Smooth Muscle Cell Markers Research Articles

Pericyte-based collagen scaffold for vascular tissue engineering

Event Abstract Back to Event Pericyte-based collagen scaffold for vascular tissue engineering Betül Çelebi-Saltik1, 2 and Beyza Gökçinar-Yagci1, 2 1 Hacettepe University, Department of Stem Cell Sciences, Graduate School of Health Sciences, Türkiye 2 Hacettepe University, Center for Stem Cell Research and Development, Türkiye Pericytes are a perivascular cell population embedded on the capillary wall in a shared basement membrane with the endothelium. During vasculogenesis and angiogenesis, endothelial cells secrete and/or present cell bound molecular cues that stimulate pericytes to proliferate, migrate and attach to nascent capillary tubes. It has been previously developed cell sheet engineering using temperature-responsive culture dishes in order to avoid traditional tissue engineering approaches. In this study our aim is to isolate human umbilical cord vein pericytes then differentiate into smooth muscle cells and fibroblasts then co-culture these cells on 3D collagen scaffold that will be used for generating tissue engineering vascular grafts. Pericytes were enzymatically isolated from human umbilical cord vein and were purified by using MACs cell separation with CD146 microbeads. They were cultured in fibroblast growth medium and smooth muscle growth medium for 21 days on poly(N-isopropylacrylamide) to obtain cell sheets. Cell differentiation was confirmed by immunofluorescence staining and flow cytometry analysis (fibroblast markers; Tenascin-C and Collagen type I, smooth muscle cell markers; Caldesmon, Calponin and Alpha smooth muscle actin). Collagen gel was prepared with human collagen type I (3mg/mL) + 10X DMEM (adjustment pH of mixture to 7.2–7.6 using sterile 0.1 M NaOH). Collagen gel was covered by cell sheets (sandwich system) and vascular graft was incubated in 37oC for 7 days (Figure 1). The results indicated that perivascular cells could differentiate into fibroblast and smooth muscle and formed vascular graft with collagen gel. As a conclusion, differentiated pericytes offer an alternative cell source for constructing tissue engineered vascular graft. The authors wish to thank The Scientific and Technological Research Council of Turkey (Project Number: 113S815) for their financial support. The Scientific and Technological Research Council of Turkey (Project Number: 113S815) Keywords: Tissue Engineering, cell, 3D scaffold, tissue niche Conference: 10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016. Presentation Type: Poster Topic: Biomaterials in mesenchymal and hematopoietic stem cell biology Citation: Çelebi-Saltik B and Gökçinar-Yagci B (2016). Pericyte-based collagen scaffold for vascular tissue engineering. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.02836 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Mar 2016; Published Online: 30 Mar 2016. Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Betül Çelebi-Saltik Beyza Gökçinar-Yagci Google Betül Çelebi-Saltik Beyza Gökçinar-Yagci Google Scholar Betül Çelebi-Saltik Beyza Gökçinar-Yagci PubMed Betül Çelebi-Saltik Beyza Gökçinar-Yagci Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Engineered small diameter vascular grafts by cell sheet engineering with human umbilical cord vein perivascular cells

