Smooth Muscle Cell Markers Research Articles

Fibronectin matrix polymerization regulates smooth muscle cell phenotype through a Rac1 dependent mechanism.

Smooth muscle cells are maintained in a differentiated state in the vessel wall, but can be modulated to a synthetic phenotype following injury. Smooth muscle phenotypic modulation is thought to play an important role in the pathology of vascular occlusive diseases. Phenotypically modulated smooth muscle cells exhibit increased proliferative and migratory properties that accompany the downregulation of smooth muscle cell marker proteins. Extracellular matrix proteins, including fibronectin, can regulate the smooth muscle phenotype when used as adhesive substrates. However, cells produce and organize a 3-dimensional fibrillar extracellular matrix, which can affect cell behavior in distinct ways from the protomeric 2-dimensional matrix proteins that are used as adhesive substrates. We previously showed that the deposition/polymerization of fibronectin into the extracellular matrix can regulate the deposition and organization of other extracellular matrix molecules in vitro. Further, our published data show that the presence of a fibronectin polymerization inhibitor results in increased expression of smooth muscle cell differentiation proteins and inhibits vascular remodeling in vivo. In this manuscript, we used an in vitro cell culture system to determine the mechanism by which fibronectin polymerization affects smooth muscle phenotypic modulation. Our data show that fibronectin polymerization decreases the mRNA levels of multiple smooth muscle differentiation genes, and downregulates the levels of smooth muscle α-actin and calponin proteins by a Rac1-dependent mechanism. The expression of smooth muscle genes is transcriptionally regulated by fibronectin polymerization, as evidenced by the increased activity of luciferase reporter constructs in the presence of a fibronectin polymerization inhibitor. Fibronectin polymerization also promotes smooth muscle cell growth, and decreases the levels of actin stress fibers. These data define a Rac1-dependent pathway wherein fibronectin polymerization promotes the SMC synthetic phenotype by modulating the expression of smooth muscle cell differentiation proteins.

Transcriptome profiling reveals that the SM22α-regulated molecular pathways contribute to vascular pathology

Smooth muscle cell marker, SM22α, was down-regulated in the pathogenesis of arterial diseases including atherosclerosis, restenosis and abdominal aortic aneurysms. However, the question still exists whether this down-regulation actively contributes to the pathogenesis of vascular diseases. In an ongoing effort to understand the role of SM22α, here we explored transcriptome profiling by RNA-Seq from arteries of SM22α−/− and SM22α+/+ mice. Analysis revealed that the most enriched pathways caused by SM22α-knockout were hematopoiesis, inflammation and lipid metabolism, respectively, and NF-κB, RXRα and PPARα were the major upstream regulators. The candidate genes involved in inflammation and lipid metabolism were clustered in atherosclerosis. Thus we suspected that the molecular basis in SM22α−/− mice was already prepared for the initiation of atherosclerosis. Further analysis suggested the up-regulated TNF caused NF-κB pathway activation. Our results showed loss of SM22α exacerbated TNF-α-mediated NF-κB activation and increased the expression levels of ApoCI in vitro, while overexpression of SM22α suppressed TNF-α-mediated NF-κB activation. In addition, disruption of SM22α enhanced injury-induced neointimal hyperplasia, and increased expression levels of molecules related with cellular adhesion and extracellular matrix degradation. Taken together, these findings not only suggested down-regulation of SM22α can actively contribute to the pathogenesis of atherosclerosis from the molecular basis, but also further confirmed that the vascular cells of SM22α−/− mice may become more sensitive to extracellular stimulation, increasing its tendency to develop vascular diseases. Meanwhile, rescuing SM22α expression may provide a novel therapeutic strategy for arterial diseases.

