Smooth Muscle Cell Differentiation Marker Research Articles

ANGIOTENSIN II PROMOTES DIFFERENTIATION OF MOUSE EMBRYONIC STEM CELLS TO VASCULAR SMOOTH MUSCLE CELLS THROUGH PI3-KINASE SIGNALLING PATHWAY AND NF-κB

ObjectivesEmbryonic stem cells (ES cells), the pluripotent derivatives of inner cell mass of blastocysts, have the capacity for unlimited growth, self-renewal and differentiate into all types of mature tissue cells....

Krüppel-like Factor 4 Contributes to High Phosphate-induced Phenotypic Switching of Vascular Smooth Muscle Cells into Osteogenic Cells

Hyperphosphatemia in chronic kidney disease is highly associated with vascular calcification. Previous studies have shown that high phosphate-induced phenotypic switching of vascular smooth muscle cells (SMCs) into osteogenic cells plays an important role in the calcification process. In the present study, we determined whether Krüppel-like factor 4 (Klf4) and phosphorylated Elk-1, transcriptional repressors of SMC differentiation marker genes activated by intimal atherogenic stimuli, contributed to this process. Rat aortic SMCs were cultured in the medium with normal (0.9 mmol/liter) or high (4.5 mmol/liter) phosphate concentration. Results showed that high phosphate concentration induced SMC calcification. Moreover, high phosphate decreased expression of SMC differentiation marker genes including smooth muscle α-actin and SM22α, whereas it increased expression of osteogenic genes, such as Runx2 and osteopontin. High phosphate also induced Klf4 expression, although it did not phosphorylate Elk-1. In response to high phosphate, Klf4 selectively bound to the promoter regions of SMC differentiation marker genes. Of importance, siRNA-mediated knockdown of Klf4 blunted high phosphate-induced suppression of SMC differentiation marker genes, as well as increases in expression of osteogenic genes and calcium deposition. Klf4 was also induced markedly in the calcified aorta of adenine-induced uremic rats. Results provide novel evidence that Klf4 mediates high phosphate-induced conversion of SMCs into osteogenic cells.

Effect of Human Muscle–derived Stem Cells on Cryoinjured Mouse Bladder Contractility

To investigate the effect of human muscle-derived stem cells (hMDSCs) on ameliorating impaired detrusor contractility in a cryoinjured bladder murine model. The hMDSCs were isolated and cultured by modified preplate technique, and only CD34-positive hMDSCs were extracted by Mini-MACS kits. Isolated hMDSCs were prelabeled with PKH26 and injected into the cryoinjured bladder to observe the pattern and characteristics. The nude mice were subdivided into three groups: normal group (N), cryoinjury bladder group with saline injection (C), and hMDSCs injection group after cryoinjury (M). At 2 weeks after injecting hMDSCs, we compared the contractility of bladder muscle strip stimulated by electrical field stimulation (EFS), acetylcholine (Ach.), and adenosine triphosphate (ATP), and the bladder smooth muscle tissue was examined by immunohistochemistry. The contractile powers of bladder muscle strip in the C group were more decreased than the N group after EFS, Ach, and ATP treatment (P < .05). The bladder contractility of the M group was more increased than in the C group (P < .05), but was lower than the N group after EFS and Ach treatment. However, there was no significant difference of contractile power between the C and M groups after ATP stimulation. In immunohistochemical staining, the thickness of the bladder smooth muscle layer in the M group was significantly increased compared with the C group, and PKH26-labeled implanted cells were positive for smooth muscle cell differentiation marker (α-SMA) in the injected region. hMDSCs injection increased cholinergic bladder contractile power but not the purinergic component of bladder contraction after cryoinjury.