Event Abstract Back to Event Engineered small diameter vascular grafts by cell sheet engineering with human umbilical cord vein perivascular cells Beyza Gökçinar-Yagci1, 2 and Betül Çelebi-Saltik1, 2 1 Hacettepe University, Department of Stem Cell Sciences,Graduate School of Health Sciences, Türkiye 2 Hacettepe University, Center for Stem Cell Research and Development, Türkiye Pericytes are perivascular cells that surround endothelial cells of capillaries, microvessels and larger vessels; arteries and veins. They have important roles in vascular development (vasculogenesis and angiogenesis), stability, maturation and remodeling, regulation of vessel diameter and blood flow, blood pressure control, contractility and tone of vascular smooth muscle cells. The cell sheet engineering is based on the capability of cells to secrete and organize their own extracellular matrix (ECM). In this study our aim is to isolate human umbilical cord vein pericytes (CD146+ cells) then differentiate into smooth muscle cells and fibroblasts that will be used for generating tissue engineering vascular grafts and also reduce the time required to produce a tissue-engineered vascular graft. Human perivascular cells were isolated from umbilical cord vein and pericytes were purified by using MACs cell separation with CD146 microbeads. Perivascular cells were cultured in FGM-2, SMCGM-2 and EGM-2 medium for 21 days to investigate fibroblast, smooth muscle cell and endothelial cell differentiation. Cell differentiation was confirmed by immunofluorescence staining and flow cytometry analysis (fibroblast markers; Tenascin-C and Collagen type I, smooth muscle cell markers; Caldesmon and alpha smooth muscle actin, endothelial cell markers; VEGFR1, VEGFR2 and CD31). Next, a tissue-engineered vascular graft was fabricated by rolling the perivascular cells derived endothelial cell, smooth muscle cell and fibroblast sheets (Rapid cell sheet detachment from Poly(N-isopropylacrylamide)) around a mandrel. Different human ECM molecules such as collagen type I and IV, fibrin, elastin, fibronectin and glycosaminoglycans were used among the vascular layers to increase the mechanical properties of the graft. The results indicated that perivascular cells could differentiate into fibroblast, smooth muscle, endothelial cells and formed tubular vascular graft with ECM proteins. As a conclusion, differentiated pericytes offer an alternative cell source for constructing tissue engineered vascular graft. The authors wish to thank The Scientific and Technological Research Council of Turkey (Project Number: 113S815) for their financial support. The Scientific and Technological Research Council of Turkey (Project Number: 113S815) Keywords: Extracellular Matrix, Regenerative Medicine, 3D scaffold, tissue niche Conference: 10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016. Presentation Type: Poster Topic: Biomaterials in constructing tissue substitutes Citation: Gökçinar-Yagci B and Çelebi-Saltik B (2016). Engineered small diameter vascular grafts by cell sheet engineering with human umbilical cord vein perivascular cells. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.01061 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Mar 2016; Published Online: 30 Mar 2016. Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Beyza Gökçinar-Yagci Betül Çelebi-Saltik Google Beyza Gökçinar-Yagci Betül Çelebi-Saltik Google Scholar Beyza Gökçinar-Yagci Betül Çelebi-Saltik PubMed Beyza Gökçinar-Yagci Betül Çelebi-Saltik Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Differences in the development of the processus vaginalis between children with undescended testis and inguinal hernia

Background: It has been shown in several investigations that smooth muscle cells (SMCs) are present on the patent processus vaginalis (PV) peritonei in cases of inguinal hernia (IH) preventing its obliteration. The PV fails to obliterate in cases of undescended testis (UT) as well, but without causing herniation. Materials and Methods: We conducted a case control study in order to compare the status of the SMCs present on the PV in UT and IH cases and correlate it with the clinical outcome of herniation. Specimens were harvested from the hernia sacs of 26 boys with IH aged from 2 days to 16 years (mean 44.31 months) and the PV of 14 children with UT, aged from 13 months to 13 years (mean 30.28 months). They were examined histologically and immunohistochemically for markers of mature SMC such as smooth muscle actin (SMA), desmin and h-caldesmon as well as for vimentin, an immature SMC marker. Results: The expression of SMA, desmin, and h-caldesmon was present in all cases of IH and UT. Vimentin was expressed in 13 out of 14 (93%) UT specimens and in 12 out of 26 IH sacs (46.1%), (P = 0.0102). In IH and UT cases, when vimentin was not expressed, SMCs were organized in bundles. Conclusion: The SMCs on the PV in UT cases reach a more advanced stage of dedifferentiation that corresponds to a status more close to that of the natural obliteration, compared to IH cases, preventing herniation to occur.

Vascular Notch proteins and Notch signaling in the peri-implantation mouse uterus.

BackgroundAngiogenesis is essential for uterine decidualization, the progesterone-mediated transformation of the uterus allowing embryo implantation and initiation of pregnancy. In the current study, we define the vasculature, expression of Notch proteins and Notch ligands, and Notch activity in both endothelial cells and vascular-associated mural cells of blood vessels in the pre-implantation endometrium and post-implantation decidua of the mouse uterus.MethodsWe used immunofluorescence to determine the expression of Notch in endothelial cells and mural cells by co-staining for the endothelial cell marker, CD31, the pan-mural cell marker, platelet-derived growth factor receptor beta (PDGFR-β), the pericyte markers, neural/glial antigen 2 (NG2) and desmin, or the smooth muscle cell marker, alpha smooth muscle actin (SMA). A fluorescein isothiocyanate-labeled dextran tracer, was used to identify functional peri-implantation vasculature. CBF:H2B-Venus Notch reporter transgenic mice were used to determine Notch activity.ResultsNotch signaling is observed in endothelial cells and pericytes in the peri-implantation uterus. Prior to implantation, Notch1, Notch2 and Notch4 and Notch ligand, Delta-like 4 (Dll4) are expressed in capillary endothelial cells, while Notch3 is expressed in the pericytes. Jagged1 is expressed in both capillary endothelial cells and pericytes. After implantation, Notch1, Notch4 and Dll4 are expressed in endothelial cells of newly formed decidual capillaries. Jagged1 is expressed in endothelial cells of spiral arteries and a subset of decidual pericytes. Notch proteins are not expressed in lymphatic vessels or macrophages in the peri-implantation uterus.ConclusionsWe show Notch activity and distinct expression patterns for Notch proteins and ligands, suggesting unique roles for Notch1, Notch4, Dll4, and Jag1 during decidual angiogenesis and early placentation. These data set the stage for loss-of-function and gain-of-function studies that will determine the cell-type specific requirements for Notch proteins in decidual angiogenesis and placentation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13221-015-0034-y) contains supplementary material, which is available to authorized users.

