SMC-like Cells Research Articles

Identification and removal of unexpected proliferative off-target cells emerging after iPSC-derived pancreatic islet cell implantation

Differentiation of pancreatic endocrine cells from human pluripotent stem cells (PSCs) has been thoroughly investigated for application in cell therapy against diabetes. In the context of induced pancreatic endocrine cell implantation, previous studies have reported graft enlargement resulting from off-target pancreatic lineage cells. However, there is currently no documented evidence of proliferative off-target cells beyond the pancreatic lineage in existing studies. Here, we show that the implantation of seven-stage induced PSC-derived pancreatic islet cells (s7-iPICs) leads to the emergence of unexpected off-target cells with proliferative capacity via in vivo maturation. These cells display characteristics of both mesenchymal stem cells (MSCs) and smooth muscle cells (SMCs), termed proliferative MSC- and SMC-like cells (PMSCs). The frequency of PMSC emergence was found to be high when 108 s7-iPICs were used. Given that clinical applications involve the use of a greater number of induced cells than 108, it is challenging to ensure the safety of clinical applications unless PMSCs are adequately addressed. Accordingly, we developed a detection system and removal methods for PMSCs. To detect PMSCs without implantation, we implemented a 4-wk-extended culture system and demonstrated that putative PMSCs could be reduced by compound treatment, particularly with the taxane docetaxel. When docetaxel-treated s7-iPICs were implanted, the PMSCs were no longer observed. This study provides useful insights into the identification and resolution of safety issues, which are particularly important in the field of cell-based medicine using PSCs.

Pericytes contribute to pulmonary vascular remodeling via HIF2α signaling

Vascular remodeling is the process of structural alteration and cell rearrangement of blood vessels in response to injury and is the cause of many of the world’s most afflicted cardiovascular conditions, including pulmonary arterial hypertension (PAH). Many studies have focused on the effects of vascular endothelial cells and smooth muscle cells (SMCs) during vascular remodeling, but pericytes, an indispensable cell population residing largely in capillaries, are ignored in this maladaptive process. Here, we report that hypoxia-inducible factor 2α (HIF2α) expression is increased in the lung tissues of PAH patients, and HIF2α overexpressed pericytes result in greater contractility and an impaired endothelial-pericyte interaction. Using single-cell RNAseq and hypoxia-induced pulmonary hypertension (PH) models, we show that HIF2α is a major molecular regulator for the transformation of pericytes into SMC-like cells. Pericyte-selective HIF2α overexpression in mice exacerbates PH and right ventricular hypertrophy. Temporal cellular lineage tracing shows that HIF2α overexpressing reporter NG2+ cells (pericyte-selective) relocate from capillaries to arterioles and co-express SMA. This novel insight into the crucial role of NG2+ pericytes in pulmonary vascular remodeling via HIF2α signaling suggests a potential drug target for PH.

Cyclic nucleotide phosphodiesterase 1C contributes to abdominal aortic aneurysm

Abdominal aortic aneurysm (AAA) is characterized by aorta dilation due to wall degeneration, which mostly occurs in elderly males. Vascular aging is implicated in degenerative vascular pathologies, including AAA. Cyclic nucleotide phosphodiesterases, by hydrolyzing cyclic nucleotides, play critical roles in regulating vascular structure remodeling and function. Cyclic nucleotide phosphodiesterase 1C (PDE1C) expression is induced in dedifferentiated and aging vascular smooth muscle cells (SMCs), while little is known about the role of PDE1C in aneurysm. We observed that PDE1C was not expressed in normal aorta but highly induced in SMC-like cells in human and murine AAA. In mouse AAA models induced by Angiotensin II or periaortic elastase, PDE1C deficiency significantly decreased AAA incidence, aortic dilation, and elastin degradation, which supported a causative role of PDE1C in AAA development in vivo. Pharmacological inhibition of PDE1C also significantly suppressed preestablished AAA. We showed that PDE1C depletion antagonized SMC senescence in vitro and/or in vivo, as assessed by multiple senescence biomarkers, including senescence-associated β-galactosidase activity, γ-H2AX foci number, and p21 protein level. Interestingly, the role of PDE1C in SMC senescence in vitro and in vivo was dependent on Sirtuin 1 (SIRT1). Mechanistic studies further showed that cAMP derived from PDE1C inhibition stimulated SIRT1 activation, likely through a direct interaction between cAMP and SIRT1, which leads to subsequent up-regulation of SIRT1 expression. Our findings provide evidence that PDE1C elevation links SMC senescence to AAA development in both experimental animal models and human AAA, suggesting therapeutical significance of PDE1C as a potential target against aortic aneurysms.