MicroRNA‐21 represses human cystathionine gamma‐lyase expression by targeting at specificity protein‐1 in smooth muscle cells

Cystathionine gamma-lyase (CSE) is the major H(2)S-generating enzyme in vascular smooth muscle cells (SMCs). CSE/H(2)S system contributes to the maintenance of SMC phenotype, and transcript factor specificity protein-1 (SP1) is a critical regulator of CSE expression during SMC differentiation. The involvements of microRNA-21 (miR-21) in cardiovascular pathophysiology have been known, however miR-21 regulation of CSE and SP1 as well as SMC phenotype are uncertain. Using quantitative real-time PCR, we demonstrated that the expression of miR-21 was upregulated in dedifferentiated human aorta SMCs (HASMCs) and injured mouse carotid arteries. To determine the potential roles of miR-21 in SP1-mediated CSE gene expression and SMC phenotypic change, we showed that miR-21 expression was upregulated by miR-21 precursor. Interestingly, miR-21 overexpression significantly repressed the protein expressions of both CSE and SP1, inhibited H(2)S production, stimulated SMC proliferation, and reduced SMC differentiation marker gene expression, respectively. The mRNA expression of CSE but not SP1 was inhibited by miR-21 precursor. Blockage of SP1 binding by mithramycin or inhibition of CSE activity by DL-propargylglycine did not change miR-21 expression. We further demonstrated that miR-21 repressed SP1 protein expression by directly targeting at SP1 3' untranslational regions, which in turn downregulated CSE mRNA expression and stimulated SMC proliferation. Take together, these results suggest that miR-21 participates in CSE/H(2)S-mediated-SMC differentiation by targeting SP1.

Abstract 176: Extracellular S100A4 Is a Key Modulator of Arterial Smooth Muscle Cell Phenotypic Transition

Background: It has been proposed that smooth muscle cells (SMCs) from the arterial wall are heterogeneous and that only a subset of medial SMCs are prone to accumulate into the intima leading to atheromatous plaque formation. We isolated 2 distinct SMC phenotypes from porcine coronary artery: spindle-shaped (S) and rhomboid (R). Biological features of R-SMCs (i.e. enhanced proliferative and migratory activities as well as poor level of differentiation) explain their capacity to accumulate into the intima. We identified S100A4 as a marker of R-SMCs in vitro and of intimal SMCs both in pig and human. S100A4 is an intracellular Ca2+ binding protein that can also be secreted; it has extracellular functions probably via the receptor for advanced glycation end products (RAGE). Objective: Investigate the role of S100A4 in SMC phenotypic change, a process characteristic of atherosclerotic plaque formation. Methods and results: Transfection of a human S100A4-containing plasmid in S-SMCs (practically devoid of S100A4) led to overexpression of S100A4 in approximately 10% of SMCs, release of S100A4, and a transition towards a R-phenotype of the whole SMC population. Moreover, treatment of S-SMCs with S100A4-rich conditioned medium collected from S100A4-transfected S-SMCs induced a transition towards a R-phenotype, which was associated with increased proliferative and migratory activities as well as downregulation of SMC differentiation markers (i.e. alpha-smooth muscle actin and smooth muscle myosin heavy chains). It yielded activation of NF-kappa B without changing RAGE mRNA baseline level. Blockade of extracellular S100A4 in R-SMCs with S100A4 neutralizing antibody induced a transition from R- to S-phenotype, decreased proliferative activity and upregulated SMC differentiation markers. In contrast, silencing of S100A4 mRNA in R-SMCs and during platelet-derived growth factor-BB-induced S-to-R-phenotypic change did not affect SMC morphology in spite of decreased proliferative activity. Conclusions: Our results show that extracellular S100A4 modulates SMC phenotypic changes and is essential for the establishment of the R-phenotype. It could be a new target to prevent SMC accumulation during atherosclerosis and restenosis.

