c-Myb regulates transcriptional activation of miR-143/145 in vascular smooth muscle cells.

BACKGROUND: MicroRNAs (miR) are small non-coding RNAs that regulate diverse biological functions. The bicistronic gene miR-143/145 determines cell fate and phenotype of vascular smooth muscle cells (VSMC), in part, by destabilizing Elk-1 mRNA. The transcription factor c-Myb also regulates differentiation and proliferation of VSMC, and here we test whether these effects may be mediated by miR-143/145. METHODS & RESULTS: Flow cytometry of cardiovascular-directed d3.75 embryoid bodies (EBs) isolated smooth muscle progenitors with specific cell surface markers. In c-myb knockout (c-myb -/-) EB, these progenitors manifest low levels of miR-143 (19%; p<0.05) and miR-145 (6%; p<0.01) expression as compared to wild-type (wt) EB. Primary VSMC isolated from transgenic mice with diminished expression (c-myblx/lx) or reduced activity (c-mybh/h) of c-Myb also manifest low levels of miR-143 (c-myblx/lx: 50%; c-mybh/h: 41%), and miR-145 (c-myblx/lx: 49%; c-mybh/h: 56%), as compared to wt (P<0.05). Sequence alignment identified four putative c-Myb binding sites (MBS1-4) in the proximal promoter (PP) of the miR-143/145 gene. PP-reporter constructs revealed that point mutations in MBS1 and MBS4 abrogated c-Myb-dependent transcription from the miR-143/145 PP (P<0.01). Chromatin immunoprecipitation (ChIP) revealed preferential c-Myb binding at MBS4 (p<0.001). By conjugating Elk-1 3'-untranslated region (UTR) to a reporter and co-transducing wt VSMC with this plus a miR-143-antagomir, and co-transducing c-myblx/lx VSMC with this plus a miR-143-mimic, we demonstrate that c-Myb's ability to repress Elk-1 is mediated by miR-143. CONCLUSION: c-Myb regulates VSMC gene expression by transcriptional activation of miR-143/145.

c-Myb Regulates Proliferation and Differentiation of Adventitial Sca1+ Vascular Smooth Muscle Cell Progenitors by Transactivation of Myocardin.

OBJECTIVE: Vascular smooth muscle cells (VSMCs) are believed to dedifferentiate and proliferate in response to vessel injury. Recently, adventitial progenitor cells were implicated as a source of VSMCs involved in vessel remodeling. c-Myb is a transcription factor known to regulate VSMC proliferation in vivo and differentiation of VSMCs from mouse embryonic stem cell-derived progenitors in vitro. However, the role of c-Myb in regulating specific adult vascular progenitor cell populations was not known. Our objective was to examine the role of c-Myb in the proliferation and differentiation of Sca1(+) adventitial VSMC progenitor cells. APPROACH AND RESULTS: Using mice with wild-type or hypomorphic c-myb (c-myb(h/h)), BrdU (bromodeoxyuridine) uptake and flow cytometry revealed defective proliferation of Sca1(+) adventitial VSMC progenitor cells at 8, 14, and 28 days post carotid artery denudation injury in c-myb(h/h) arteries. c-myb(h/h) cKit(+)CD34(-)Flk1(-)Sca1(+)CD45(-)Lin(-) cells failed to proliferate, suggesting that c-myb regulates the activation of specific Sca1(+) progenitor cells in vivo and in vitro. Although expression levels of transforming growth factor-ß1 did not vary between wild-type and c-myb(h/h) carotid arteries, in vitro differentiation of c-myb(h/h) Sca1(+) cells manifested defective transforming growth factor-ß1-induced VSMC differentiation. This is mediated by reduced transcriptional activation of myocardin because chromatin immunoprecipitation revealed c-Myb binding to the myocardin promoter only during differentiation of Sca1(+) cells, myocardin promoter mutagenesis identified 2 specific c-Myb-responsive binding sites, and adenovirus-mediated expression of myocardin rescued the phenotype of c-myb(h/h) progenitors. CONCLUSIONS: These data support a role for c-Myb in the regulation of VSMC progenitor cells and provide novel insight into how c-myb regulates VSMC differentiation through myocardin.

Glucagon-Like Peptide 1 Receptor Activation Attenuates Platelet Aggregation and Thrombosis.