Pro-Epicardial Cells are Heterogeneous with Specified Smooth Muscle-Like and Pacemaker Progenitor Cells

The heterogeneity and specification of pro-epicardial cells (pro-EpiCs) to fibroblast and smooth muscle cell (SMC) are unknown. We applied single-cell RNA sequencing (scRNA-seq) paired with RNAScope, bioinformatics, and lineage tracing in an unbiased manner to identify the previously uncharacterized molecular heterogeneity of the pro-EpiCs isolated from pro-epicardium (PE). We found that pro-EpiCs labeled by Tbx18 Cre/+ are heterogeneous, with three clusters displaying differential expression profiles and distinct spatial locations. Cluster 1 are mesothelial cells, and Cluster 2 express SMC markers. Surprisingly, Cluster 3 express Isl1 and markers of pacemaker progenitor cells (PMPC) but not marker of atrial cardiomyocytes. Our studies conclude that pro-EpiCs are heterogeneous and SMC-like cells are specified in the PE, while fibroblasts are not specified in PE but epicardium. We identified a region in PE that contains PMPCs, which translocate through the inflow tract to the sinoatrial node. The expression profile of Cluster 3 cells unifies previous studies regarding the origins and markers of PMPCs.

Disease-Relevant Single Cell Photonic Signatures Identify S100\u03b2 Stem Cells and their Myogenic Progeny in Vascular Lesions

A hallmark of subclinical atherosclerosis is the accumulation of vascular smooth muscle cell (SMC)-like cells leading to intimal thickening and lesion formation. While medial SMCs contribute to vascular lesions, the involvement of resident vascular stem cells (vSCs) remains unclear. We evaluated single cell photonics as a discriminator of cell phenotype in vitro before the presence of vSC within vascular lesions was assessed ex vivo using supervised machine learning and further validated using lineage tracing analysis. Using a novel lab-on-a-Disk(Load) platform, label-free single cell photonic emissions from normal and injured vessels ex vivo were interrogated and compared to freshly isolated aortic SMCs, cultured Movas SMCs, macrophages, B-cells, S100β+ mVSc, bone marrow derived mesenchymal stem cells (MSC) and their respective myogenic progeny across five broadband light wavelengths (λ465 - λ670 ± 20 nm). We found that profiles were of sufficient coverage, specificity, and quality to clearly distinguish medial SMCs from different vascular beds (carotid vs aorta), discriminate normal carotid medial SMCs from lesional SMC-like cells ex vivo following flow restriction, and identify SMC differentiation of a series of multipotent stem cells following treatment with transforming growth factor beta 1 (TGF- β1), the Notch ligand Jagged1, and Sonic Hedgehog using multivariate analysis, in part, due to photonic emissions from enhanced collagen III and elastin expression. Supervised machine learning supported genetic lineage tracing analysis of S100β+ vSCs and identified the presence of S100β+vSC-derived myogenic progeny within vascular lesions. We conclude disease-relevant photonic signatures may have predictive value for vascular disease.Graphical abstract

Detrusor bioengineering using a cell-enriched compressed collagen hydrogel.