Abstract 83: Molecular Mechanism of Aortic Aneurysm Formation in Fibulin-4--Deficient Mice

Background: Fibulin-4 (Fbln4) is a secreted extracellular matrix protein, which is expressed in blood vessels and associated with microfibrils surrounding elastic fibers. Fbln4-null mice exhibit perinatal lethality with rupture of aortic aneurysm and a marked disruption of elastic fibers, and these phenotypes were recapitulated by smooth muscle cell (SMC)-specific deletion of Fbln4 in vivo (Fbln4SMKO). Severe aneurysms developed exclusively in the ascending aorta at postnatal day 90 (P90) and vascular SMCs were hyper-proliferative and failed to fully differentiate in the Fbln4SMKO aorta. Objective: To elucidate the molecular mechanism that initiates the aneurysmal change in Fbln4SMKO mice, we performed proteomics analysis of Fbln4-deficient (Fbln4-/-) SMCs and compared with wild-type SMCs. Methods: The early stage of ascending aneurysm was evaluated in Fbln4SMKO mice using microscopic methods and immunohistochemistry. The protein expression profiles were obtained from postnatal day (P) 7 wild-type and Fbln4 -/- SMCs by two-dimensional fluorescence difference gel electrophoresis. Several differentially expressed proteins were identified by mass spectrometry and validated using qPCR and Western blot analysis. Results: Macroscopic observation revealed that the ascending aorta dilatation was not observed at P7 but became detectable at P14. Histologically, however, focal lesion of SMC proliferation was already noticeable in the sub-endothelial region at P7 and became evident at P14 with a thickened aortic media. Differential proteomic analysis showed that molecules involved in actin dynamics, including gelsolin, ezrin/radixin/moesin (ERM) protein, cofilin-2, and Rho GDI were upregulated in Fbln4 -/- SMCs. In addition, phosphorylation of cofilin and ERM was increased in Fbln4 -/- SMCs. Interestingly, SMC differentiation markers were normally expressed in Fbln4SMKO aortae until P7. Conclusion: Our study indicated that the phenotypic change of SMCs occurred after P7 in the Fbln4SMKO aorta and Fbln4 deficiency contributed to aneurysm formation by facilitating the alteration in actin dynamics. This information will shed light on the molecular mechanism of aneurysm initiation in the Fbln4 deficient SMCs.

Abstract 550: Nuclear Targeting of Apelin in Vascular Smooth Muscle Cells: Implications in Cell Proliferation and Atherosclerotic Plaque Formation?

Background: The main circulatory form of Apelin is a highly conserved 13 amino acid hypotensive peptide derived from a 77 amino acid prepropeptide. The very low plasma levels suggest autocrine, paracrine and possibly intracrine functions of Apelin. It is synthesized in smooth muscle cells (SMCs), and we found a nuclear expression in rhomboid type (R), poorly differentiated SMCs. The biological features of R-SMCs (i.e. enhanced proliferative and migratory activities) explain their capacity to accumulate in the intima. Objectives: Investigate the role of intracrine Apelin in SMC phenotypic change, a process characteristic of atherosclerotic plaque formation. Methods and Results: As shown in our previous work, a 3’-shift in translation can lead to N-truncated peptides with new address sequences (FASEB J. 20(6): 732-4, 2006). P-SORT software analysis of preproApelin sequence suggests that N-terminal truncated Apelin may target the nucleus, and we confirm this in many cell types by overexpression of mutated preproApelin-EGFP and preproApelin-His-tag. Transfection of mutated preproApelin-His-tag encoding plasmid in differentiated spindle-shaped (S) SMCs induces a transition towards a R-phenotype associated with increased proliferative activity, as well as downregulation of SMC differentiation markers (i.e. α-smooth muscle actin) and increased nuclear expression of S100A4 (a marker typical of R-SMCs). In contrast, transfection of S-SMCs with wild type preproApelin-His-tag encoding plasmid does not induce nuclear targeting of Apelin or S100A4, and does not change the S-phenotype. Stimulation of S-SMCs with PDGF-BB also induces nuclear targeting of both Apelin and S100A4 and transition to the R-phenotype. In vivo, Apelin is expressed in SMC nuclei of stent-induced intimal thickening, while its expression in SMCs of the media is mainly cytoplasmic. Conclusions: Altogether, theses preliminary studies provide evidence for nuclear targeting of Apelin in SMCs and suggest actions on gene expression, proliferation, and differentiation. The pathophysiological consequences of this retargeting could be of importance in the understanding of artherosclerosis.

