Smooth Muscle Cell Marker Genes Research Articles

CircDiaph3 influences PASMC apoptosis by regulating PI3K/AKT/mTOR pathway through IGF1R

The pathogenesis of pulmonary hypertension has not been elucidated. We investigated the role of a circular ribonucleic acid, circDiaph3, in the proliferation and migration of pulmonary artery smooth muscle cells during pulmonary hypertension. CircDiaph3 overexpression in blood samples of patients with pulmonary hypertension was analyzed by real-time quantitative polymerase chain reaction. Subsequently, a rat model of pulmonary arterial hypertension was established under hypoxic conditions. Pulmonary artery smooth muscle cells were harvested from the rat model for subsequent experiments with small interfering ribonucleic acid-mediated knockdown of circDiaph3. In cell model, we found that PI3K, AKT, mTOR and insulin-like growth factor 1 signaling pathway (IGF1R) and smooth muscle cell marker genes (α-SMA, Vcam1) were significantly downregulated. The overexpression of Igf1r in pulmonary artery smooth muscle cells rescued the downregulated smooth muscle cell genes, IGF1R signaling pathway proteins, increased smooth muscle cell proliferation, and reduced apoptosis. CircDiaph3 regulates the PI3K/AKT/mTOR signaling pathway via IGF1R to inhibit apoptosis and promote proliferation of smooth muscle cells. Additionally, adenovirus-mediated in vivo inhibition of circDiaph3 was carried out in rats with pulmonary arterial hypertension, followed by harvesting of their pulmonary artery smooth muscle cells for subsequent experiments. Excessive proliferation of smooth muscle cells in the pulmonary artery has narrowed the pulmonary artery lumen, thereby causing pulmonary hypertension, and our results suggest that circDiaph3 has important value in the treatment of pulmonary hypertension.

Abstract 158: The Histone Demethylase JMJD3 Regulates Smooth Muscle Differentiation And Is Associated With Blood Pressure

Hypertension is associated with significant morbidity and mortality in the United States, and despite its prevalence an unmet need exists for more personalized targeted therapies. Blood pressure is regulated by environmental and genetic factors, and genome wide association studies (GWAS) have identified numerous genes in the human population associated with hypertension. Recently, one GWAS identified the single nucleotide polymorphism rs62059712 that is associated with systolic blood pressure in humans. rs62059712 is located upstream of the gene JMJD3, a histone demethylase that our lab has studied in a variety of disease contexts, and the rs62059712 major C allele (MAF 0.92) is associated with increased blood pressure in humans. Based on our analysis of data from the UCSC genome browser, we found that rs62059712 is located in a DNase Hypersensitive Site marked by H3K27Ac, suggesting it may regulate JMJD3 transcription. In luciferase assays, the fragment containing rs62059712 displayed transcription activity in smooth muscle cells (SMC), and the major C allele exhibited increased luciferase activity compared to the minor T allele in mouse aortic SMC (p<0.05). Notably, rs62059712 is in linkage disequilibrium with rs74480102, which did not display allele-specific transcription activity in SMC. siRNA knockdown of JMJD3 in mouse aortic SMC significantly decreased SMC-specific gene expression (p<0.05), and SMC marker gene expression was also reduced by treatment of SMC with the JMJD3 selective inhibitor GSK4J (p<0.05). Likewise, SMC contractile gene expression was reduced in aortas from SM22Cre;JMJD3 flox/flox mice harboring SMC-specific deletion of JMJD3. Further, angiotensin II increased JMJD3 expression in human aortic SMC. Finally, in chromatin immunoprecipitation assays in mouse aortic SMC, JMJD3 was significantly enriched at smooth muscle gene promoters (p<0.05). Our results identify the blood pressure-associated gene JMJD3 as a key epigenetic regulator of smooth muscle differentiation. Additionally, we show that the rs62059712 major C allele, which is associated with increased blood pressure, increases JMJD3 transcription activity, and likely expression, to drive increased downstream contractile smooth muscle gene expression.

Protein Tyrosine Phosphatase 1B Deficiency in Vascular Smooth Muscle Cells Promotes Perivascular Fibrosis following Arterial Injury.