Abstract 13154: Modulation of Stem Cell Niche and Stem Cell Migration Promotes Functional Angiogenesis by Enhancing Stem Cell Homing in Ischemic Rat Myocardium: A Novel Concept of Cell-free Regeneration Therapy

Background: The microenvironments composed of extracellular matrix (ECM) components and cytokines are considered to be involved in the decision of the stem cell fate and behavior. In addition, some cytokines induced by ONO1301 has been reported to enhance cell migration contributing to myocardial regeneration. In this study, we hypothesized that combined administration of cardiac specific ECM such as laminins and ONO1301 may enhance regeneration process by modulation of stem cell activity and homing. Methods and Results: In vitro study revealed that MSCs showed strong adhesion to laminin (LM) 511. ONO1301 promoted the production of several cytokines in smooth muscle cells, and it enhanced MSCs migration in vitro assay. To examine the therapeutic effect of ONO1301 combined with LM511, we transplanted ONO1301 or ONO1301 with LM511 (n=5-12) in a rat acute myocardial infarction model. Four weeks later, the number of PDGFRα- and CD90-positive cells tended to be higher in the combination (ONO1301 with LM511) group than in the ONO1301 alone group. Furthermore the gene expression of Sca-1 and c-kit was higher in the combination group than the control group (Sca-1, ONO1301 with LM511: 2.16- (p<0.05), ONO1301 alone: 2.01-fold to the control, c-kit, ONO1301 with LM511: 2.75- (p<0.05), ONO1301 alone: 1.45-fold to the control). Histological examination revealed that the number of ILB4 (endothelial cell marker) positive cells was significantly higher in the combination group than in the ONO1301 group (ONO1301 with LM511: 521±68 cells/mm 2 , ONO1301 alone: 281±10 cells/mm 2 , control: 195±29 cells/mm 2 ; p<0.05). Moreover, the number of CD31 (endothelial cell marker) and SMA (smooth muscle cell marker) double positive cell was significantly higher in the combination group compared with the control group (ONO1301 with LM511: 616±44 cells/mm 2 , control: 226±60 cells/mm 2 ; p<0.05). Real time PCR analysis revealed that SDF-1, PGF, FGF2 and HGF were higher in the combination group. Moreover, infarct size tended to be smaller in the combination group than in the ONO1301 group. Conclusion: The cell free regeneration therapy using both ONO1301 and LM511 may contribute to enhanced stem cell homing to implanted site, leading to improve functional angiogenesis in rat ischemic heart.

Effect of Polyelectrolyte Film Stiffness on Endothelial Cells During Endothelial-to-Mesenchymal Transition.

Endothelial-to-mesenchymal transition (EndMT), during which endothelial cells (ECs) transdifferentiate into mesenchymal phenotype, plays a key role in the development of vascular implant complications such as endothelium dysfunction and in-stent restenosis. Substrate stiffness has been confirmed as a key factor to influence EC behaviors; however, so far, the relationship between substrate stiffness and EndMT has been rarely studied. Here, ECs were cultured on the (poly(L-lysine)/hyaluronate acid) (PLL/HA) multilayer films with controlled stiffness for 2 weeks, and their EndMT behaviors were studied. We demonstrated that ECs lost their markers (vWf and CD31) in a stiffness-dependent manner even without supplement of growth factors, and the softer film favored the maintaining of EC phenotype. Further, induced by transforming growth factor β1 (TGF-β1), ECs underwent EndMT, as characterized by losing their typical cobblestone morphology and markers and gaining smooth muscle cell markers (α-smooth muscle actin and calponin). Interestingly, stronger EndMT was observed when ECs were cultured on the stiffer film. Collectively, our findings suggest that substrate stiffness has significant effects on EndMT, and a softer substrate is beneficial to ECs by keeping their phenotype and inhibiting EndMT, which presents a new strategy for surface design of vascular implant materials.