The Role ofNotch 1Activation in Cardiosphere Derived Cell Differentiation

Cardiosphere derived cells (CDC) are present in the human heart and include heterogeneous cell populations of cardiac progenitor cells, multipotent progenitors that play critical roles in the physiological and pathological turnover of heart tissue. Little is known about the molecular pathways that control the differentiation of CDC. In this study, we examined the role of Notch 1/J kappa-recombining binding protein (RBPJ) signaling, a critical cell-fate decision pathway, in CDC differentiation. We isolated CDC from mouse cardiospheres and analyzed the differentiation of transduced cells expressing the Notch1 intracellular domain (N1-ICD), the active form of Notch1, using a terminal differentiation marker polymerase chain reaction (PCR) array. We found that Notch1 primarily supported the differentiation of CDC into smooth muscle cells (SMC), as demonstrated by the upreguation of key SMC proteins, including smooth muscle myosin heavy chain (Myh11) and SM22α (Tagln), in N1-ICD expressing CDC. Conversely, genetic ablation of RBPJ in CDC diminished the expression of SMC differentiation markers, confirming that SMC differentiation CDC is dependent on RBPJ. Finally, in vivo experiments demonstrate enhanced numbers of smooth muscle actin-expressing implanted cells after an injection of N1-ICD-expressing CDC into ischemic myocardium (44±8/high power field (hpf) vs. 11±4/high power field (hpf), n=7 sections, P<0.05). Taken together, these results provide strong evidence that Notch1 promotes SMC differentiation of CDC through an RBPJ-dependent signaling pathway in vitro, which may have important implications for progenitor cell-mediated angiogenesis.

Interleukin-1β modulates smooth muscle cell phenotype to a distinct inflammatory state relative to PDGF-DD via NF-κB-dependent mechanisms.

Smooth muscle cell (SMC) phenotypic modulation in atherosclerosis and in response to PDGF in vitro involves repression of differentiation marker genes and increases in SMC proliferation, migration, and matrix synthesis. However, SMCs within atherosclerotic plaques can also express a number of proinflammatory genes, and in cultured SMCs the inflammatory cytokine IL-1β represses SMC marker gene expression and induces inflammatory gene expression. Studies herein tested the hypothesis that IL-1β modulates SMC phenotype to a distinct inflammatory state relative to PDGF-DD. Genome-wide gene expression analysis of IL-1β- or PDGF-DD-treated SMCs revealed that although both stimuli repressed SMC differentiation marker gene expression, IL-1β distinctly induced expression of proinflammatory genes, while PDGF-DD primarily induced genes involved in cell proliferation. Promoters of inflammatory genes distinctly induced by IL-1β exhibited over-representation of NF-κB binding sites, and NF-κB inhibition in SMCs reduced IL-1β-induced upregulation of proinflammatory genes as well as repression of SMC differentiation marker genes. Interestingly, PDGF-DD-induced SMC marker gene repression was not NF-κB dependent. Finally, immunofluorescent staining of mouse atherosclerotic lesions revealed the presence of cells positive for the marker of an IL-1β-stimulated inflammatory SMC, chemokine (C-C motif) ligand 20 (CCL20), but not the PDGF-DD-induced gene, regulator of G protein signaling 17 (RGS17). Results demonstrate that IL-1β- but not PDGF-DD-induced phenotypic modulation of SMC is characterized by NF-κB-dependent activation of proinflammatory genes, suggesting the existence of a distinct inflammatory SMC phenotype. In addition, studies provide evidence for the possible utility of CCL20 and RGS17 as markers of inflammatory and proliferative state SMCs within atherosclerotic plaques in vivo.