Stem Cells Accumulate on a Decellularized Arterial Xenograft In Vivo

Surgical interest in complete arterial revascularization is expanding, but some patients lack suitable conduits for this goal. The field of stem cell biology is rapidly expanding and, together with the concepts of tissue engineering, offers the promise of growing autologous grafts in the laboratory. We aim to develop a model using human arteries as vascular grafts in a murine model and to assess the cellular accumulation on these grafts. Human arterial samples were collected and decellularized using an ionic detergent. These vessel scaffolds were then used as grafts in an invivo mouse model, and the cellular accumulation on them was examined histologically and by cell culture with assessment of their physiologic properties. Left internal mammary artery branches werefully decellularized and successfully implanted intoa murine model. Grafts were repopulated by cells expressing stem cell markers cluster of differentiation 34 and stage-specific embryonic antigen, and subsequently expressed markers of mature endothelial and smooth muscle cells (cluster of differentiation 31, calponin, and myosin heavy chain). The migratory capacity of the cultured cells was significantly higher than that of mouse smooth muscle cells (p < 0.001). We describe the successful use of human arteries in a murine graft model, allowing the study of repopulation. Decellularized grafts are repopulated by cells expressing stem cell markers and subsequently express smooth muscle and endothelial cell markers. This model has the potential to be used for further development of laboratory-grown vascular grafts.

Olfactomedin 2, a novel regulator for transforming growth factor‐β‐induced smooth muscle differentiation (866.1)

Olfactomedin 2 (Olfm2) is a developmentally regulated gene and mediates the early nervous system development. The role of Olfm2 in smooth muscle cell (SMC) differentiation, however, remains elusive. In this study, we found that Olfm2 was induced in TGF‐β‐stimulated SMC differentiation of human embryonic mesenchymal cells. Olfm2 knockdown suppressed TGF‐β‐induced expression of SMC markers including smooth muscle α‐actin (α‐SMA), SM22α and smooth muscle myosin heavy chain (SMMHC). Olfm2 overexpression, on the other hand, promoted SMC marker expression. TGF‐β induced Olfm2 nuclear accumulation, indicating that Olfm2 may function as a nuclear factor and be involved in transcriptional activation of SMC marker genes. Indeed, Olfm2 regulated SMC marker promoter activity in a serum response factor (SRF)‐CArG dependent manner. Mechanistically, Olfm2 physically and functionally interacted with SRF, but did not alter its binding affinity to CArG elements. Instead, Olfm2 facilitated the interaction between myocardin and SRF, which is required for SMC marker gene transcription. Taken together, our data demonstrate that Olfm2 is a novel regulator for SMC differentiation and regulates SMC differentiation through enhancing the interaction of myocardin with SRF.

0312: Characterization of human valvular interstitial cells isolated from normal and fibrocalcified aortic valves

Purpose Aortic Valve Stenosis (AVS) affects 2% to 6% of population over 65 years in industrialized countries. This atherosclerosis-like pathology involves Valve Interstitial Cell (VIC) proliferation and commitment to osteoblast- like cells. This prevalent cell type of aortic valve presents five identifiable phenotypes: embryonic progenitor endothelial/mesenchymal cells, progenitor, quiescent, activated and osteoblastic VICs. To study the pathophysiology of AVS, their in vitro cultures are frequently used. Our purpose is to characterize VICs isolated from normal and fibrocalcified human aortic valves and analyze their in vitro behavior. Methods We collected 5 normal and 5 fibrocalcified human aortic valves. VICs were isolated by collagenase digestion. Characterization is assessed at different passages (2 to 5) by immunofluorescence. Analyzed markers consist of progenitor cell markers (SSEA4, ABCG2, CD90, NG2 and OsteoBlast CaDHerin (OB-CDH)), fibroblast markers (vimentin and HSP47) and smooth muscle cell (SMC) marker (α-actin). By blue trypan and MTS, we compared the viability and proliferation of VICs in standard and starvation medium at 48 hours. Results Independently of their origin, VICs express all progenitor cell markers. Fibroblasts markers are expressed twice more by pathological VICs and four times more for SMC marker. In standard medium, VICs viability is similar (96,7±2,4% vs 96,4±2,3% ; normal vs pathological ± SEM). Pathological VICs proliferate more than normal VICs (2,2±0,7 vs 1,6±0,4 ; OD/OD control). In starvation medium, viability is significantly reduced for pathological VICs (89,6±7,9% vs 76,5±5,3%) but still proliferate in opposition with normal VICs (1,7±0,6 vs 1,2±0,3). Conclusion All VICs phenotypes are found in vitro with no culture selection but in different ratios according to their origin. These new data in VICs isolated from normal or pathological human aortic valves allow us to approve their use in vitro .