Background Smooth muscle cell (SMC) phenotype switching plays a central role during vascular remodeling. Growth factor receptors are negatively regulated by protein tyrosine phosphatases (PTPs), including its prototype PTP1B. Here, we examine how reduction of PTP1B in SMCs affects the vascular remodeling response to injury. Methods Mice with inducible PTP1B deletion in SMCs (SMC.PTP1B-KO) were generated by crossing mice expressing Cre.ER T2 recombinase under the Myh11 promoter with PTP1B flox/flox mice and subjected to FeCl 3 carotid artery injury. Results Genetic deletion of PTP1B in SMCs resulted in adventitia enlargement, perivascular SMA + and PDGFRβ + myofibroblast expansion, and collagen accumulation following vascular injury. Lineage tracing confirmed the appearance of Myh11 -Cre reporter cells in the remodeling adventitia, and SCA1 + CD45 - vascular progenitor cells increased. Elevated mRNA expression of transforming growth factor β (TGFβ) signaling components or enzymes involved in extracellular matrix remodeling and TGFβ liberation was seen in injured SMC.PTP1B-KO mouse carotid arteries, and mRNA transcript levels of contractile SMC marker genes were reduced already at baseline. Mechanistically, Cre recombinase (mice) or siRNA (cells)-mediated downregulation of PTP1B or inhibition of ERK1/2 signaling in SMCs resulted in nuclear accumulation of KLF4, a central transcriptional repressor of SMC differentiation, whereas phosphorylation and nuclear translocation of SMAD2 and SMAD3 were reduced. SMAD2 siRNA transfection increased protein levels of PDGFRβ and MYH10 while reducing ERK1/2 phosphorylation, thus phenocopying genetic PTP1B deletion. Conclusion Chronic reduction of PTP1B in SMCs promotes dedifferentiation, perivascular fibrosis, and adverse remodeling following vascular injury by mechanisms involving an ERK1/2 phosphorylation-driven shift from SMAD2 to KLF4-regulated gene transcription.

Surfactant Protein A, a Novel Regulator for Smooth Muscle Phenotypic Modulation and Vascular Remodeling-Brief Report.

The objective of this study is to determine the role of SPA (surfactant protein A) in vascular smooth muscle cell (SMC) phenotypic modulation and vascular remodeling. Approach and Results: PDGF-BB (Platelet-derived growth factor-BB) and serum induced SPA expression while downregulating SMC marker gene expression in SMCs. SPA deficiency increased the contractile protein expression. Mechanistically, SPA deficiency enhanced the expression of myocardin and TGF (transforming growth factor)-β, the key regulators for contractile SMC phenotype. In vivo, SPA was induced in medial and neointimal SMCs following mechanical injury in both rat and mouse carotid arteries. SPA knockout in mice dramatically attenuated the wire injury-induced intimal hyperplasia while restoring SMC contractile protein expression in medial SMCs. These data indicate that SPA plays an important role in SMC phenotype modulation and vascular remodeling in vivo. SPA is a novel protein factor modulating SMC phenotype. Blocking the abnormal elevation of SPA may be a potential strategy to inhibit the development of proliferative vascular diseases.

AT2 receptor stimulation inhibits phosphate-induced vascular calcification

Vascular calcification is a common finding in atherosclerosis and in patients with chronic kidney disease. The renin-angiotensin system plays a role in the pathogenesis of cardiovascular remodeling. Here, we examined the hypothesis that angiotensin II type 2 receptor (AT2) stimulation has inhibitory effects on phosphate-induced vascular calcification. Invivo, calcification of the thoracic aorta induced by an adenine and high-phosphate diet was markedly attenuated in smooth muscle cell-specific AT2-overexpressing mice (smAT2-Tg) compared with wild-type and AT2-knockout mice (AT2KO). Similarly, mRNA levels of relevant osteogenic and vascular smooth muscle cell marker genes were unchanged in smAT2-Tg mice, while their expression was significantly altered in wild-type mice in response to high dietary phosphate. Exvivo, sections of thoracic aorta were cultured in media supplemented with inorganic phosphate. Aortic rings from smAT2-Tg mice showed less vascular calcification compared with those from wild-type mice. Invitro, calcium deposition induced by high-phosphate media was markedly attenuated in primary vascular smooth muscle cells derived from smAT2-Tg mice compared with the two other mouse groups. To assess the underlying mechanism, we investigated the effect of PPAR-γ, which we previously reported as one of the possible downstream effectors of AT2 stimulation. Treatment with a PPAR-γ antagonist attenuated the inhibitory effects on vascular calcification observed in smAT2-Tg mice fed an adenine and high-phosphate diet. Our results suggest that AT2 activation represents an endogenous protective pathway against vascular calcification. Its stimulation may efficiently reduce adverse cardiovascular events in patients with chronic kidney disease.