The gold standard for bladder regeneration in end-stage bladder disease is the use of intestinal tissue, which is however associated with significant long-term complications. Our study aims to bioengineer functional detrusor muscle combining bladder smooth muscle cells (SMC) and SMC-like adipose-derived stem cells (pADSC) in compressed collagen (CC) hydrogels and to investigate biocompatibility and tissue regeneration of such detrusor-equivalents in a rat detrusorectomy model. Compressed collagen hydrogels seeded with 1 × 106 or 4 × 106 SMC alone or in combination with pADSC in a 1:1 ratio were investigated. Morphology, phenotype, and viability as well as proteomic secretome analysis were assessed in the 1:1 co-cultures and the respective monocultures. The hydrogels were implanted into rat bladders after partial detrusorectomy. Bladders were harvested 8 weeks after transplantation, and assessed for tissue morphology, detrusor regeneration, neo-vascularization and -innervation. Co-cultured cells exhibited native SMC morphology, high viability and proliferated to form microtissues in vitro. The pro-angiogenic factors angiogenin, vascular endothelial growth factor (VEGF)-A and -D were increased in the secretome of the pADSC samples. After 8 weeks of in vivo, the regenerated bladder wall showed a multilayered structure containing all bladder wall components. The overall performance of the bladder wall regeneration of CC seeded with 4 × 106 cells was significantly better than with 1 × 106 cells and the combination SMC:pADCS performed slightly better than SMC alone. Compressed collagen possesses an adequate regenerative potential to promote regeneration of bladder wall tissue in vivo. Seeded with a combination of pADSC and SMC this may well be the first step towards a functional bladder reconstruction especially in patients suffering of end-stage bladder diseases.

Abstract 16591: Exosome-Mediated Endothelial Cell Apoptosis and Network Disruption by Lymphangioleiomyomatosis-Smooth Muscle Cells (LAM-SMCs) Derived from Human Induced Pluripotent Stem Cell (iPSC)

Introduction: In LAM, SMC-like cells invade the lung thus compromising respiratory function which is dependent on normal vascular and alveoli structure. We posit that while healthy mural SMCs stabilize fragile neovessels and maintain vascular homeostasis, LAM-SMCs promote disruption of lung microvasculature by inducing endothelial cell (EC) apoptosis. Methods/Results: iPSCs derived from LAM patients or healthy subjects were differentiated into SMCs using a teratoma protocol. Co-culture of healthy iSMCs with HUVECs on Matrigel improved EC network persistence for ≥72Hrs, while LAM-SMC co-culture led to rapid network collapse in <15Hrs. Culture of ECs with LAM-SMC conditioned media (24-72Hrs) increased EC apoptosis as assessed by Annexin V/PI flow cytometry, confirmed by increases in cleaved Caspase-3 protein expression. To further elucidate mechanism and kinetics of EC apoptosis, Annexin V/PI flow cytometry and Western Blot with Caspases 8,9,12 were performed in parallel at 3hr intervals from 0-12 Hrs. Increase in EC apoptosis at 9hrs, corresponded with a 6-fold increase in Cas-12 protein expression, followed by a 3-fold increase in Cas-9 at 12hrs with little change in Cas-8. This is in line with our RNAseq study showing dysregulation in endoplasmic reticulum and mitochondria related genes in LAM-SMC. Culture of HUVECs with LAM-SMC exosomes similarly induced EC apoptosis, with a profound decrease in EC gene expression (eNOS,TIE2,TAL1; 5-7 fold; p<0.05) but increase expression of vessel-destabilizing, ANGPT2 (6-fold; p<0.05). Scratch-wound assay also showed ~60% reduction (p<0.05) in EC migratory capacity. Intriguingly, apoptotic ECs released exosomes containing translationally controlled tumor protein, TCTP, which were taken up by LAM-SMCs, as evidenced by immunofluorescence studies, associated with increases in the levels of proliferation-associated phospho-mTOR-and-S6k protein. Conclusions: LAM-SMCs derived exosomes resulted in EC apoptosis and network destabilization. Conversely, apoptotic ECs enhanced LAM-SMC growth, possibly by Caspase-dependent exosome release of TCTP, a positive regulator of mTOR signaling. These novel mechanisms may promote both microvasculature disruption and uncontrolled growth of LAM-SMCs.