Short-term studies in subjects with diabetes receiving glucagon-like peptide 1 (GLP-1)-targeted therapies have suggested a reduced number of cardiovascular events. The mechanisms underlying this unexpectedly rapid effect are not known. We cloned full-length GLP-1 receptor (GLP-1R) mRNA from a human megakaryocyte cell line (MEG-01), and found expression levels of GLP-1Rs in MEG-01 cells to be higher than those in the human lung but lower than in the human pancreas. Incubation with GLP-1 and the GLP-1R agonist exenatide elicited a cAMP response in MEG-01 cells, and exenatide significantly inhibited thrombin-, ADP-, and collagen-induced platelet aggregation. Incubation with exenatide also inhibited thrombus formation under flow conditions in ex vivo perfusion chambers using human and mouse whole blood. In a mouse cremaster artery laser injury model, a single intravenous injection of exenatide inhibited thrombus formation in normoglycemic and hyperglycemic mice in vivo. Thrombus formation was greater in mice transplanted with bone marrow lacking a functional GLP-1R (Glp1r(-/-)), compared with those receiving wild-type bone marrow. Although antithrombotic effects of exenatide were partly lost in mice transplanted with bone marrow from Glp1r(-/-) mice, they were undetectable in mice with a genetic deficiency of endothelial nitric oxide synthase. The inhibition of platelet function and the prevention of thrombus formation by GLP-1R agonists represent potential mechanisms for reduced atherothrombotic events.

Cardioprotective Signature of Short-Term Caloric Restriction.

OBJECTIVE: To understand the molecular pathways underlying the cardiac preconditioning effect of short-term caloric restriction (CR). BACKGROUND: Lifelong CR has been suggested to reduce the incidence of cardiovascular disease through a variety of mechanisms. However, prolonged adherence to a CR life-style is difficult. Here we reveal the pathways that are modulated by short-term CR, which are associated with protection of the mouse heart from ischemia. METHODS: Male 10-12 wk old C57bl/6 mice were randomly assigned to an ad libitum (AL) diet with free access to regular chow, or CR, receiving 30% less food for 7 days (d), prior to myocardial infarction (MI) via permanent coronary ligation. At d8, the left ventricles (LV) of AL and CR mice were collected for Western blot, mRNA and microRNA (miR) analyses to identify cardioprotective gene expression signatures. In separate groups, infarct size, cardiac hemodynamics and protein abundance of caspase 3 was measured at d2 post-MI. RESULTS: This short-term model of CR was associated with cardio-protection, as evidenced by decreased infarct size (18.5±2.4% vs. 26.6±1.7%, N=10/group; P=0.01). mRNA and miR profiles pre-MI (N=5/group) identified genes modulated by short-term CR to be associated with circadian clock, oxidative stress, immune function, apoptosis, metabolism, angiogenesis, cytoskeleton and extracellular matrix (ECM). Western blots pre-MI revealed CR-associated increases in phosphorylated Akt and GSK3ß, reduced levels of phosphorylated AMPK and mitochondrial related proteins PGC-1α, cytochrome C and cyclooxygenase (COX) IV, with no differences in the levels of phosphorylated eNOS or MAPK (ERK1/2; p38). CR regimen was also associated with reduced protein abundance of cleaved caspase 3 in the infarcted heart and improved cardiac function.

Serum-free differentiation of functional human coronary-like vascular smooth muscle cells from embryonic stem cells.

AIMS: Despite the diverse developmental origins of vascular smooth muscle cells (VSMCs), recent attempts to generate VSMCs from human embryonic stem cells (hESCs) differentiated along various lineages did not yield distinct cell phenotypes. The aim of this study was to derive and characterize functional coronary-like VSMCs from hESCs using serum-free cardiac-directed differentiation. METHODS AND RESULTS: Embryoid bodies (EBs) from three pluripotent stem cell lines subjected to cardiac-directed differentiation in defined media were characterized over 30 days for VSMC-specific gene expression by qRT-PCR, immunofluorescence microscopy and fluorescence-activated cell sorting (FACS). EBs composed of cardiomyocytes, endothelial cells (ECs), fibroblasts, and VSMCs underwent FACS on d28 to reveal that the VSMCs form a distinct subpopulation, which migrate with ECs in an in vitro angiogenesis assay. To enrich for VSMCs, d28 EBs were dissociated and cultured as monolayers. Over several passages, mRNA and protein levels of cardiomyocyte, endothelial, and fibroblast markers were abolished, whereas those of mature VSMCs were unchanged. Vascular endothelial growth factor and basic fibroblast growth factor were critical for the separation of the cardiac and VSMC lineages in EBs, and for the enrichment of functional VSMCs in monolayer cultures. Calcium cycling and cell shortening responses to vasoconstrictors in hESC-derived VSMCs in vitro were indistinguishable from primary human coronary artery SMCs, and distinct from bladder and aorta SMCs. VSMCs identically derived from green fluorescent protein -expressing hESCs integrated in and contributed to new vessel formation in vivo. CONCLUSION: The ability to generate hESC-derived functional human coronary-like VSMCs in serum-free conditions has implications for disease modelling, drug screening, and regenerative therapies.