Brain cytoplasmic RNA 1 suppresses smooth muscle differentiation and vascular development in mice

The cardiovascular system develops during the early stages of embryogenesis, and differentiation of smooth muscle cells (SMCs) is essential for that process. SMC differentiation is critically regulated by transforming growth factor (TGF)-β/SMAD family member 3 (SMAD3) signaling, but other regulators may also play a role. For example, long noncoding RNAs (lncRNAs) regulate various cellular activities and events, such as proliferation, differentiation, and apoptosis. However, whether long noncoding RNAs also regulate SMC differentiation remains largely unknown. Here, using the murine cell line C3H10T1/2, we found that brain cytoplasmic RNA 1 (BC1) is an important regulator of SMC differentiation. BC1 overexpression suppressed, whereas BC1 knockdown promoted, TGF-β-induced SMC differentiation, as indicated by altered cell morphology and expression of multiple SMC markers, including smooth muscle α-actin (αSMA), calponin, and smooth muscle 22α (SM22α). BC1 appeared to block SMAD3 activity and inhibit SMC marker gene transcription. Mechanistically, BC1 bound to SMAD3 via RNA SMAD-binding elements (rSBEs) and thus impeded TGF-β-induced SMAD3 translocation to the nucleus. This prevented SMAD3 from binding to SBEs in SMC marker gene promoters, an essential event in SMC marker transcription. In vivo, BC1 overexpression in mouse embryos impaired vascular SMC differentiation, leading to structural defects in the artery wall, such as random breaks in the elastic lamina, abnormal collagen deposition on SM fibers, and disorganized extracellular matrix proteins in the media of the neonatal aorta. Our results suggest that BC1 is a suppressor of SMC differentiation during vascular development.

Unspliced XBP1 Confers VSMC Homeostasis and Prevents Aortic Aneurysm Formation via FoxO4 Interaction.

Although not fully understood, the phenotypic transition of vascular smooth muscle cells exhibits at the early onset of the pathology of aortic aneurysms. Exploring the key regulators that are responsible for maintaining the contractile phenotype of vascular smooth muscle cells (VSMCs) may confer vascular homeostasis and prevent aneurysmal disease. XBP1 (X-box binding protein 1), which exists in a transcriptionally inactive unspliced form (XBP1u) and a spliced active form (XBP1s), is a key component in response to endoplasmic reticular stress. Compared with XBP1s, little is known about the role of XBP1u in vascular homeostasis and disease. We aim to investigate the role of XBP1u in VSMC phenotypic switching and the pathogenesis of aortic aneurysms. XBP1u, but not XBP1s, was markedly repressed in the aorta during the early onset of aortic aneurysm in both angiotensin II-infused apolipoprotein E knockout (ApoE-/-) and CaPO4 (calcium phosphate)-induced C57BL/6J murine models, in parallel with a decrease in smooth muscle cell contractile apparatus proteins. In vivo studies revealed that XBP1 deficiency in smooth muscle cells caused VSMC dedifferentiation, enhanced vascular inflammation and proteolytic activity, and significantly aggravated both thoracic and abdominal aortic aneurysms in mice. XBP1 deficiency, but not an inhibition of XBP1 splicing, induced VSMC switching from the contractile phenotype to a proinflammatory and proteolytic phenotype. Mechanically, in the cytoplasm, XBP1u directly associated with the N terminus of FoxO4 (Forkhead box protein O 4), a recognized repressor of VSMC differentiation via the interaction and inhibition of myocardin. Blocking the XBP1u-FoxO4 interaction facilitated nuclear translocation of FoxO4, repressed smooth muscle cell marker genes expression, promoted proinflammatory and proteolytic phenotypic transitioning in vitro, and stimulated aortic aneurysm formation in vivo. Our study revealed the pivotal role of the XBP1u-FoxO4-myocardin axis in maintaining the VSMC contractile phenotype and providing protection from aortic aneurysm formation.