Nrf3-Pla2g7 Interaction Plays an Essential Role in Smooth Muscle Differentiation From Stem Cells

Phospholipase A2, group 7 (Pla2g7) is an important mediator in cardiovascular development and diseases because of its divergent physiological and pathological functions in inflammation and oxidative stress. However, little is known about the functional role of Pla2g7 in smooth muscle cell (SMC) differentiation from stem cells. In the present study, embryonic stem cells were cultivated on collagen IV-coated plates to allow SMC differentiation. Pla2g7 gene expression and activity were upregulated significantly following 4 to 14 days of cell differentiation and colocalized with SMC differentiation markers in the differentiated SMCs. Knockdown of Pla2g7 resulted in downregulation of smooth muscle-specific markers in vitro and impairment of SMC differentiation in vivo, whereas enforced expression of Pla2g7 enhanced SMC differentiation and increased reactive oxygen species generation. Importantly, enforced expression of Pla2g7 significantly increased the binding of serum response factor to SMC differentiation gene promoters, resulting in SMC differentiation, which was abolished by free radical scavenger and flavoprotein inhibitor of NADPH oxidase but not hydrogen peroxide inhibitor. Moreover, we demonstrated that nuclear factor erythroid 2-related factor 3 (Nrf3) regulates Pla2g7 gene expression through direct binding to the promoter regions of Pla2g7 gene. Our findings demonstrated that Pla2g7 plays a crucial physiological role in SMC differentiation from stem cells, and the fine interactions between Nrf3 and Pla2g7 are essential for SMC differentiation.

Downregulation of transforming growth factor, beta receptor 2 and Notch signaling pathway in human abdominal aortic aneurysm

ObjectiveMutations in FBN1 and TGFBR2 genes are the main causative mutations identified in Marfan syndrome (MFS). The major vascular complication of MFS is aneurysm formation. Abdominal aortic aneurysm (AAA) is an acquired disease of later life of unknown etiology. The aim of this study was to examine if genetic aberrations in MFS-related genes FBN1 and TGFBR2 are present in patients with AAA. MethodsWe assessed the presence of copy number variation (CNV) in FBN1 and TGFBR2 genes in AAA biopsies from twelve patients. We also analyzed the expression of these genes in AAA biopsies compared to control biopsies from six organ donors. In addition we assessed the expression of two members of the Notch signaling pathway NOTCH3 and HEY2 as well as aortic smooth muscle cell (AoSMC) differentiation marker TAGLN in AAA and control biopsies. ResultsLoss of one copy (deletion) of the FBN1 exon 66 sequence and TGFBR2 exon 8 was identified in 7 (58%) and 11 (92%) of the 12 AAA biopsies. No copy number amplifications (duplications) were detected. Patients carrying TGFBR2 exon 8 deletion showed marked downregulation of this gene in AAA biopsies compared to control biopsies (0.699 vs. 1.765, p=0.038). Notch signaling components NOTCH3 and HEY2 were markedly downregulated in AAA, while expression of the AoSMC differentiation marker TAGLN did not differ between AAA and control biopsies (0.468 vs. 0.486, p=0.546). ConclusionThis study suggests an acquired impairment in TGF-β signaling that along with downregulation of the Notch signaling pathway may contribute to the pathogenesis of AAA.

Vascular differentiation of multipotent spermatogonial stem cells derived from neonatal mouse testis

We previously reported the successful establishment of embryonic stem cell (ESC)-like multipotent spermatogonial stem cells (mSSCs) from neonatal mouse testis. Here, we examined the ability of mSSCs to differentiate into vascular endothelial cells and smooth muscle cells, and compared to that of mouse ESCs. We used real-time reverse transcriptase polymerase chain reaction and immunohistochemistry to examine gene expression profiles of mSSCs and ESCs during in vitro vascular differentiation. Both mSSCs and ESCs exhibited substantial increase in the expression of mesodermal markers, such as Brachyury, Flk1, Mesp1, Nkx2.5, and Islet1, and a decrease in the expression of pluripotency markers, such as Oct3/4 and Nanog during the early stage of differentiation. The mRNA levels of vascular endothelial (VE)-cadherin and CD31 gradually increased in both differentiated mSSCs and ESCs. VE-cadherin- or CD31-positive cells formed sprouting branch-like structures, as observed during embryonic vascular development. At the same time, vascular smooth muscle cell-specific markers, such as myocardin and α-smooth muscle actin (SMA), were also highly expressed in differentiated mSSCs and ESCs. Immunocytochemical analysis revealed that the differentiated cells expressed both α-SMA and SM22-α proteins, and exhibited the intracellular fibril structure typical of smooth muscle cells. Overall, our findings showed that mSSCs have similar vascular differentiation abilities to those of ESCs, suggesting that mSSCs may be an alternative source of autologous pluripotent stem cells for vascular regeneration.