Regulated expression and role of c-Myb in the cardiovascular-directed differentiation of mouse embryonic stem cells.

RATIONALE: c-myb null (knockout) embryonic stem cells (ESC) can differentiate into cardiomyocytes but not contractile smooth muscle cells (SMC) in embryoid bodies (EB). OBJECTIVE: To define the role of c-Myb in SMC differentiation from ESC. METHODS AND RESULTS: In wild-type (WT) EB, high c-Myb levels on days 0-2 of differentiation undergo ubiquitin-mediated proteosomal degradation on days 2.5-3, resurging on days 4-6, without changing c-myb mRNA levels. Activin-A and bone morphogenetic protein 4-induced cardiovascular progenitors were isolated by FACS for expression of vascular endothelial growth factor receptor (VEGFR)2 and platelet-derived growth factor receptor (PDGFR)α. By day 3.75, hematopoesis-capable VEGFR2+ cells were fewer, whereas cardiomyocyte-directed VEGFR2+/PDGFRα+ cells did not differ in abundance in knockout versus WT EB. Importantly, highest and lowest levels of c-Myb were observed in VEGFR2+ and VEGFR2+/PDGFRα+ cells, respectively. Proteosome inhibitor MG132 and lentiviruses enabling inducible expression or knockdown of c-myb were used to regulate c-Myb in WT and knockout EB. These experiments showed that c-Myb promotes expression of VEGFR2 over PDGFRα, with chromatin immunopreciptation and promoter-reporter assays defining specific c-Myb-responsive binding sites in the VEGFR2 promoter. Next, FACS-sorted VEGFR2+ cells expressed highest and lowest levels of SMC- and fibroblast-specific markers, respectively, at days 7-14 after retinoic acid (RA) as compared with VEGFR2+/PDGFRα+ cells. By contrast, VEGFR2+/PDGFRα+ cells cultured without RA beat spontaneously, like cardiomyocytes between days 7 and 14, and expressed cardiac troponin. Notably, RA was required to more fully differentiate SMC from VEGFR2+ cells and completely blocked differentiation of cardiomyocytes from VEGFR2+/PDGFRα+ cells. CONCLUSIONS: c-Myb is tightly regulated by proteosomal degradation during cardiovascular-directed differentiation of ESC, expanding early-stage VEGFR2+ progenitors capable of RA-responsive SMC formation.

Directed differentiation of skin-derived precursors into functional vascular smooth muscle cells.

OBJECTIVE: The goal of this study was to characterize the factors and conditions required for smooth muscle cell (SMC)-directed differentiation of Sox2(+) multipotent rat and human skin-derived precursors (SKPs) and to define whether they represent a source of fully functional vascular SMCs for applications in vivo. METHODS AND RESULTS: We found that rat SKPs can differentiate almost exclusively into SMCs by reducing serum concentrations to 0.5% to 2% and plating them at low density. Human SKPs derived from foreskin required the addition of transforming growth factor-ß1 or -ß3 to differentiate into SMCs, but they did so even in the absence of serum. SMC formation was confirmed by quantitative reverse transcription-polymerase chain reaction, immunocytochemistry, and fluorescence-activated cell sorting, with increased expression of smoothelin-B and little to no expression of telokin or smooth muscle γ-actin, together indicating that SKPs differentiated into vascular rather than visceral SMCs. Rat and human SKP-derived SMCs were able to contract in vitro and also wrap around and support new capillary and larger blood vessel formation in angiogenesis assays in vivo. CONCLUSIONS: SKPs are Sox2(+) progenitors that represent an attainable autologous source of stem cells that can be easily differentiated into functional vascular SMCs in defined serum-free conditions without reprogramming. SKPs represent a clinically viable cell source for potential therapeutic applications in neovascularization.

The SWI/SNF chromatin remodeling complex regulates myocardin-induced smooth muscle-specific gene expression.