The long non-coding RNA GAS5 regulates transforming growth factor β (TGF-β)–induced smooth muscle cell differentiation via RNA Smad–binding elements

Smooth muscle cell (SMC) differentiation is essential for vascular development, and TGF-β signaling plays a critical role in this process. Although long non-coding RNAs (lncRNAs) regulate various cellular events, their functions in SMC differentiation remain largely unknown. Here, we demonstrate that the lncRNA growth arrest-specific 5 (GAS5) suppresses TGF-β/Smad3 signaling in smooth muscle cell differentiation of mesenchymal progenitor cells. We found that forced expression of GAS5 blocked, but knockdown of GAS5 increased, the expression of SMC contractile proteins. Mechanistically, GAS5 competitively bound Smad3 protein via multiple RNA Smad-binding elements (rSBEs), which prevented Smad3 from binding to SBE DNA in TGF-β-responsive SMC gene promoters, resulting in suppression of SMC marker gene transcription and, consequently, in inhibition of TGF-β/Smad3-mediated SMC differentiation. Importantly, other lncRNAs or artificially synthesized RNA molecules that contained rSBEs also effectively inhibited TGF-β/Smad3 signaling, suggesting that lncRNA-rSBE may be a general mechanism used by cells to fine-tune Smad3 activity in both basal and TGF-β-stimulated states. Taken together, our results have uncovered an lncRNA-based mechanism that modulates TGF-β/Smad3 signaling during SMC differentiation.

Uremia does not affect neointima formation in mice

Atherosclerotic cardiovascular disease is a major complication of chronic kidney disease (CKD). CKD leads to uremia, which modulates the phenotype of aortic smooth muscle cells (SMCs). Phenotypic modulation of SMCs plays a key role in accelerating atherosclerosis. We investigated the hypothesis that uremia potentiates neointima formation in response to vascular injury in mice. Carotid wire injury was performed on C57BL/6 wt and apolipoprotein E knockout (Apoe−/−) mice two weeks after induction of uremia by 5/6 nephrectomy. Wire injury led to neointima formation and downregulation of genes encoding classical SMC markers (i.e., myocardin, α-smooth muscle actin, SM22-alpha, and smooth muscle myosin heavy chain) in both wt and Apoe−/− mice. Contrary to our expectations, uremia did not potentiate neointima formation, nor did it affect intimal lesion composition as judged from magnetic resonance imaging and histological analyses. Also, there was no effect of uremia on SMC marker gene expression in the injured carotid arteries, suggesting that there may be different effects of uremia on SMCs in different vascular beds. In conclusion, uremia does not accelerate neointima formation in response to wire injury of the carotid artery in mice.

The geometrical shape of mesenchymal stromal cells measured by quantitative shape descriptors is determined by the stiffness of the biomaterial and by cyclic tensile forces.