A novel role of Meox1 in regulating TGFβ‐induced smooth muscle cell differentiation (866.3)

Mesenchyme homeobox 1 (MEOX1) is expressed in the early developing somite and derivatives during embryogenesis. Somite has been shown to be one of the origins or progenitors for SMC. Therefore, in this study, we studied the role of MEOX1 in SMC differentiation. Smooth muscle cell (SMC) differentiation is an important process during vasculogenesis and angiogenesis. It is well known that alterations of VSMC phenotype play a role in the progression of several cardiovascular disorders including atherosclerosis, hypertension, and restenosis. However, the molecular mechanisms controlling the differentiation and development of VSMC are largely unknown. We found that transforming growth factor‐β (TGF‐β) induces MEOX1 expression in the initial phase of SMC differentiation of pluripotent mesenchymal C3H10T1/2 cells. MEOX1 expression appears to be mediated by both PI3 kinase and Smad3 signaling pathways because blockade of these two pathways by specific inhibitors leads to a decreased expression of MEOX1 and SMC markers. Importantly, knockdown of MEOX1 by specific shRNA suppresses TGF‐β‐induced expression of SMC early markers including SM22α and calponin. MEOX1 overexpression, on the other hand, increases SMC marker expression. These results indicate that MEOX1 is a novel regulator for TGF‐β‐induced SMC differentiation.

Microparticles stimulated by hypoxic pulmonary hypertension have differing effects on vascular cell growth than microparticles from severe occlusive pulmonary arterial hypertension (1090.7)

The release of microparticles (MPs), intact, sub‐micron vesicles containing signaling molecules such as proteins, lipids, mRNAs, and miRNAs, is significantly increased in pulmonary arterial hypertension (PAH). MPs contribute to vascular cell proliferation, and the mechanism of increased proliferation in the arteriopathy of PAH is still unclear. Thus, we propose that circulating MPs from pulmonary hypertensive rats will stimulate pulmonary microvascular endothelial (PMVEC) and/or pulmonary artery smooth muscle cell (PASMC) proliferation. PMVECs and PASMCs were treated with MPs from controls, 3 wk hypoxia only (Hx) rats, or 3 wk Su/Hx/Nx rats. Interestingly, although the Su/Hx/Nx model shows severe vascular remodeling, MPs from the hypoxic rat had the most significant effects on PASMC proliferation by shifting the log phase of growth. Western blot analysis shows corresponding changes in smooth muscle cell markers. PMVECs were treated similarly with 5 wk MPs from the Su/Hx/Nx progressive model, resulting in a shift of log growth phase. Our data suggest that circulating MPs isolated from 3 wk Hx‐treated rats may signal to PASMCs to increase proliferation, whereas MPs from a severe occlusive model of PAH stimulate changes in proliferation rates of PMVECs. Combined these data suggest that hypoxic pulmonary hypertension and severe occlusive PAH result in different signaling MPs with unique vascular cell effects.Grant Funding Source: Supported by NHLBI T32 HL076125 to TMY and AHA 11SDG7390037 to NNB.

Expression of enzymes of sphingolipid metabolism in the blood vessel. (LB697)

Background: Sphingolipids represent a class of diverse bioactive lipid molecules that are increasingly appreciated as key modulators of diverse physiologic and pathophysiologic processes that include cell growth, cell death, autophagy, angiogenesis, and stress and inflammatory responses. The cellular contents of the various sphingolipid species are controlled by enzymes involved in their metabolic pathways: sphingomyelinase (neutral‐SMase), Ceramidase (neutral‐CDase), Sphingosine kinase 1 (SPHK1) and Sphingosine kinase 2 (SPHK2). Therefore, the purpose of this study was to determine the expression of enzymes of sphingolipid metabolism in the blood vessel of kidney and heart as well as in aorta.Methods: The expression of neutral‐SMase, neutral‐CDase, SPHK1, SPHK2, α‐actin (Smooth muscle cells marker) and PECAM (endothelial cell marker) were measured by Immunofluorescence and Confocal Microscopy.Results: neutral‐SMase, neutral‐CDase, SPHK1 and SPHK2 immunoreactivity were found to have a widespread distribution in the blood vessel of kidney and heart as well as in aorta.Conclusions: The blood vessel express neutral‐SMase, neutral‐CDase, SPHK1 and SPHK2, enzymes involved in the sphingolipid metabolism and can therefore endogenously form sphingomyelin metabolites in the vasculature.