Development and evaluation of in vivo tissue engineered blood vessels in a porcine model

BackgroundThere's a large clinical need for novel vascular grafts. Tissue engineered blood vessels (TEBVs) have great potential to improve the outcome of vascular grafting procedures. Here, we present a novel approach to generate autologous TEBV in vivo. Polymer rods were engineered and implanted, evoking an inflammatory response that culminates in encapsulation by a fibrocellular capsule. We hypothesized that, after extrusion of the rod, the fibrocellular capsule differentiates into an adequate vascular conduit once grafted into the vasculature. Methods and resultsRods were implanted subcutaneously in pigs. After 4 weeks, rods with tissue capsules grown around it were harvested. Tissue capsules were grafted bilaterally as carotid artery interposition. One and 4-week patency were evaluated by angiography whereupon pigs were sacrificed. Tissue capsules before and after grafting were evaluated on tissue remodeling using immunohistochemistry, RNA profiling and mechanical testing. Rods were encapsulated by thick, well-vascularized tissue capsules, composed of circumferentially aligned fibroblasts, collagen and few leukocytes, with adequate mechanical strength. Patency was 100% after 1 week and 87.5% after 4 weeks. After grafting, tissue capsules remodeled towards a vascular phenotype. Gene profiles of TEBVs gained more similarity with carotid artery. Wall thickness and αSMA-positive area significantly increased. Interestingly, a substantial portion of (myo)fibroblasts present before grafting expressed smooth muscle cell markers. While leukocytes were hardly present anymore, the lumen was largely covered with endothelial cells. Burst pressure remained stable after grafting. ConclusionsAutologous TEBVs were created in vivo with sufficient mechanical strength enabling vascular grafting. Grafts differentiated towards a vascular phenotype upon grafting.

Inhibition of Diaphanous Formin Signaling In Vivo Impairs Cardiovascular Development and Alters Smooth Muscle Cell Phenotype.

We and others have previously shown that RhoA-dependent stimulation of myocardin-related transcription factor nuclear localization promotes smooth muscle cell (SMC) marker gene expression. The goal of this study was to provide direct in vivo evidence that actin polymerization by the diaphanous-related formins contributes to the regulation of SMC differentiation and phenotype. Conditional Cre-based genetic approaches were used to overexpress a well-characterized dominant-negative variant of mDia1 (DNmDia) in SMC. DNmDia expression in SM22-expressing cells resulted in embryonic and perinatal lethality in ≈20% of mice because of defects in myocardial development and SMC investment of peripheral vessels. Although most DNmDia(+)/SM22Cre(+) mice exhibited no overt phenotype, the re-expression of SMC differentiation marker gene expression that occurs after carotid artery ligation was delayed, and this effect was accompanied by a significant decrease in myocardin-related transcription factor-A nuclear localization. Interestingly, neointima growth was inhibited by expression of DNmDia in SMC and this was likely because of a defect in directional SMC migration and not to defects in SMC proliferation or survival. Finally, by using the tamoxifen-inducible SM MHC-CreER(T2) line, we showed that SMC-specific induction of DNmDia in adult mice decreased SMC marker gene expression. Our demonstration that diaphanous-related formin signaling plays a role in heart and vascular development and the maintenance of SMC phenotype provides important new evidence that Rho/actin/myocardin-related transcription factor signaling plays a critical role in cardiovascular function.

Expression of V3 Versican by Rat Arterial Smooth Muscle Cells Promotes Differentiated and Anti-inflammatory Phenotypes

Arterial smooth muscle cells (ASMCs) undergo phenotypic changes during development and pathological processes in vivo and during cell culture in vitro. Our previous studies demonstrated that retrovirally mediated expression of the versican V3 splice variant (V3) by ASMCs retards cell proliferation and migration in vitro and reduces neointimal thickening and macrophage and lipid accumulation in animal models of vascular injury and atherosclerosis. However, the molecular pathways induced by V3 expression that are responsible for these changes are not yet clear. In this study, we employed a microarray approach to examine how expression of V3 induced changes in gene expression and the molecular pathways in rat ASMCs. We found that forced expression of V3 by ASMCs affected expression of 521 genes by more than 1.5-fold. Gene ontology analysis showed that components of the extracellular matrix were the most significantly affected by V3 expression. In addition, genes regulating the formation of the cytoskeleton, which also serve as markers of contractile smooth muscle cells (SMCs), were significantly up-regulated. In contrast, components of the complement system, chemokines, chemokine receptors, and transcription factors crucial for regulating inflammatory processes were among the genes most down-regulated. Consistently, we found that the level of myocardin, a key transcription factor promoting contractile SMC phenotype, was greatly increased, and the proinflammatory transcription factors NFκB1 and CCAAT/enhancer-binding protein β were significantly attenuated in V3-expressing SMCs. Overall, these findings demonstrate that V3 expression reprograms ASMCs promoting differentiated and anti-inflammatory phenotypes.