Controlling mesenchymal stromal cell (MSC) shape is a novel method for investigating and directing MSC behaviour in vitro. it was hypothesized that specifigc MSC shapes can be generated by using stiffness-defined biomaterial surfaces and by applying cyclic tensile forces. Biomaterials used were thin and thick silicone sheets, fibronectin coating, and compacted collagen type I sheets. The MSC morphology was quantified by shape descriptors describing dimensions and membrane protrusions. Nanoscale stiffness was measured by atomic force microscopy and the expression of smooth muscle cell (SMC) marker genes (ACTA2, TAGLN, CNN1) by quantitative reverse-transcription polymerase chain reaction. Cyclic stretch was applied with 2.5% or 5% amplitudes. Attachment to biomaterials with a higher stiffness yielded more elongated MSCs with fewer membrane protrusions compared with biomaterials with a lower stiffness. For cyclic stretch, compacted collagen sheets were selected, which were associated with the most elongated MSC shape across all investigated biomaterials. As expected, cyclic stretch elongated MSCs during stretch. One hour after cessation of stretch, however, MSC shape was rounder again, suggesting loss of stretch-induced shape. Different shape descriptor values obtained by different stretch regimes correlated significantly with the expression levels of SMC marker genes. Values of approximately 0.4 for roundness and 3.4 for aspect ratio were critical for the highest expression levels of ACTA2 and CNN1. Thus, specific shape descriptor values, which can be generated using biomaterial-associated stiffness and tensile forces, can serve as a template for the induction of specific gene expression levels in MSC. Copyright © 2017 John Wiley & Sons, Ltd.

Inhibition of Smooth Muscle β-Catenin Hinders Neointima Formation After Vascular Injury.

Smooth muscle cells (SMCs) contribute to neointima formation after vascular injury. Although β-catenin expression is induced after injury, whether its function is essential in SMCs for neointimal growth is unknown. Moreover, although inhibitors of β-catenin have been developed, their effects on SMC growth have not been tested. We assessed the requirement for SMC β-catenin in short-term vascular homeostasis and in response to arterial injury and investigated the effects of β-catenin inhibitors on vascular SMC growth. We used an inducible, conditional genetic deletion of β-catenin in SMCs of adult mice. Uninjured arteries from adult mice lacking SMC β-catenin were indistinguishable from controls in terms of structure and SMC marker gene expression. After carotid artery ligation, however, vessels from mice lacking SMC β-catenin developed smaller neointimas, with lower neointimal cell proliferation and increased apoptosis. SMCs lacking β-catenin showed decreased mRNA expression of Mmp2, Mmp9, Sphk1, and S1pr1 (genes that promote neointima formation), higher levels of Jag1 and Gja1 (genes that inhibit neointima formation), decreased Mmp2 protein expression and secretion, and reduced cell invasion in vitro. Moreover, β-catenin inhibitors PKF118-310 and ICG-001 limited growth of mouse and human vascular SMCs in a dose-dependent manner. SMC β-catenin is dispensable for maintenance of the structure and state of differentiation of uninjured adult arteries, but is required for neointima formation after vascular injury. Pharmacological β-catenin inhibitors hinder growth of human vascular SMCs. Thus, inhibiting β-catenin has potential as a therapy to limit SMC accumulation and vascular obstruction.

Olfactomedin 2 Regulates Smooth Muscle Phenotypic Modulation and Vascular Remodeling Through Mediating Runt-Related Transcription Factor 2 Binding to Serum Response Factor.

The objective of this study is to investigate the role and underlying mechanism of Olfactomedin 2 (Olfm2) in smooth muscle cell (SMC) phenotypic modulation and vascular remodeling. Platelet-derived growth factor-BB induces Olfm2 expression in primary SMCs while modulating SMC phenotype as shown by the downregulation of SMC marker proteins. Knockdown of Olfm2 blocks platelet-derived growth factor-BB-induced SMC phenotypic modulation, proliferation, and migration. Conversely, Olfm2 overexpression inhibits SMC marker expression. Mechanistically, Olfm2 promotes the interaction of serum response factor with the runt-related transcription factor 2 that is induced by platelet-derived growth factor-BB, leading to a decreased interaction between serum response factor and myocardin, causing a repression of SMC marker gene transcription and consequently SMC phenotypic modulation. Animal studies show that Olfm2 is upregulated in balloon-injured rat carotid arteries. Knockdown of Olfm2 effectively inhibits balloon injury-induced neointima formation. Importantly, knockout of Olfm2 in mice profoundly suppresses wire injury-induced neointimal hyperplasia while restoring SMC contractile protein expression, suggesting that Olfm2 plays a critical role in SMC phenotypic modulation in vivo. Olfm2 is a novel factor mediating SMC phenotypic modulation. Thus, Olfm2 may be a potential target for treating injury-induced proliferative vascular diseases.