24 - The x-factor involved in apoptosis and proliferation—reciprocal exosome cross-talk between lymphangioleiomyomatosis-derived smooth muscle cells (LAM-SMCs) and endothelial cells (ECs) mediates EC network disruption/apoptosis and promotes LAM-SMC growth

Introduction: Lymphangioleiomyomatosis (LAM) is a progressive and incurable disease caused by mutations in the TSC2 gene, which is involved in mTOR pathway regulation. Fragile endothelial cell (EC) neovessels are stabilized by mural smooth muscle cells (SMCs). However, SMC-like LAM cells invade and destroy the lung through unknown mechanism(s). We postulate that LAM-SMCs will disrupt EC capillary-like networks and promote EC apoptosis.

MP24-18 DETRUSOR BIOENGINEERING USING COMPRESSED COLLAGEN, ADIPOSE-DERIVED STEM CELLS AND SMOOTH MUSCLE CELLS

You have accessJournal of UrologyTrauma/Reconstruction/Diversion: Ureter (including Pyeloplasty) and Bladder Reconstruction (including fistula), Augmentation, Substitution, Diversion I1 Apr 2017MP24-18 DETRUSOR BIOENGINEERING USING COMPRESSED COLLAGEN, ADIPOSE-DERIVED STEM CELLS AND SMOOTH MUSCLE CELLS Jakub Smolar, Maya Horst, and Daniel Eberli Jakub SmolarJakub Smolar More articles by this author , Maya HorstMaya Horst More articles by this author , and Daniel EberliDaniel Eberli More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.3312AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Conditions impairing bladder function in children and adults often need urinary diversion or augmentation cystoplasty as when untreated they may cause severe bladder dysfunction and kidney failure. Currently, the gold standard therapy of end-stage bladder disease refractory to conservative management is enterocystoplasty, which despite providing functional improvement is associated with significant long-term complications. Therefore, there is a strong clinical need for alternative therapies for these reconstructive procedures. The aim of this study is to develop functional smooth muscle tissue for the detrusor muscle repair combining various cell types in hydrogel scaffolds. METHODS Rat bladder smooth muscle (SMC) and adipose-derived stem cells (ADSC) were isolated, expanded and characterized using flow cytometry. ADSC were pre-differentiated into SMC-like cells (pADSC). Cells were combined in ratios 1:1, 1:2 and 1:3 (SMC:pADSC) and embedded in compressed collagen (CC). After 1, 2 and 3 weeks, cells in CC scaffolds, direct and indirect 2D co-cultures were analyzed for viability, proliferation, morphology, SMC-marker expression and functionality. RESULTS Cell growth conditions have been optimized and cells have shown high viability and good proliferation in the CC scaffolds. Interconnected microtissues and cell layers have developed all over the CC already after 1 week of co-culture. At 1 and 2 week timepoints cells in CC showed strong expression of the SMC markers calponin, MyH11 and smoothelin. Direct cell co-culture resulted in significantly increased cellular proliferation. Microtissues consisted of a SMC-core surrounded by pADSC. Indirect co-culture resulted in an increased pADSC survival and ratio-dependent increase in SMC-proliferation. pADSC proliferation rate also improved, but remained unaffected by the cell ratio, with 1:1 showing the most consistent results. SMC-marker expression normalized between the different ratios after 2 weeks of co-culture and reached almost the SMC monoculture expression levels. The 1:1 co-culture contracted significantly better than the other ratios after 24h. CONCLUSIONS We have shown that a SMC–pADSC co-culture results in an improved cell survival, proliferation, microtissue and cell layer formation without any significant changes in phenotype and functionality. The combination of SMC and pADSC with CC may help to engineer functional detrusor muscle tissue by solving the major issues of tissue engineering, namely poor cell survival, proliferation, phenotype instability and functionality. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e308-e309 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Jakub Smolar More articles by this author Maya Horst More articles by this author Daniel Eberli More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

"Attack of the Clones": Commonalities Between Cancer and Atherosclerosis.