Transforming Growth Factor-β Signaling in Myogenic Cells Regulates Vascular Morphogenesis, Differentiation, and Matrix Synthesis

Transforming growth factor-β (TGF-β) signaling is required for normal vascular development. We aimed to discover the role of TGF-β signaling in embryonic smooth muscle cells (SMCs). We bred mice with smooth muscle (SM) 22α-Cre and Tgfbr2(flox) alleles to generate embryos in which the type II TGF-β receptor (TGFBR2; required for TGF-β signaling) was deleted in SMCs. Embryos were harvested between embryonic day (E) 9.5 and E18.5 and examined grossly, microscopically, and by histochemical and RNA analyses. SM22α-Cre(+/0) Tgfbr2(flox/flox) (knockout [KO]) embryos died before E15.5 with defects that included cardiac outflow tract abnormalities, persistence of the right dorsal aorta, and dilation of the distal aorta. Histological analyses suggested normal expression of SMC differentiation markers in KO aortas; however, RNA analyses showed that SMC differentiation markers were increased in KO cardiac outflow vessels but decreased in the descending aorta. KO aortas had only rare mature elastin deposits and contained abnormal aggregates of extracellular matrix proteins. Expression of several matrix proteins was significantly decreased in KO descending aortas but not in cardiac outflow vessels. TGF-β signaling in SMCs controls differentiation, matrix synthesis, and vascular morphogenesis. Effects of TGF-β on SMC gene expression appear to differ depending on the location of SMCs in the aorta.

51 PLA2g7 mediates smooth muscle cell differentiation from stem cells

RationalePhospholipase A2, group 7 (PLA2g7), the plasma isoform of a platelet-activating factor acetylhydrolases, also known as lipoprotein associated phospholipase A2, is a circulating enzyme that catalyse hydrolysis of the sn-2...

Nuclear Factor of Activated T Cells 5 Regulates Vascular Smooth Muscle Cell Phenotypic Modulation

The tonicity-responsive transcription factor, nuclear factor of activated T cells 5 (NFAT5/tonicity enhancer binding protein [TonEBP]), has been well characterized in numerous cell types; however, NFAT5 function in vascular smooth muscle cells (SMCs) is unknown. Our main objective was to determine the role of NFAT5 regulation in SMCs. We showed that NFAT5 is regulated by hypertonicity in SMCs and is upregulated in atherosclerosis and neointimal hyperplasia. RNAi knockdown of NFAT5 inhibited basal expression of several SMC differentiation marker genes, including smooth muscle α actin (SMαA). Bioinformatic analysis of SMαA revealed 7 putative NFAT5 binding sites in the first intron, and chromatin immunoprecipitation analysis showed NFAT5 enrichment of intronic DNA. Overexpression of NFAT5 increased SMαA promoter-intron activity, which requires an NFAT5 cis element at +1012, whereas dominant-negative NFAT5 decreased SMαA promoter-intron activity. Because it is unlikely that SMCs experience extreme changes in tonicity, we investigated other stimuli and uncovered 2 novel NFAT5-inducing factors: angiotensin II, a contractile agonist, and platelet-derived growth factor-BB (PDGF-BB), a potent mitogen in vascular injury. Angiotensin II stimulated NFAT5 translocation and activity, and NFAT5 knockdown inhibited an angiotensin II-mediated upregulation of SMαA mRNA. PDGF-BB increased NFAT5 protein, and loss of NFAT5 inhibited PDGF-BB-induced SMC migration. We have identified NFAT5 as a novel regulator of SMC phenotypic modulation and have uncovered the role of NFAT5 in angiotensin II-induced SMαA expression and PDGF-BB-stimulated SMC migration.