Tracking chromatin signatures in individual cells to trace vascular smooth muscle cell lineage in human atherosclerotic lesion (278.4)

Atherosclerosis is a chronic disease of the arterial wall responsible for nearly 50% of deaths in developed countries. There is a strong correlation between ratio of smooth muscle cells (SMC) to macrophages and stability of atherosclerotic lesions; a high macrophage to SMC content associated with high risk of plaque rupture. Herein, we reevaluated the contribution of SMC within lesions that was underestimated due to lack of identification of SMC undergoing phenotypic switching (i.e loss of SMC marker gene expression). Using a SMC‐lineage tracing mouse, we show that traditional staining for SMC is unable to detect 86% of the SMC population within lesions of Apoe‐/‐ mice and that 25% of macrophages identified by traditional macrophage markers are of SMC origin. To validate these data in human, we developed a novel method of detection of histone modifications on gene loci in single cells using tissue sections. Our assay was based on studies showing that the histone modification H3K4dime on the SMC marker genes is a specific and stable epigenetic marker of the SMC lineage in vitro. First, H3K4dime on the SMC marker genes (PLA+ cells) was strictly restricted to SMC lineage in vivo. Then, we identified PLA+/macrophage markers+ cells in human lesions. In conclusion, we rigorously identified lesion cells of SMC origin and we provide exciting evidence that a fraction of macrophage‐like cells in human lesions are derived from SMC.Grant Funding Source: American Heart Association

Platelets and Smooth Muscle Cells Affecting the Differentiation of Monocytes

BackgroundAtherosclerosis is characterised by the formation of plaques. Monocytes play a pivotal role in plaque development as they differentiate into foam cells, a component of the lipid core whilst smooth muscle cells (SMC) are the principal cell identified in the cap. Recently, the ability of monocytes to differentiate into a myriad of other cell types has been reported. In lieu of these findings the ability of monocytes to differentiate into SMCs/smooth muscle (SM)-like cells was investigated.Method and ResultsHuman monocytes were co-cultured with platelets or human coronary aortic SMCs and then analysed to assess their differentiation into SMCs/SM-like cells. The differentiated cells expressed a number of SMC markers and genes as determined by immunofluorescence staining and quantitative polymerase chain reaction (qPCR). CD array analysis identified marker expression profiles that discriminated them from monocytes, macrophages and foam cells as well as the expression of markers which overlapped with fibroblast and mesenchymal cells. Electron microscopy studies identified microfilaments and increased amounts of rough endoplasmic reticulum indicative of the SM- like cells, fibroblasts.ConclusionsIn the appropriate environmental conditions, monocytes can differentiate into SM-like cells potentially contributing to cap formation and plaque stability. Thus, monocytes may play a dual role in the development of plaque formation and ultimately atherosclerosis.

Overexpression of myocardin induces partial transdifferentiation of human-induced pluripotent stem cell-derived mesenchymal stem cells into cardiomyocytes.

Mesenchymal stem cells (MSCs) derived from human‐induced pluripotent stem cells (iPSCs) show superior proliferative capacity and therapeutic potential than those derived from bone marrow (BM). Ectopic expression of myocardin further improved the therapeutic potential of BM‐MSCs in a mouse model of myocardial infarction. The aim was of this study was to assess whether forced myocardin expression in iPSC‐MSCs could further enhance their transdifferentiation to cardiomyocytes and improve their electrophysiological properties for cardiac regeneration. Myocardin was overexpressed in iPSC‐MSCs using viral vectors (adenovirus or lentivirus). The expression of smooth muscle cell and cardiomyocyte markers, and ion channel genes was examined by reverse transcription‐polymerase chain reaction (RT‐PCR), immunofluorescence staining and patch clamp. The conduction velocity of the neonatal rat ventricular cardiomyocytes cocultured with iPSC‐MSC monolayer was measured by multielectrode arrays recording plate. Myocardin induced the expression of α‐MHC, GATA4, α‐actinin, cardiac MHC, MYH11, calponin, and SM α‐actin, but not cTnT, β‐MHC, and MLC2v in iPSC‐MSCs. Overexpression of myocardin in iPSC‐MSC enhanced the expression of SCN9A and CACNA1C, but reduced that of KCa3.1 and Kir2.2 in iPSC‐MSCs. Moreover, BKCa, IKir, ICl, Ito and INa.TTX were detected in iPSC‐MSC with myocardin overexpression; while only BKCa, IKir, ICl, IKDR, and IKCa were noted in iPSC‐MSC transfected with green florescence protein. Furthermore, the conduction velocity of iPSC‐MSC was significantly increased after myocardin overexpression. Overexpression of myocardin in iPSC‐MSCs resulted in partial transdifferentiation into cardiomyocytes phenotype and improved the electrical conduction during integration with mature cardiomyocytes.