Many studies have noted similarities between atherosclerosis and cancer including pronounced cellular plasticity, clonal expansion of cellular subtypes, increased DNA mutations, defective efferocytosis pathways, and an important role for proto-oncogenes in disease development. Although it is clear that these 2 diseases have disparate causes, noting the parallels between atherosclerosis and cancer may help us identify unique, targeted therapeutic strategies. Early articles described atheromas as benign neoplasms of the blood vessel comprised primarily of fibrous smooth muscle cells (SMCs). However, additional studies soon noted the presence of macrophages and other cell types, thereby promoting controversy as to which cells actually give rise to the atherosclerotic plaque. Much of this controversy results from the promiscuity of the so-called lineage-specific markers.1 For example, bone marrow–derived cells can migrate into plaques and begin to express certain SMC markers,2 whereas SMCs can upregulate macrophage-specific markers such as galectin-3, CD11b, and F4/80 as they migrate into the plaque.3,4 In addition, recent lineage tracing studies suggest that endothelial cells can acquire mesenchymal cell characteristics, and some adventitial cells may have the capability of forming SMC-like cells. Collectively, these studies demonstrate the impressive capacity of many vascular cell types to dedifferentiate or transdifferentiate in response to the atherosclerotic environment and suggest that plaques may arise from multiple vascular and circulating cell types. Clearly, cellular plasticity is a major theme in atherosclerotic plaque development—as it is in cancer—and methods to reprogram proatherosclerotic cells could have tremendous therapeutic potential. The clonal evolution theory of cancer development posits that certain tumors arise from a single cell that expands in response to a series of acquired mutations. In 1973, Benditt and Benditt5 performed X-inactivation studies on human atherosclerotic lesions and found evidence that atherosclerotic plaques could be derived from a single cell that underwent monoclonal expansion, …

Transdifferentiation of Human Dermal Fibroblasts to Smooth Muscle-Like Cells to Study the Effect of MYH11 and ACTA2 Mutations in Aortic Aneurysms.

Mutations in genes encoding proteins of the smooth muscle cell (SMC) contractile apparatus contribute to familial aortic aneurysms. To investigate the pathogenicity of these mutations, SMC are required. We demonstrate a novel method to generate SMC-like cells from human dermal fibroblasts by transdifferentiation to study the effect of variants in genes encoding proteins of the SMC contractile apparatus (ACTA2 and MYH11) in patients with aortic aneurysms. Dermal fibroblasts from seven healthy donors and cells from seven patients with MYH11 or ACTA2 variants were transdifferentiated into SMC-like cells within a 2-week duration using 5 ng/ml TGFβ1 on a scaffold containing collagen and elastin. The induced SMC were comparable to primary human aortic SMC in mRNA expression of SMC markers which was confirmed on the protein level by immunofluorescence quantification analysis and Western blotting. In patients with MYH11 or ACTA2 variants, the effect of intronic variants on splicing was demonstrated on the mRNA level in the induced SMC, allowing classification into pathogenic or nonpathogenic variants. In conclusion, direct conversion of human dermal fibroblasts into SMC-like cells is a highly efficient method to investigate the pathogenicity of variants in proteins of the SMC contractile apparatus.

Responses of adventitial CD34+ vascular wall-resident stem/progenitor cells and medial smooth muscle cells to carotid injury in rats

Cell culture and carotid injury studies with SD rats were performed to investigate the roles of CD34+ vascular wall-resident stem/progenitor cells (VRS/Pcs) and vascular smooth muscle cells (SMCs) in neointimal formation. In vitro, the media-isolated SM MHC+ SMCs occupied 93.92±8.62% of total BrdU+ cells, whereas the CD34+ cells, only 2.61±0.82%, indicating that the cell expansion in SMC culture was attributed to SM MHC+ SMCs. The adventitia-isolated CD34+ VRS/Pcs responded to PDGF-BB by differentiating into SMC-like cells which expressed SM22α (an early stage SMC marker), but seldom SM MHC (a late stage SMC marker). In carotid injury model, the CD34+ VRS/Pcs differentiated SMC-like cells migrated in very few numbers into only the outer layer of the media, and this was further confirmed by a cell tracking analysis. While the neointimal cells were consistently SM MHC+ and CD34− SMCs during whole course of the post-injury remodeling. Thus it is speculated that the adventitial CD34+ VRS/Pcs, at least in rat model, do not directly participate in neointimal formation, but function to maintain homeostasis of the media during injury-induced vascular wall remodeling.