Specificity Protein-1 as a Critical Regulator of Human Cystathionine γ-Lyase in Smooth Muscle Cells

Cystathionine γ-lyase (CSE) is the major enzyme in vascular smooth muscle cells (SMCs) that catalyzes the endogenous production of H(2)S. Phenotypic switching of SMCs is affected by endogenous H(2)S level and alterations of this switching may result in vascular disorders. To date, the mechanisms underlying the alteration of CSE expression and H(2)S production in vascular proliferative diseases have been unclear. In the present study, we found that serum deprivation induced SMC differentiation marker gene expressions and increased CSE expression and H(2)S production in cultured human aorta SMCs (HASMCs). Carotid artery ligation in mice resulted in enhanced neointima formation and down-regulation of CSE expression, suggesting an important role of CSE in SMC differentiation. Transient transfection of HASMCs with human CSE (hCSE) promoter/luciferase reporter revealed that the region between -226 to +140 base pair contains the core promoter for the hCSE gene. Deletion and mutation analysis demonstrated that two specificity protein-1 (Sp1) consensus binding sites were present in the core promoter region of the hCSE gene. Incubation of HASMCs with Sp1 binding inhibitor mithramycin inhibited CSE mRNA expression in a dose-dependent manner. Overexpression of Sp1 alone was sufficient to increase the activity of the hCSE core promoter and CSE protein expression. Chromatin immunoprecipitation assay showed that the binding of Sp1 to the hCSE promoter was increased in differentiated HASMCs compared with that in proliferated HASMCs. Exogenously applied H(2)S at 100 μM stimulated SMC differentiation, which was reversed by p38 MAPK inhibitor SB203580. These results suggest that transcript factor Sp1 is a critical regulator of the hCSE expression during SMC differentiation, and CSE/H(2)S system is essential for maintenance of SMC phenotype.

Cilostazol Promotes Vascular Smooth Muscles Cell Differentiation Through the cAMP Response Element-Binding Protein-Dependent Pathway

Cilostazol, a potent type 3 phosphodiesterase inhibitor, has recently been found to reduce neointimal formation by inhibiting vascular smooth muscle cell (VSMC) proliferation. The aim of this study is to investigate whether cilostazol exerts an action on phenotypic modulation of VSMCs, another important process in the pathogenesis of neointimal formation. Cilostazol may convert VSMCs from a serum-induced dedifferentiation state to a differentiated state, as indicated by a spindle-shaped morphology and an increase in the expression of smooth muscle cell differentiation marker contractile proteins. The upregulation of contractile proteins by cilostazol involves the cAMP/protein kinase A (PKA) signaling pathway, because the cAMP analog mimicked and specific cAMP/PKA inhibitors opposed the effect of cilostazol. Furthermore, cilostazol-activated cAMP response element (CRE)-binding protein (CREB), including phosphorylation at Ser133 and its nuclear translocation. Deletion and mutational analysis of the contractile protein promoters along with chromatin immunoprecipitation using anti-CREB antibody showed that CRE is essential for cilostazol-induced contractile protein expression. Transfection of dominant-negative CREB (mutated Ser133) plasmid in VSMCs blocked cilostazol-stimulated contractile protein expression. In vivo, cilostazol upregulated contractile proteins and induced the activation of CREB in the neointima of balloon-injured arteries. Cilostazol promotes VSMC differentiation through the cAMP/PKA/CREB signaling cascade.

Chromatin Remodeling Pathways in Smooth Muscle Cell Differentiation, and Evidence for an Integral Role for p300

