while inhibited pluripotent maker gene expression level during the in vitro culture. Meanwhile, after being implanted subcutaneously into nude mice, the implanted cells maintained the positive LacZ staining within the constructs and no teratoma formation was observed. In conclusion, our results demonstrated the potential of SMCs derived from ESCs as a promising cell source for therapeutic vascular tissue engineering and disease model applications.     

Uniaxial mechanical strain modulates the differentiation of neural crest stem cells into smooth muscle lineage on micropatterned surfaces

Neural crest stem cells (NCSCs) play an important role in the development and represent a valuable cell source for tissue engineering. However, how mechanical factors in vivo regulate NCSC differentiation is not understood. Here NCSCs were derived from induced pluripotent stem cells and used as a model to determine whether vascular mechanical strain modulates the differentiation of NCSCs into smooth muscle (SM) lineage. NCSCs were cultured on micropatterned membranes to mimic the organization of smooth muscle cells (SMCs), and subjected to cyclic uniaxial strain. Mechanical strain enhanced NCSC proliferation and ERK2 phosphorylation. In addition, mechanical strain induced contractile marker calponin-1 within 2 days and slightly induced SM myosin within 5 days. On the other hand, mechanical strain suppressed the differentiation of NCSCs into Schwann cells. The induction of calponin-1 by mechanical strain was inhibited by neural induction medium but further enhanced by TGF-β. For NCSCs pre-treated with TGF-β, mechanical strain induced the gene expression of both calponin-1 and SM myosin. Our results demonstrated that mechanical strain regulates the differentiation of NCSCs in a manner dependent on biochemical factors and the differentiation stage of NCSCs. Understanding the mechanical regulation of NCSC differentiation will shed light on the development and remodeling of vascular tissues, and how transplanted NCSCs respond to mechanical factors.