Fibroblast Growth Factor 12 Is a Novel Regulator of Vascular Smooth Muscle Cell Plasticity and Fate.

Vascular smooth muscle cells (VSMCs) modulate their phenotype between synthetic and contractile states in response to environmental changes; this modulation plays a crucial role in the pathogenesis of restenosis and atherosclerosis. Here, we identified fibroblast growth factor 12 (FGF12) as a novel key regulator of the VSMC phenotype switch. Using murine models and human specimens, we found that FGF12 was highly expressed in contractile VSMCs of normal vessel walls but was downregulated in synthetic VSMCs from injured and atherosclerotic vessels. In human VSMCs, FGF12 expression was inhibited at the transcriptional level by platelet-derived growth factor-BB. Gain- and loss-of-function experiments showed that FGF12 was both necessary and sufficient for inducing and maintaining the quiescent and contractile phenotypes of VSMCs. FGF12 inhibited cell proliferation through the p53 pathway and upregulated the key factors involved in VSMC lineage differentiation, such as myocardin and serum response factor. Such FGF12-induced phenotypic change was mediated by the p38 MAPK (mitogen-activated protein kinase) pathway. Moreover, FGF12 promoted the differentiation of mouse embryonic stem cells and the transdifferentiation of human dermal fibroblasts into SMC-like cells. Furthermore, adenoviral infection of FGF12 substantially decreased neointima hyperplasia in a rat carotid artery injury model. In general, FGF family members induce a synthetic VSMC phenotype. Interestingly, the present study showed the unanticipated finding that FGF12 belonging to FGF family, strongly induced the quiescent and contractile VSMC phenotypes and directly promoted VSMC lineage differentiation. These novel findings suggested that FGF12 could be a new therapeutic target for treating restenosis and atherosclerosis.

Myocardin Overexpression Is Sufficient for Promoting the Development of a Mature Smooth Muscle Cell-Like Phenotype from Human Embryonic Stem Cells

BackgroundMyocardin 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/PrincipalFindings 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/SignificanceMyocardin 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.

Bone Marrow–Derived Smooth Muscle Cells Are Breaking Bad in Atherogenesis

Vascular smooth muscle cells (SMCs) are highly specialized in contraction, thus regulating the vascular tone. In contrast to other muscle cells, however, SMCs can reversibly undergo quite drastic phenotypic changes in response to environmental signals.1 Following vascular injury, for instance, the expression of contractile proteins in SMCs substantially declines and these cells adopt a “synthetic” phenotype.2 The origin of cells expressing SMC-specific markers in neointimal lesions or atherosclerotic plaques is known to be heterogeneous. The long-standing paradigm that these SMC-like cells migrate from the media into the intima on injury has been challenged on the grounds that it may not be the only explanation of their provenience. Namely, seminal work by Sata et al3 has provided evidence that—at least partly and depending on the degree of injury—SMC-like cells in different forms of arterial plaque development are derived from the bone marrow (BM). The extent to which BM-derived SMCs (BM-SMCs) contribute to the cellularity …

Bone marrow-derived smooth muscle-like cells are infrequent in advanced primary atherosclerotic plaques but promote atherosclerosis.