BackgroundPhenotypic alteration of vascular smooth muscle cells (SMC) in response to injury or inflammation is an essential component of vascular disease. Evidence suggests that this process is dependent on epigenetic regulatory processes. P300, a histone acetyltransferase (HAT), activates crucial muscle-specific promoters in terminal (non-SMC) myocyte differentiation, and may be essential to SMC modulation as well.ResultsWe performed a subanalysis examining transcriptional time-course microarray data obtained using the A404 model of SMC differentiation. Numerous chromatin remodeling genes (up to 62% of such genes on our array platform) showed significant regulation during differentiation. Members of several chromatin-remodeling families demonstrated involvement, including factors instrumental in histone modification, chromatin assembly-disassembly and DNA silencing, suggesting complex, multi-level systemic epigenetic regulation. Further, trichostatin A, a histone deacetylase inhibitor, accelerated expression of SMC differentiation markers in this model. Ontology analysis indicated a high degree of p300 involvement in SMC differentiation, with 60.7% of the known p300 interactome showing significant expression changes. Knockdown of p300 expression accelerated SMC differentiation in A404 cells and human SMCs, while inhibition of p300 HAT activity blunted SMC differentiation. The results suggest a central but complex role for p300 in SMC phenotypic modulation.ConclusionsOur results support the hypothesis that chromatin remodeling is important for SMC phenotypic switching, and detail wide-ranging involvement of several epigenetic modification families. Additionally, the transcriptional coactivator p300 may be partially degraded during SMC differentiation, leaving an activated subpopulation with increased HAT activity and SMC differentiation-gene specificity.

Formin Homology Domain–Containing Protein 1 Regulates Smooth Muscle Cell Phenotype

Our goal was to test whether formin homology protein 1 (FHOD1) plays a significant role in the regulation of smooth muscle cell (SMC) differentiation and, if so, whether Rho kinase (ROCK)-dependent phosphorylation in the diaphanous autoinhibitory domain is an important signaling mechanism that controls FHOD1 activity in SMC. FHOD1 is highly expressed in aortic SMCs and in tissues with a significant SMC component. Exogenous expression of constitutively active FHOD1, but not wild-type, strongly activated SMC-specific gene expression in 10T1/2 cells. Treatment of SMC with the RhoA activator sphingosine-1-phosphate increased FHOD1 phosphorylation at Thr1141, and this effect was completely prevented by inhibition of ROCK with Y-27632. Phosphomimetic mutations to ROCK target residues enhanced FHOD1 activity, suggesting that phosphorylation interferes with FHOD1 autoinhibition. Importantly, knockdown of FHOD1 in SMC strongly inhibited sphingosine-1-phosphate-dependent increases in SMC differentiation marker gene expression and actin polymerization, suggesting that FHOD1 plays a major role in RhoA-dependent signaling in SMC. Our results indicate that FHOD1 is a critical regulator of SMC phenotype and is regulated by ROCK-dependent phosphorylation. Thus, additional studies on the role of FHOD1 during development and the progression of cardiovascular disease will be important.

Sphingosine 1-Phosphate Receptor 2 Signals Through Leukemia-Associated RhoGEF (LARG), to Promote Smooth Muscle Cell Differentiation

The goals of this study were to identify the signaling pathway by which sphingosine 1-phosphate (S1P) activates RhoA in smooth muscle cells (SMC) and to evaluate the contribution of this pathway to the regulation of SMC phenotype. Using a combination of receptor-specific agonists and antagonists we identified S1P receptor 2 (S1PR2) as the major S1P receptor subtype that regulates SMC differentiation marker gene expression. Based on the known coupling properties of S1PR2 and our demonstration that overexpression of Galpha(12) or Galpha(13) increased SMC-specific promoter activity, we next tested whether the effects of S1P in SMC were mediated by the regulator of G protein-signaling-Rho guanine exchange factors (RGS-RhoGEFs) (leukemia-associated RhoGEF [LARG], PDZ-RhoGEF [PRG], RhoGEF [p115]). Although each of the RGS-RhoGEFs enhanced actin polymerization, myocardin-related transcription factor-A nuclear localization, and SMC-specific promoter activity when overexpressed in 10T1/2 cells, LARG exhibited the most robust effect and was the only RGS-RhoGEF activated by S1P in SMC. Importantly, siRNA-mediated depletion of LARG significantly inhibited the activation of RhoA and SMC differentiation marker gene expression by S1P. Knockdown of LARG had no effect on SMC proliferation but promoted SMC migration as measured by scratch wound and transwell assays. These data indicate that S1PR2-dependent activation of RhoA in SMC is mediated by LARG and that this signaling mechanism promotes the differentiated SMC phenotype.