Normal endothelial but impaired arterial development in MAP-Kinase activated protein kinase 2 (MK2) deficient mice.

Angiogenesis is a fundamental process during development and disease, and many details of the underlying molecular and cellular mechanisms are incompletely understood. Mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MK2), a major downstream target of p38 MAPK, has recently been identified as a regulator of Interleukin 1β dependent angiogenesis in vivo, and in vitro data suggest a role of MK2 for VEGF-dependent angiogenic processes in endothelial cells. We thus hypothesized that MK2 plays a role during physiological vascular development in vivo.Vascular development was investigated in the retina of MK2-deficient mice. Retinal angiogenesis such as sprouting, branching and pruning was unchanged in MK2-/- mice compared to wildtype littermates. Early arterial development was also comparable between genotypes. However, with further expansion of vascular smooth muscle cells (SMC) during maturation of the arterial network at later time points, the number of arterial branch points was significantly lower in MK2-/- mice, resulting in a reduced total arterial area in adult mice. Isolated aortic smooth muscle cells from MK2-/- mice showed a more dedifferentiated phenotype in vitro and downregulation of central SMC marker genes, consistent with the known impaired migration of MK2-/- SMC.In conclusion, MK2 is not required for physiological retinal angiogenesis. However, its loss is associated with an altered genetic profile of SMC and an impaired arterial network in adult mice, indicating a distinct and probably cell-specific role of MK2 in arteries.Electronic supplementary materialThe online version of this article (doi:10.1186/s13221-016-0038-2) contains supplementary material, which is available to authorized users.

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.

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.

Abstract 60: Histone Modification H3k4me2 Controls Vascular Smooth Muscle Cell Lineage Memory during Vascular Injury

Smooth muscle cells (SMC) have the ability to undergo reversible phenotypic switch (i.e. dedifferentiation/redifferentiation) characterized by loss and re-expression of SMC marker genes, a process occurring during blood vessel repair and adaptive remodeling. Using SMC lineage-tracing mice, we showed that newly generated SMC derive from pre-existing SMC that transiently de-differentiate, participate in repair, and then redifferentiate following wire-induced femoral injury. However, a critical unresolved question is how dedifferentiated SMC retain their lineage identity. We previously demonstrated that SMC acquire a unique epigenetic signature during differentiation, H3K4me2 on the SMC marker genes, retained in SMC during PDGF-BB-induced phenotypic switch. Our hypothesis is that H3K4me2 on SMC genes biases SMC re-differentiation and functions as a lineage memory mechanism during vascular injury-repair. We demonstrated that H3K4me2 on SMC genes is retained in dedifferentiated and redifferentiated SMC in vivo following vascular injury using our unique ISH-PLA epigenetic assay. We also found that H3K4me2 is present and retained on the SMC regulatory gene Myocardin during PDGF-BB treatment, reinforcing significance of H3K4me2 as a regulator of SMC identity. To directly assess the functional role of H3K4me2 on the SMC repertoire, we generated a fusion protein consisting of the H3K4 demethylase LSD1 coupled to the SRF-CArG binding domain of Myocardin (Myocd-LSD1WT) to perform SMC-specific locus-selective removal of H3K4me2 on the SMC marker genes. We also generated loss-of-function mutant of Myocd-LSD1 to ensure that effects are due to H3K4me2 removal and not steric hindrance. Stable expression of Myocd-LSD1WT in SMC induced locus selective removal of H3K4me2 on SMC CArG genes without impacting non-SMC CArG-dependent genes like c-fos. Myocd-LSD1WT also inhibits expression of the SMC marker genes and abolishes the ability of SMC to redifferentiate in PDGF-BB induced phenotypic switch. None of these effects were observed in Myocd-LSD1Mut SMC. Results are the first to our knowledge to provide direct functional evidence that locus specific epigenetic modifications regulate cell lineage memory during reversible phenotypic transitions.

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.

From nerve to blood vessel: a new role of Olfm2 in smooth muscle differentiation from human embryonic stem cell-derived mesenchymal cells.