Although vascular smooth muscle cells (VSMCs) provide the major structural integrity of atherosclerotic plaques, their origin has been questioned. In particular, although some studies identified plaque VSMCs originating from bone marrow or peripheral blood, their frequency is controversial and their function unknown. We used genetic tracking of cell fate through smooth muscle cell (SMC)-specific LacZ reporter activity and VSMC-selective apoptosis to investigate the frequency, distribution, and role of marrow-derived VSMCs in atherogenesis. Cultured mouse bone marrow-derived smooth muscle-like cells expressed SMC markers and functional SMC promoter-driven transgenes over time. Transplantation of apolipoprotein E (ApoE)(-/-) mice with smooth muscle myosin heavy chain-Cre/ROSA26R/ApoE(-/-) marrow showed that 0.7±0.14% cells expressed LacZ in atherosclerotic plaques, located superficially in early plaques, and in necrotic cores but not fibrous caps of advanced lesions. Cells expressing both progenitor and SMC markers showed a similar distribution and frequency. Apoptosis of marrow-derived SMC-like cells transplanted from SM22α-human diphtheria toxin receptor/ApoE(-/-) mice retarded atherogenesis, with reduced plaque macrophage content. Cultured marrow-derived SMC-like cells secreted proinflammatory cytokines and promoted macrophage migration, VSMC proliferation, and collagen synthesis. Bone marrow-derived SMC-like cells are infrequent in advanced primary atherosclerotic plaques and absent in fibrous caps. However, these cells secrete proinflammatory cytokines and mitogens and promote atherosclerosis.

Endothelial progenitor cells undergo an endothelial-to-mesenchymal transition-like process mediated by TGFβRI

Endothelial progenitor cells (EPC) have been shown to repair pulmonary endothelium, although they can also migrate into the arterial intima and differentiate into smooth muscle-like (mesenchymal) cells contributing to intimal hyperplasia. The molecular mechanisms by which this process proceeds have not been fully elucidated. Here, we study whether genes involved in the endothelial-to-mesenchymal transition (EnMT) may contribute to the mesenchymal phenotype acquisition of EPC and we evaluate whether transforming growth factor β1 (TGFβ1) is involved in this process. Our results show that co-culture of EPC with smooth muscle cells (SMC) increases the expression of the mesenchymal cell markers α-smooth muscle actin, sm22-α, and myocardin, and decreases the expression of the endothelial cell marker CD31. In the same conditions, we also observed a concomitant increase in the gene expression of the EnMT-related transcription factors: slug, snail, zeb1, and endothelin-1. This indicates that mesenchymal phenotype acquisition occurred through an EnMT-like process. Inhibition of TGFβ receptor I (TGFβRI) downregulated snail gene expression, blocked the EnMT, and facilitated the differentiation of EPC to the endothelial cell lineage. Furthermore, TGFβRI inhibition decreased migration of EPC stimulated by SMC without affecting their functionality and adhesion capacity. These results indicate that EPC may differentiate into SMC-like cells through an EnMT-like process and that TGFβI plays an important role in the fate of EPC.

Modulation of Myocardin Function by the Ubiquitin E3 Ligase UBR5

Fully differentiated mature smooth muscle cells (SMCs) are characterized by the presence of a unique repertoire of smooth muscle-specific proteins. Although previous studies have shown myocardin to be a critical transcription factor for stimulating expression of smooth muscle-specific genes, the mechanisms regulating myocardin activity are still poorly understood. We used a yeast two-hybrid screen with myocardin as bait to search for factors that may regulate the transcriptional activity of the myocardin. From this screen we identified a HECT domain-containing protein UBR5 (ubiquitin protein ligase E3 component n-recognin 5) as a myocardin-binding protein. Previous studies have shown that HECT domain-containing proteins are ubiquitin E3 ligases that play an important role in protein degradation. UBR5 has, however, also been shown to regulate transcription independent of its E3 ligase activity. In the current study we demonstrated that UBR5 localized in the nuclei of SMCs and forms a complex with myocardin in vivo and in vitro. We also show that UBR5 specifically enhanced trans-activation of smooth muscle-specific promoters by the myocardin family of proteins. In addition, UBR5 significantly augmented the ability of myocardin to induce expression of endogenous SMC marker genes independent on its E3 ligase function. Conversely, depletion of endogenous UBR5 by small interfering RNA in fibroblast cells attenuated myocardin-induced smooth muscle-specific gene expression, and UBR5 knockdown in SMCs resulted in down-regulation of smooth muscle-specific genes. Furthermore, we found that UBR5 can attenuate myocardin protein degradation resulting in increased myocardin protein expression without affecting myocardin mRNA expression. The effects of UBR5 on myocardin requires only the HECT and UBR1 domains of UBR5. This study reveals an unexpected role for the ubiquitin E3 ligase UBR5 as an activator of smooth muscle differentiation through its ability to stabilize myocardin protein.