Dickkopf Homolog 3 Induces Stem Cell Differentiation into Smooth Muscle Lineage via ATF6 Signalling

Smooth muscle cells (SMCs) are a key component of healthy and tissue engineered vessels and play a crucial role in vascular development and the pathogenic events of vascular remodeling i.e. restenosis. However, the cell source from which they can be isolated is limited. Embryonic stem (ES) cells that have the remarkable capability to differentiate into vascular SMCs in response to specific stimuli provide a useful model for studying SMC differentiation. Previous studies suggested that dickkopf homolog 3 (DKK3) has a role in human partially induced pluripotent stem cell to SMC differentiation. Here, we demonstrate that the expression of DKK3 is essential for the expression of SMC markers and myocardin at both the mRNA and protein levels during mouse ES cell differentiation into SMCs (ESC-SMC differentiation). Overexpression of DKK3 leads to further up-regulation of the aforementioned markers. Further investigation indicates that DKK3 added as a cytokine activates activating transcription factor 6 (ATF6), leading to the increased binding of ATF6 on the myocardin promoter and increased its expression. In addition, inhibition of extracellular signal-regulated kinases 1/2 (ERK1/2) promotes the expression of ATF6 and leads to further increase of myocardin transcription. Our findings offer a novel mechanism by which DKK3 regulates ESC-SMC differentiation by activating ATF6 and promoting myocardin expression.

Abstract 42: Differentiation of Human Multipotent Vascular Stem Cells Contributes to Vascular Diseases

Vascular disease, such as neointimal hyperplasia and atherosclerosis, involves in migration and proliferation of vascular cells in blood vessel wall. It is generally accepted that the de-differentiation of vascular smooth muscle cells (SMCs) contributes to vascular diseases. However, previous studies suggest that resident multipotent vascular stem cells (MVSCs) are also involved in neointima formation. In this study, we isolated MVSCs from the medial layer of human carotid artery and thoracic aorta by explant cultures, characterized these cells, and identified the origin of these vascular cells populated in lesion areas. Human MVSCs could be isolated from healthy and diseased blood vessels, were cloneable, and were positive for stem cell markers SOX10 and SOX17 but not SMC markers α-actin and calponin-1. MVSCs were able to differentiate into neural cells, SMCs and other mesenchymal lineages in vitro. In addition, we examined the location of MVSCs in diseased vascular wall by immunohistochemistry. The majority of cells within tunica media were calponin-positive cells, whereas a small population of cells was proliferative and double positive for SOX10 and Ki67 in the border between tunica intima and media. The number of SOX10+ cells in atherosclerotic lesions was more than that in healthy blood vessels, and some of these cells were double positive for SOX10 and chondrogenic markers. Furthermore, we performed in situ PCR and proximity ligation assays to stain for the epigenetic markers of SMC lineage. The results suggested that SOX10+ cells in cell cultures and tissue sections were not derived from de-differentiated SMCs. In conclusion, these findings support that MVSCs contributes to human vascular diseases.

Expansion and characterization of neonatal cardiac pericytes provides a novel cellular option for tissue engineering in congenital heart disease.

BackgroundLiving grafts produced by combining autologous heart-resident stem/progenitor cells and tissue engineering could provide a new therapeutic option for definitive correction of congenital heart disease. The aim of the study was to investigate the antigenic profile, expansion/differentiation capacity, paracrine activity, and pro-angiogenic potential of cardiac pericytes and to assess their engrafting capacity in clinically certified prosthetic grafts.Methods and ResultsCD34pos cells, negative for the endothelial markers CD31 and CD146, were identified by immunohistochemistry in cardiac leftovers from infants and children undergoing palliative repair of congenital cardiac defects. Following isolation by immunomagnetic bead-sorting and culture on plastic in EGM-2 medium supplemented with growth factors and serum, CD34pos/CD31neg cells gave rise to a clonogenic, highly proliferative (>20 million at P5), spindle-shape cell population. The following populations were shown to expresses pericyte/mesenchymal and stemness markers. After exposure to differentiation media, the expanded cardiac pericytes acquired markers of vascular smooth muscle cells, but failed to differentiate into endothelial cells or cardiomyocytes. However, in Matrigel, cardiac pericytes form networks and enhance the network capacity of endothelial cells. Moreover, they produce collagen-1 and release chemo-attractants that stimulate the migration of c-Kitpos cardiac stem cells. Cardiac pericytes were then seeded onto clinically approved xenograft scaffolds and cultured in a bioreactor. After 3 weeks, fluorescent microscopy showed that cardiac pericytes had penetrated into and colonized the graft.ConclusionsThese findings open new avenues for cellular functionalization of prosthetic grafts to be applied in reconstructive surgery of congenital heart disease.

TMEM16A and myocardin form a positive feedback loop that is disrupted by KLF5 during Ang II-induced vascular remodeling.