Interferon Regulatory Factor 8 Modulates Phenotypic Switching of Smooth Muscle Cells by Regulating the Activity of Myocardin

Interferon regulatory factor 8 (IRF8), a member of the IRF transcription factor family, was recently implicated in vascular diseases. In the present study, using the mouse left carotid artery wire injury model, we unexpectedly observed that the expression of IRF8 was greatly enhanced in smooth muscle cells (SMCs) by injury. Compared with the wild-type controls, IRF8 global knockout mice exhibited reduced neointimal lesions and maintained SMC marker gene expression. We further generated SMC-specific IRF8 transgenic mice using an SM22α-driven IRF8 plasmid construct. In contrast to the knockout mice, mice with SMC-overexpressing IRF8 exhibited a synthetic phenotype and enhanced neointima formation. Mechanistically, IRF8 inhibited SMC marker gene expression through regulating serum response factor (SRF) transactivation in a myocardin-dependent manner. Furthermore, a coimmunoprecipitation assay indicated a direct interaction of IRF8 with myocardin, in which a specific region of myocardin was essential for recruiting acetyltransferase p300. Altogether, IRF8 is crucial in modulating SMC phenotype switching and neointima formation in response to vascular injury via direct interaction with the SRF/myocardin complex.

Macrophages Control Vascular Stem/Progenitor Cell Plasticity Through Tumor Necrosis Factor-α–Mediated Nuclear Factor-κB Activation

Vascular lineage differentiation of stem/progenitor cells can contribute to both tissue repair and exacerbation of vascular diseases such as in vein grafts. The role of macrophages in controlling vascular progenitor differentiation is largely unknown and may play an important role in graft development. This study aims to identify the role of macrophages in vascular stem/progenitor cell differentiation and thereafter elucidate the mechanisms that are involved in the macrophage- mediated process. We provide in vitro evidence that macrophages can induce endothelial cell (EC) differentiation of the stem/progenitor cells while simultaneously inhibiting their smooth muscle cell differentiation. Mechanistically, both effects were mediated by macrophage-derived tumor necrosis factor-α (TNF-α) via TNF-α receptor 1 and canonical nuclear factor-κB activation. Although the overexpression of p65 enhanced EC (or attenuated smooth muscle cell) differentiation, p65 or TNF-α receptor 1 knockdown using lentiviral short hairpin RNA inhibited EC (or rescued smooth muscle cell) differentiation in response to TNF-α. Furthermore, TNF-α-mediated EC differentiation was driven by direct binding of nuclear factor-κB (p65) to specific VE-cadherin promoter sequences. Subsequent experiments using an ex vivo decellularized vessel scaffold confirmed an increase in the number of ECs and reduction in smooth muscle cell marker expression in the presence of TNF-α. The lack of TNF-α in a knockout mouse model of vein graft decreased endothelialization and significantly increased thrombosis formation. Our study highlights the role of macrophages in directing vascular stem/progenitor cell lineage commitment through TNF-α-mediated TNF-α receptor 1 and nuclear factor-κB activation that is likely required for endothelial repair in vascular diseases such as vein graft.

Expression of smooth muscle cell markers and co-activators in calcified aortic valves

Similar risk factors and mediators are involved in calcific aortic stenosis (CAS) and atherosclerosis. Since normal valves harbour a low percentage of smooth muscle cells (SMCs), we hypothesize that the SMC phenotype participates in the pathogenesis of CAS. We analysed 12 normal and 22 calcified aortic valves for SMC markers and the expression of co-activators of SMC gene expression, myocardin and myocardin-related transcription factors (MRTF-A/B). Transforming growth factor β (TGFβ1) was used to upregulate SMC markers and co-activators in valve interstitial cells (VICs) and transmission electron microscopy (TEM) was used to detect the presence of SMC in atypical regions of the valve leaflets. Smooth muscle cell markers and co-activators, myocardin, MRTF-A, and MRTF-B, demonstrated an increased incidence and aberrant expression around calcified nodules in all 22 calcified valves as well as in surface and microvessel endothelial cells. Smooth muscle cell markers and MRTF-A were significantly increased in calcified valves. Transforming growth factor β1 (TGFβ1) (10 ng/mL) was able to significantly upregulate the expression of some SMC markers and MRTF-A in VICs. Transmission electron microscopy of the fibrosa layer of calcified valves demonstrated the presence of bundles of SMCs and smooth muscle-derived foam cells. Smooth muscle cell markers and co-activators, myocardin and MRTFs, were aberrantly expressed in calcified valves. Transforming growth factor β1 was able to significantly upregulate SMC markers and MRTF-A in VICs. Transmission electron microscopy unequivocally identified the presence of SMCs in calcified regions of valve leaflets. These findings provide evidence that the SMC phenotype plays a role in the development of CAS.