A Specific Subset of Mouse Bone Marrow Cells Has Smooth Muscle Cell Differentiation Capacity–Brief Report

To determine whether CX(3)CR1(+) bone marrow cells have the capacity for smooth muscle cell (SMC) differentiation. CX(3)CR1(+) and CX(3)CR1(-) cells were isolated from marrow of CX(3)CR1 transgenic mice and cultured in SMC differentiation media. Phenotypic and functional analyses showed only CX(3)CR1(+) bone marrow cells exhibit colony cell outgrowth with SMC-specific protein expression, calcium signaling, and contraction responses similar to mature SMC. CX(3)CR1 marks a bone marrow cells population that enriches for progenitors with capacity to differentiate in vitro into SMC-like cells.

Abstract 5547: CXCR4 Signaling Exacerbates Vein Graft Neointimal Hyperplasia

Vein graft neointimal hyperplasia can cause vein graft failure directly or, by promoting accelerated atherosclerosis, indirectly. Because most vein graft neointimal cells originate from outside the vein graft, and because CXCR4, the SDF-1α receptor, contributes significantly to recruitment of vascular progenitor cells, we tested the hypothesis that CXCR4 activity exacerbates vein graft neointimal hyperplasia. We performed carotid artery interposition vein grafting in congenic WT and cxcr4 −/+ mice, using the inferior vena cava of WT and cxcr4 −/+ mice as vein grafts. We created 3 surgically chimeric groups, designated by vein graft donor/vein graft recipient: WT/WT, cxcr4 −/+ /WT, and cxcr4 −/+ / cxcr4 −/+ . Immunofluorescence for SDF-1, the CXCR4 agonist, was evident in 2-wk-old vein grafts (with expanding neointimas), but not in 6-wk-old vein grafts (in which neointimal hyperplasia had reached steady state). In 6-wk-old WT/WT, cxcr4 −/+ /WT, and cxcr4 −/+ / cxcr4 −/+ vein grafts, respectively, neointimal areas were 0.12 ± 0.01, 0.118 ± 0.008, and 0.066 ± 0.008 mm 2 , while medial areas were 0.20 ± 0.02, 0.26 ± 0.01, and 0.32 ± 0.02 mm 2 . Thus, relative to WT/WT grafts, neointimal area was reduced only in cxcr4 −/+ / cxcr4 −/+ vein grafts, by 47% ( p <0.01), while medial area was increased in cxcr4 −/+ /WT and cxcr4 −/+ / cxcr4 −/+ vein grafts, by 26% and 56%, respectively ( p <0.05). However, vein graft re-endothelialization was equivalent in all 3 vein graft groups (judged by immunostaining for Tie2). SMC-like cells comprised 100% of neointimal cells, but < 50% of medial cells. In cxcr4 −/+ / cxcr4 −/+ vein grafts, however, the prevalence of medial SMC actin-positive cells was 45±20% greater than in WT/WT grafts (p<0.04), while the prevalence of collagen I-positive cells was 40±5% less (p<0.01), as assessed by quantitative immunofluorescence. We conclude that CXCR4 contributes to vein graft neointimal hyperplasia through endothelium-independent mechanisms that alter homing of graft-extrinsic cells to the vein graft. Moreover, while CXCR4 activity augments neointimal hyperplasia, it reduces the medial hypertrophy that is believed to be desirable for reducing vein graft wall stress. These results suggest that CXCR4 antagonist therapy may alleviate vein graft disease.