The TMEM16A protein is an important component of Ca(2+)-dependent Cl(-) channels (CaCCs) in vascular smooth muscle cells. A recent study showed that TMEM16A inhibits angiotensin II-induced proliferation in rat basilar smooth muscle cells. However, whether and how TMEM16A is involved in vascular remodeling characterized by vascular smooth muscle cell proliferation remains largely unclear. In this study, luciferase reporter, Western blotting, and qRT-PCR assays were performed. The results suggested that myocardin promotes TMEM16A expression by forming a complex with serum response factor (SRF) on the TMEM16A promoter in human aortic smooth muscle cells (HASMCs). In turn, upregulated TMEM16A promotes expression of myocardin and vascular smooth muscle cell marker genes, thus forming a positive feedback loop that induces cell differentiation and inhibits cell proliferation. Angiotensin II inhibits TMEM16A expression via Krüppel-like factor 5 (KLF5) in cultured HASMCs. Moreover, in vivo experiments show that infusion of angiotensin II into mice causes a marked reduction in TMEM16A expression and vascular remodeling, and angiotensin II-induced effects are largely reversed in KLF5 null (KLF5(-/-)) mice. KLF5 competes with SRF to interact with myocardin, thereby limiting myocardin binding to SRF and the synergistic activation of the TMEM16A promoter by myocardin and SRF. Our studies demonstrated that angiotensin II induces KLF5 expression and facilitates KLF5 association with myocardin to disrupt the myocardin-SRF complex, subsequently leading to inhibition of TMEM16A transcription. Blocking the positive feedback loop between myocardin and TMEM16A may be a novel therapeutic approach for vascular remodeling.

Bone marrow mesenchymal cells improve muscle function in a skeletal muscle re-injury model.

Skeletal muscle injury is the most common problem in orthopedic and sports medicine, and severe injury leads to fibrosis and muscle dysfunction. Conventional treatment for successive muscle injury is currently controversial, although new therapies, like cell therapy, seem to be promise. We developed a model of successive injuries in rat to evaluate the therapeutic potential of bone marrow mesenchymal cells (BMMC) injected directly into the injured muscle. Functional and histological assays were performed 14 and 28 days after the injury protocol by isometric tension recording and picrosirius/Hematoxilin & Eosin staining, respectively. We also evaluated the presence and the fate of BMMC on treated muscles; and muscle fiber regeneration. BMMC treatment increased maximal skeletal muscle contraction 14 and 28 days after muscle injury compared to non-treated group (4.5 ± 1.7 vs 2.5 ± 0.98 N/cm2, p<0.05 and 8.4 ± 2.3 vs. 5.7 ± 1.3 N/cm2, p<0.05 respectively). Furthermore, BMMC treatment increased muscle fiber cross-sectional area and the presence of mature muscle fiber 28 days after muscle injury. However, there was no difference in collagen deposition between groups. Immunoassays for cytoskeleton markers of skeletal and smooth muscle cells revealed an apparent integration of the BMMC within the muscle. These data suggest that BMMC transplantation accelerates and improves muscle function recovery in our extensive muscle re-injury model.

Smooth Muscle Stiffness Sensitivity is Driven by Soluble and Insoluble ECM Chemistry.

Smooth muscle cell (SMC) invasion into plaques and subsequent proliferation is a major factor in the progression of atherosclerosis. During disease progression, SMCs experience major changes in their microenvironment, such as what integrin-binding sites are exposed, the portfolio of soluble factors available, and the elasticity and modulus of the surrounding vessel wall. We have developed a hydrogel biomaterial platform to examine the combined effect of these changes on SMC phenotype. We were particularly interested in how the chemical microenvironment affected the ability of SMCs to sense and respond to modulus. To our surprise, we observed that integrin binding and soluble factors are major drivers of several critical SMC behaviors, such as motility, proliferation, invasion, and differentiation marker expression, and these factors modulated the effect of stiffness on proliferation and migration. Overall, modulus only modestly affected behaviors other than proliferation, relative to integrin binding and soluble factors. Surprisingly, pathological behaviors (proliferation, motility) are not inversely related to SMC marker expression, in direct conflict with previous studies on substrates coupled with single extracellular matrix (ECM) proteins. A high-throughput bead-based ELISA approach and inhibitor studies revealed that differentiation marker expression is mediated chiefly via focal adhesion kinase (FAK) signaling, and we propose that integrin binding and FAK drive the transition from a migratory to a proliferative phenotype. We emphasize the importance of increasing the complexity of in vitro testing platforms to capture these subtleties in cell phenotypes and signaling, in order to better recapitulate important features of in vivo disease and elucidate potential context-dependent therapeutic targets.