From nerve to blood vessel: a new role of Olfm2 in smooth muscle differentiation from human embryonic stem cell-derived mesenchymal cells.

DNA Methylation of a GC Repressor Element in the Smooth Muscle Myosin Heavy Chain Promoter Facilitates Binding of the Notch-Associated Transcription Factor, RBPJ/CSL1

The goal of the present study was to identify novel mechanisms that regulate smooth muscle cell (SMC) differentiation marker gene expression. We demonstrate that the CArG-containing regions of many SMC-specific promoters are imbedded within CpG islands. A previously identified GC repressor element in the SM myosin heavy chain (MHC) promoter was highly methylated in cultured aortic SMC but not in the aorta, and this difference was inversely correlated with SM MHC expression. Using an affinity chromatography/mass spectroscopy-based approach, we identified the multifunctional Notch transcription factor, recombination signal binding protein for immunoglobulin κ J region (RBPJ), as a methylated GC repressor-binding protein. RBPJ protein levels and binding to the endogenous SM MHC GC repressor were enhanced by platelet-derived growth factor-BB treatment. A methylation mimetic mutation to the GC repressor that facilitated RBPJ binding inhibited SM MHC promoter activity as did overexpression of RBPJ. Consistent with this, knockdown of RBPJ in phenotypically modulated human aortic SMC enhanced endogenous SMC marker gene expression, an effect likely mediated by increased recruitment of serum response factor and Pol II to the SMC-specific promoters. In contrast, the depletion of RBPJ in differentiated transforming growth factor-β-treated SMC inhibited SMC-specific gene activation, supporting the idea that the effects of RBPJ/Notch signaling are context dependent. Our results indicate that methylation-dependent binding of RBPJ to a GC repressor element can negatively regulate SM MHC promoter activity and that RBPJ can inhibit SMC marker gene expression in phenotypically modulated SMC. These results will have important implications on the regulation of SMC phenotype and on Notch-dependent transcription.

Abstract 664: The Coronary Heart Disease--Associated Transcription Factor TCF21 Regulates Disease-Related Genes and May Contribute to the Migration of SMC Progenitors to the Fibrous Cap

Introduction: Recent genome-wide association studies have identified a number of genes that contribute to the risk for coronary heart disease, including TCF21, a bHLH transcription factor believed to serve a critical role in the differentiation of epicardial progenitor cells that give rise to coronary smooth muscle. Methods and Results: Results of immunolocalization and in situ hybridization studies showed that TCF21 is not expressed in medial smooth muscle cells (SMC) of adult human coronary arteries but primarily within the adventitia and within cells of unknown origin in atherosclerotic lesions. LacZ reporter animal studies with ApoE null background showed a shift in Tcf21 expression from adventitial and medial cells to the neointima, notably into the area below and within the forming fibrous cap, suggesting that Tcf21 expressing cells migrate into the forming atherosclerotic lesion. To investigate disease-related functions of TCF21 in SMCs, knockdown and overexpression studies were performed in human coronary artery smooth muscle cells, revealing an effect on migration, proliferation and SMC marker gene expression. Genome-wide RNA-seq studies in these cells with siRNA treatment identified putative TCF21 downstream genes and pathways involved in vascular development as well as atherosclerosis. Supporting ChIP-seq data suggests that these - as over half of all differentially expressed genes - are direct transcriptional targets. To elucidate the behavior of Tcf21 expressing cells in the development of disease lesions, we are employing an in vivo Tcf21 lineage tracing model. Preliminary data from these studies suggests that Tcf21 expressing cells give rise to fibrous cap SMCs, and ongoing studies are aimed at the further characterization of molecular pathways that are active in these Tcf21 expressing cells. Conclusions: The gene regulatory and expression profile of Tcf21 during disease progression strongly implies a key role in Coronary Heart Disease. Tcf21 is likely to play a role in the migration of SMC progenitors from the adventitia into the forming atherosclerotic plaque. As a source of differentiated SMCs of the fibrous cap Tcf21 may therefore promote plaque stability and protect from lesion rupture.