Vascular Calcification in Chronic Kidney Disease is Induced by Bone Morphogenetic Protein-2 via a Mechanism Involving the Wnt/β-Catenin Pathway

Background: Vascular calcification (VC), in which vascular smooth muscle cells (VSMCs) undergo a phenotypic transformation into osteoblast-like cells, is one of the emergent risk factors for the accelerated atherosclerosis process characteristic of chronic kidney disease (CKD). Phosphate is an important regulator of VC. Methods: The expression of different smooth muscle cell or osteogenesis markers in response to high concentrations of phosphate or exogenous bone morphogenetic protein 2 (BMP-2) was examined by qRT-PCR and western blotting in rat VSMCs. Osteocalcin secretion was measured by radioimmunoassay. Differentiation and calcification of VSMCs were examined by alkaline phosphatase (ALP) activity assay and Alizarin staining. Short hairpin RNA-mediated silencing of β-catenin was performed to examine the involvement of Wnt/β-catenin signaling in VSMC calcification and osteoblastic differentiation induced by high phosphate or BMP-2. Apoptosis was determined by TUNEL assay and immunofluorescence imaging. Results: BMP-2 serum levels were significantly higher in CKD patients than in controls. High phosphate concentrations and BMP-2 induced VSMC apoptosis and upregulated the expression of β-catenin, Msx2, Runx2 and the phosphate cotransporter Pit1, whereas a BMP-2 neutralization antibody reversed these effects. Knockdown of β-catenin abolished the effect of high phosphate and BMP-2 on VSMC apoptosis and calcification. Conclusions: BMP-2 plays a crucial role in calcium deposition in VSMCs and VC in CKD patients via a mechanism involving the Wnt/β-catenin pathway.

EC4, a truncation of soluble N-cadherin, reduces vascular smooth muscle cell apoptosis and markers of atherosclerotic plaque instability

Atherosclerotic plaque instability is precipitated by vascular smooth muscle cell apoptosis in the fibrous cap, weakening it and leading to plaque rupture. We previously showed that reducing smooth muscle cell apoptosis with soluble N-cadherin (SNC) increased features of plaque stability. We have now identified the active site of SNC and examined whether a truncated form containing this site retains the antiapoptotic effect. SNC was mutated to prevent interaction with N-cadherin or fibroblast growth factor receptor (FGFR). Interaction with FGFR in the extracellular (EC) 4 domain of SNC was essential for the antiapoptotic effect. Therefore, we made a truncated form consisting of the EC4 domain. EC4 significantly reduced smooth muscle cell, macrophage, and endothelial cell apoptosis in vitro by ~70%, similar to SNC. Elevation of plasma levels of EC4 in male apolipoprotein E–deficient mice with existing atherosclerosis significantly reduced apoptosis in brachiocephalic artery plaques by ~50%. EC4 reduced plaque size and the incidence of buried fibrous layers and the macrophage:smooth muscle cell ratio (surrogate markers of plaque instability). Interaction of EC4 with FGFR induced potent antiapoptotic signaling in vitro and in vivo. EC4 modulates atherosclerosis in mice demonstrating its therapeutic potential for retarding plaque size and instability.