Coronary Artery Disease Associated Transcription Factor TCF21 Regulates Smooth Muscle Precursor Cells That Contribute to the Fibrous Cap.

Recent genome wide association studies have identified a number of genes that contribute to the risk for coronary heart disease. One such gene, TCF21, encodes a basic-helix-loop-helix transcription factor believed to serve a critical role in the development of epicardial progenitor cells that give rise to coronary artery smooth muscle cells (SMC) and cardiac fibroblasts. Using reporter gene and immunolocalization studies with mouse and human tissues we have found that vascular TCF21 expression in the adult is restricted primarily to adventitial cells associated with coronary arteries and also medial SMC in the proximal aorta of mouse. Genome wide RNA-Seq studies in human coronary artery SMC (HCASMC) with siRNA knockdown found a number of putative TCF21 downstream pathways identified by enrichment of terms related to CAD, including “vascular disease,” “disorder of artery,” and “occlusion of artery,” as well as disease-related cellular functions including “cellular movement” and “cellular growth and proliferation.” In vitro studies in HCASMC demonstrated that TCF21 expression promotes proliferation and migration and inhibits SMC lineage marker expression. Detailed in situ expression studies with reporter gene and lineage tracing revealed that vascular wall cells expressing Tcf21 before disease initiation migrate into vascular lesions of ApoE-/- and Ldlr-/- mice. While Tcf21 lineage traced cells are distributed throughout the early lesions, in mature lesions they contribute to the formation of a subcapsular layer of cells, and others become associated with the fibrous cap. The lineage traced fibrous cap cells activate expression of SMC markers and growth factor receptor genes. Taken together, these data suggest that TCF21 may have a role regulating the differentiation state of SMC precursor cells that migrate into vascular lesions and contribute to the fibrous cap and more broadly, in view of the association of this gene with human CAD, provide evidence that these processes may be a mechanism for CAD risk attributable to the vascular wall.

KLF4-dependent phenotypic modulation of smooth muscle cells has a key role in atherosclerotic plaque pathogenesis.

Herein we employ Myh11-CreERT2 ROSA floxed STOP eYFP Apoe−/− smooth muscle cell (SMC) lineage tracing mice to show that traditional methods for detecting SMCs based on immuno-staining fail to detect > 80% of SMC-derived cells within advanced atherosclerotic lesions. These unidentified SMC-derived cells exhibit phenotypes of other cell lineages including macrophages (Mϕs), and mesenchymal stem cells (MSCs). SMC-specific conditional knockout (KO) of Krüppel-like factor 4 (KLF4) resulted in reduced numbers of SMC-derived MSC-, and Mϕ-like cells, marked reductions in lesion size, and increases in multiple indices of plaque stability, including an increase in fibrous cap thickness. Results of in vivo KLF4 ChIP-Seq analyses, and studies in cultured SMC treated with cholesterol identified > 800 KLF4 target genes including many that regulate pro-inflammatory responses of SMC. Results indicate that the contribution of SMCs within atherosclerotic plaques has been greatly underestimated, and that KLF4-dependent transitions in SMC phenotype are critical in lesion pathogenesis.

Abstract 150: Identification of SYNPO2, SYNM, LMOD1, PDLIM7 and PLN as Novel Markers of Smooth Muscle Cells in Atherosclerosis

Smooth muscle cell (SMC) activation is a hallmark of vascular remodeling associated with downregulation of SMC specific genes and altered cell function. Our aim was to identify a novel set of genes silenced following SMC activation in vascular disease. We compared microarrays from carotid plaques (n=177) vs. undiseased arteries and symptomatic vs. asymptomatic patients and found that ‘muscle contraction’, ‘muscle differentiation’ and ‘cytoskeleton based migration’ were the most significantly downregulated pathways (p=6.06e-23; 5.22e-11; 4.62e-11 respectively). Genes clustered in these categories were known SMC markers together with a novel set of genes functionally coupled to actin cytoskeleton: SYNPO2, SYNM, LMOD1, PDLIM7 and PLN. Transcription factors regulating the expression of these genes showed enrichment of SRF, MYOD1 and MYOGENIN, known to control muscle differentiation. Downregulation of these genes was validated in independent microarrays from carotid (n=21) and coronary plaques (n=38). Their expression was positively correlated to typical SMC markers both in human plaques (Pearson r&gt;0.8, p&lt;0.0001) and rat carotid intimal hyperplasia (r&gt;0.9, p&lt;0.0001). In microarrays from rat carotid balloon injury, these genes were downregulated in the early phases of tissue remodeling but gradually reappeared in the mature intima. By immunohistochemistry, the proteins localized to SMCs in normal human vessels, but were mostly absent in plaques and restenosis tissues. In vitro, SYNPO2 and PDLIM7 localized to actin filaments, SYNM to cortical cytoskeleton, PLN to nucleus while LMOD1 was rapidly lost in cultured human and primary rat SMCs. Polymorphisms in some of these genes associated with carotid intima-media thickness in high-risk coronary artery disease subjects (n=3400). By eQTL analyses, rs11746443 influenced PDLIM7 expression in plaques. Interestingly, this polymorphism constitutes the binding site for HEY1 transcription factor implicated in vascular development. We identified a panel of novel SMC proteins, which are lost in vascular disease and reflect the altered phenotype of SMCs in vascular remodeling. Our results indicate that these could be early and sensitive markers of the SMCs dedifferentiation in atherosclerosis.