OBJECTIVE: Regulatory complexes comprising myocardin and serum response factor (SRF) are critical for the transcriptional regulation of many smooth muscle-specific genes. However, little is known about the epigenetic mechanisms that regulate the activity of these complexes. In the current study, we investigated the role of SWI/SNF ATP-dependent chromatin remodeling enzymes in regulating the myogenic activity of myocardin. METHODS AND RESULTS: We found that both Brg1 and Brm are required for maintaining expression of several smooth muscle-specific genes in primary cultures of aortic smooth muscle cells. Furthermore, the ability of myocardin to induce expression of smooth muscle-specific genes is abrogated in cells expressing dominant negative Brg1. In SW13 cells, which lack endogenous Brg1 and Brm1, myocardin is unable to induce expression of smooth muscle-specific genes. Whereas, reconstitution of wild-type, or bromodomain mutant forms Brg1 or Brm1, into SW13 cells restored their responsiveness to myocardin. SWI/SNF complexes were found to be required for myocardin to increase SRF binding to the promoters of smooth muscle-specific genes. Brg1 and Brm directly bind to the N terminus of myocardin, in vitro, through their ATPase domains and Brg1 forms a complex with SRF and myocardin in vivo in smooth muscle cells. CONCLUSIONS: These data demonstrate that the ability of myocardin to induce smooth muscle-specific gene expression is dependent on its interaction with SWI/SNF ATP-dependent chromatin remodeling complexes.

Regulation of myosin light chain kinase and telokin expression in smooth muscle tissues.

The mylk1 gene is a large gene spanning approximately 250 kb and comprising at least 31 exons. The mylk1 gene encodes at least four protein products: two isoforms of the 220-kDa myosin light chain kinase (MLCK), a 130-kDa MLCK, and telokin. Transcripts encoding these products are derived from four independent promoters within the mylk1 gene. The kinases expressed from the mylk1 gene have been extensively characterized and function to regulate the activity of nonmuscle and smooth muscle myosin II. Activation of these myosin motors by MLCK modulates a variety of contractile processes, including smooth muscle contraction, cell adhesion, migration, and proliferation. Dysregulation of these processes contributes to a number of diseases. The noncatalytic gene product telokin also has been shown to modulate contraction in smooth muscle cells through its ability to inhibit myosin light chain phosphatase. Given the crucial role of the products of the mylk1 gene in regulating numerous contractile processes, it seems intuitive that alterations in the transcriptional activity of the mylk1 gene also will have a significant impact on many physiological and pathological processes. In this review we highlight some of the recent studies that have described the transcriptional regulation of mylk1 gene products in smooth muscle tissues and discuss the implications of these findings for regulation of expression of other smooth muscle-specific genes.

Regulation of smooth muscle-specific gene expression by homeodomain proteins, Hoxa10 and Hoxb8.

Smooth muscle cells arise from different populations of precursor cells during embryonic development. The mechanisms that specify the smooth muscle cell phenotype in each of these populations of cells are largely unknown. In many tissues and organs, homeodomain transcription factors play a key role in directing cell specification. However, little is known about how these proteins regulate smooth muscle differentiation. Using degenerate reverse transcription-PCR coupled to cDNA library screening we identified two homeodomain proteins, Hoxa10 and Hoxb8, which are expressed in adult mouse smooth muscle tissues. All three of the previously described transcripts of the Hoxa10 gene, Hoxa10-1, Hoxa10-2, and Hoxa10-3, were identified. Hoxa10-1 directly activated the smooth muscle-specific telokin promoter but did not activate the SM22alpha, smooth muscle alpha-actin, or smooth muscle myosin heavy chain promoters. Small interfering RNA-mediated knock-down of Hoxa10-1 demonstrated that Hoxa10-1 is required for high levels of telokin expression in smooth muscle cells from uterus and colon. On the other hand, Hoxb8 inhibited the activity of the telokin, SM22alpha, and smooth muscle alpha-actin promoters. Cotransfection of Hoxa10-1 together with Hoxa10-2 or Hoxb8 suggested that Hoxa10-2 and Hoxb8 act as competitive inhibitors of Hoxa10-1. Results from gel mobility shift assays demonstrated that Hoxa10-1, Hoxa10-2, and Hoxb8 bind directly to multiple sites in the telokin promoter. Mutational analysis of telokin promoter reporter genes demonstrated that the three homeodomain protein binding sites located between -80 and -75, +2 and +6, and +14 and +17 were required for maximal promoter activation by Hoxa10-1 and maximal inhibition by Hoxb8. Together these data demonstrate that the genes encoding smooth muscle-restricted proteins are direct transcriptional targets of clustered homeodomain proteins and that different homeodomain proteins have distinct effects on the promoters of these genes.