Basic Fibroblast Growth Factor Is Essential to Maintain Endothelial Progenitor Cell Phenotype in TR-BME2 Cells

Endothelial progenitor cells (EPC) can differentiate into both endothelial cells and contractile smooth muscle cells (SMC). Previously we reported that TR-BME2 cells, a model for EPC, developed contractile SMC-like characteristics in culture medium deprived of endothelial cell growth factors (ECGF). The aim of the present study was to clarify the effect of one of these factors, basic fibroblast growth factor (bFGF) on differentiation of EPC. First it was confirmed that bFGF receptor (FGFR-1) mRNA is expressed in TR-BME2 cultured in both ECGF-rich and ECGF-deprived medium. When TR-BME2 cells were cultured in ECGF-deprived medium, they differentiated into contractile SMC. Expression of an undifferentiated state marker, CD133, and proliferation of TR-BME2 were both reduced by ECGF deprivation, but these changes were diminished in the presence of bFGF. mRNA expression of smooth muscle α-actin (SMA) and smooth muscle protein 22 (SM22), which are contractile SMC markers, was induced by deprivation of ECGF and the induction was suppressed by bFGF. In vascular endothelial cell growth factor (VEGF)-induced tube formation assay, TR-BME2 cells formed tube structures in the presence of bFGF, but not in its absence. Our results indicate that bFGF is essential for the maintenance of EPC phenotype, serving to suppress differentiation to contractile SMC.

Low Intensity Laser Irradiation and Growth Factors Influence Differentiation of Adipose Derived Stem Cells into Smooth Muscle Cells in a Coculture Environment over a Period of 72 Hours

Stem cells have the ability to self-renew and differentiate into several specialised cells. Low intensity laser irradiation (LILI) has been shown to have positive effects on cells including adipose derived stem cells (ADSCs). Growth factors such as retinoic acid and transforming growth factor (TGF-β1) play significant roles in the differentiation of cells. This study aimed at investigating the role of LILI and growth factors on differentiation of adipose derived stem cells cocultured with smooth muscle cells (SMCs). The study used isolated human adipose derived stem cells and smooth muscle commercial cells (SKUT-1). The cells were cocultured directly in the ratio 1 : 1 using the established methods with and without growth factors (retinoic acid and TGF-β1) and then exposed to LILI at a wavelength of 636 nm with 5 J/cm2using a diode laser. The cellular proliferation and expression of the both cell type markers were assessed using optical density and flow cytometry at 24 h and 72 h. The study showed that LILI increased the proliferation of cocultured cells. The expression of the smooth muscle cell markers increased in the coculture groups that were exposed to LILI in the presence of growth factors while those of the ADSCs decreased.

Hox genes are involved in vascular wall-resident multipotent stem cell differentiation into smooth muscle cells

Human vascular wall-resident CD44+ multipotent stem cells (VW-MPSCs) within the vascular adventitia are capable to differentiate into pericytes and smooth muscle cells (SMC). This study demonstrates HOX-dependent differentiation of CD44(+) VW-MPSCs into SMC that involves epigenetic modification of transgelin as a down-stream regulated gene. First, HOXB7, HOXC6 and HOXC8 were identified to be differentially expressed in VW-MPSCs as compared to terminal differentiated human aortic SMC, endothelial cells and undifferentiated pluripotent embryonic stem cells. Silencing these HOX genes in VW-MPSCs significantly reduced their sprouting capacity and increased expression of the SMC markers transgelin and calponin and the histone gene histone H1. Furthermore, the methylation pattern of the TAGLN promoter was altered. In summary, our findings suggest a role for certain HOX genes in regulating differentiation of human VW-MPSC into SMCs that involves epigenetic mechanisms. This is critical for understanding VW-MPSC–dependent vascular disease processes such as neointima formation and tumor vascularization.

Small diameter vascular graft engineered using human embryonic stem cell-derived mesenchymal cells

Despite the progress made thus far in the generation of small-diameter vascular grafts, cell sourcing still remains a problem. Human embryonic stem cells (hESCs) present an exciting new cell source for the regeneration applications due to their high proliferative and differentiation capabilities. In this study, the feasibility of creating small-diameter vascular constructs using smooth muscle cells (SMCs) differentiated from hESC-derived mesenchymal cells was evaluated. In vitro experiments confirmed the ability of these cells to differentiate into smooth muscle actin- and calponin-expressing SMCs in the presence of known inducers, such as transforming growth factor beta. Human vessel walls were constructed by culturing these cells in a bioreactor system under pulsatile conditions for 8 weeks. Histological analysis showed that vessel grafts had similarities to their native counterparts in terms of cellularity and SMC marker expression. However, markers of cartilage and bone tissue were also detected, thus raising questions about stable lineage commitment during differentiation and calling for more stringent analysis of differentiating cell populations.