Abstract 649: Dedicator of Cytokinesis 2 is Essential for the Initiation of Transforming Growth Factor-Beta-Induced Smooth Muscle Differentiation

Smooth muscle cell (SMC) differentiation is an important process during embryonic development. The underlying mechanisms regulating the SMC differentiation process, however, remain largely unknown. The present study identified dedicator of cytokinesis 2 (DOCK2) as one of the factors mediating the SMC differentiation. DOCK2 was important in mediating the transforming growth factor-β (TGF-β)-induced SMC differentiation from human embryonic stem cell derived mesenchymal cells (hES-MCs) and 10T 1/2 pluripotent embryonic mesenchymal cells. TGF-β induced DOCK2 expression during SMC differentiation as shown by the SMC marker expression including α-SMA, SM22α and calponin. DOCK2 overexpression induced while DOCK2 knockout inhibited SMC early marker gene expression. Mechanistically, DOCK2 induced SMC differentiation by activating phosphorylated p38 MAPK. DOCK2 overexpression induced while DOCK2 knockdown inhibited the level of phosphorylated p38. P38 pathway inhibitor dramatically decreased DOCK2-induced SMC marker gene expression. In vivo, reduced blood vessel formation was observed in the DOCK2-/- embryo yolk sac compared to the wild type yolk sac. Hemorrhage was also evident in the DOCK2-/- embryo. Histological analysis revealed defective expression of calponin and α-SMA in dorsal aorta of DOCK2-/- embryo. Taken together, our studies demonstrated that DOCK2 is an important novel factor regulating SMC differentiation.

Abstract 679: Modeling Thoracic Aortic Aneurysm of Bicuspid Aortic Valve Patients with Induced Pluripotent Stem Cells Reveals Distinct Phenotypes of Smooth Muscle Cells from Different LIneages

Ascending aortic aneurysm occurs in 35%-68% patients with bicuspid aortic valves (BAV), which is more frequently than it does in patients with normal trileaflet aortic valves (TAV). In contrast to recurrent aneurysms of the ascending aorta, descending aortic aneurysms is very rare in patients with BAV. Vascular Smooth Muscle Cells (SMCs) make up the media of the aortic walls, and their contractile properties help sustain vessel tone. Interestingly, ascending aorta is populated by SMCs arising from neural crest and descending aorta by SMCs from paraxial mesoderm. An intriguing hypothesis is that the distinct susceptibility of aneurysm in the ascending and descending aorta in patients with BAV is due in part to disparities in SMC embryonic origins. To prove our hypothesis, we used an induced Pluripotent Stem Cells (iPS) model system to generate SMCs from neural crest and paraxial mesoderm. Firstly, iPSCs were established from a patient with BAV and ascending thoracic aortic aneurysm (BAV/TAA) and two control patients with normal aorta and TAV. The iPSCs pluripotency was verified by standard stem cell markers and teratoma formation. Neural crest stem cells (NCSCs) were differentiated from iPSCs and expressed neural crest markers, including P75, HNK1, AP2 and Sox9. In parallel, paraxial mesoderm cells (PMCs) were differentiated from iPSCs and expressed specific markers, including TCF15, TBX6, Meox1 and Pax1. The SMCs differentiated from both lineages had expression of SMC markers including MYH11, CNN1, ACTA2, SM22α. Interestingly, SMCs derived from BAV-NCSCs had significantly decreased in MYH11 and ACTA2 gene expression and impaired contractile property by gel contraction assay compared to SMCs derived from control-NCSCs. However, SMCs from BAV-PMCs had similar level of expression of MYH11 and ACTA2, and strong contractile activity with Carbacol treatment compared to SMCs derived from control PMCs. In conclusion, the iPS model system revealed decreased expression of contractile proteins and impairment of contractility in SMCs derived from NCSCs in BAV patient, but not in the SMCs derived from paraxial mesoderm cells. The high susceptibility of aneurysm in the ascending aorta in patients with BAV might be partly due to embryonic origin of the SMCs.