Smooth Muscle Myosin Heavy Chain Expression Research Articles

Investigation of electrical stimulation on phenotypic vascular smooth muscle cells differentiation in tissue-engineered small-diameter vascular graft

In the development of vascular tissue engineering, particularly in the case of small diameter vessels, one of the key obstacles is the blockage of these veins once they enter the in vivo environment. One of the contributing factors to this problem is the aberrant proliferation and migration of vascular smooth muscle cells (VSMCs) from the media layer of the artery to the interior of the channel. Two distinct phenotypes have been identified for smooth muscle cells, namely synthetic and contractile. Since the synthetic phenotype plays an essential role in the unusual growth and migration, the aim of this study was to convert the synthetic phenotype into the contractile one, which is a solution to prevent the abnormal growth of VSMCs. To achieve this goal, these cells were subjected to electrical signals, using a 1000 μA sinusoidal stimulation at 10 Hz for four days, with 20 min duration per 24 h. The morphological transformations and changes in the expression of vimentin, nestin, and β-actin proteins were then studied using ICC and flow cytometry assays. Also, the expression of VSMC specific markers such as smooth muscle myosin heavy chain (SMMHC) and smooth muscle alpha-actin (α-SMA) were evaluated using RT-PCR test. In the final phase of this study, the sheep decellularized vessel was employed as a scaffold for seeding these cells. Based on the results, electrical stimulation resulted in some morphological alterations in VSMCs. Furthermore, the observed reductions in the expression levels of vimentin, nestin and β-actin proteins and increase in the expression of SMMHC and α-SMA markers showed that it is possible to convert the synthetic phenotype to the contractile one using the studied regime of electrical stimulation. Finally, it can be concluded that electrical stimulation can significantly affect the phenotype of VSMCs, as demonstrated in this study.

Neonatal Streptococcus Pneumoniae pneumonia induces airway SMMHC expression through HMGB1/TLR4/ERK

BackgroundOur previous study showed that neonatal S. pneumoniae pneumonia promoted airway smooth muscle myosin heavy chain (SMMHC) expression and AHR development. Researches demonstrated HMGB1, TLR4 and ERK are involved in smooth muscle contractile protein expression, so we hypothesis that HMGB1/TLR4/ERK pathway participated in airway SMMHC overexpression in neonatal S. pneumoniae pneumonia model. MethodNeonatal (1-week-old) BALB/c mice were intranasal inoculated with D39 to establish non-lethal S. pneumoniae pneumonia model. TLR4 was inhibited 2 weeks after infection with TLR4 specific inhibitor (TAK-242). Five weeks after infection, the bronchoalveolar lavage fluid (BALF) and lungs of neonatal S. pneumoniae pneumonia and mock infection mice with or without TLR4 inhibition were collected to assess the expressions of HMGB1, TLR4 and p-ERK1/2. Airway Hyperresponsiveness (AHR) of the three groups was determined by whole-body plethysmograph. ResultsOur results demonstrated that neonatal S. pneumoniae pneumonia promoted HMGB1/TLR4 production, SMMHC expression and AHR development significantly, with ERK1/2 phosphorylation decreased remarkably. TLR4 inhibition after pneumonia significantly increased ERK1/2 phosphorylation, reversed airway SMMHC overexpression and alleviated AHR. ConclusionNeonatal S. pneumoniae pneumonia promotes airway SMMHC expression and AHR through HMGB1/TLR4/ERK.

Decreased vascular smooth muscle contractility in Hutchinson\u2013Gilford Progeria Syndrome linked to defective smooth muscle myosin heavy chain expression

Children with Hutchinson–Gilford Progeria Syndrome (HGPS) suffer from multiple cardiovascular pathologies due to the expression of progerin, a mutant form of the nuclear envelope protein Lamin A. Progerin expression has a dramatic effect on arterial smooth muscle cells (SMCs) and results in decreased viability and increased arterial stiffness. However, very little is known about how progerin affects SMC contractility. Here, we studied the LaminAG609G/G609G mouse model of HGPS and found reduced arterial contractility at an early age that correlates with a decrease in smooth muscle myosin heavy chain (SM-MHC) mRNA and protein expression. Traction force microscopy on isolated SMCs from these mice revealed reduced force generation compared to wild-type controls; this effect was phenocopied by depletion of SM-MHC in WT SMCs and overcome by ectopic expression of SM-MHC in HGPS SMCs. Arterial SM-MHC levels are also reduced with age in wild-type mice and humans, suggesting a common defect in arterial contractility in HGPS and normal aging.

TSP-1 (Thrombospondin-1) Deficiency Protects ApoE-/- Mice Against Leptin-Induced Atherosclerosis.

Hyperleptinemia, hallmark of obesity, is a putative pathophysiologic trigger for atherosclerosis. We previously reported a stimulatory effect of leptin on TSP-1 (thrombospondin-1) expression, a proatherogenic matricellular protein implicated in atherogenesis. However, a causal role of TSP-1 in leptin-driven atherosclerosis remains unknown. Approach and Results: Seventeen-weeks-old ApoE-/- and TSP-1-/-/ApoE-/- double knockout mice, on normocholesterolemic diet, were treated with or without murine recombinant leptin (5 µg/g bwt, IP) once daily for 3 weeks. Using aortic root morphometry and en face lesion assay, we found that TSP-1 deletion abrogated leptin-stimulated lipid-filled lesion burden, plaque area, and collagen accumulation in aortic roots of ApoE-/- mice, shown via Oil red O, hematoxylin and eosin, and Masson trichrome staining, respectively. Immunofluorescence microscopy of aortic roots showed that TSP-1 deficiency blocked leptin-induced inflammatory and smooth muscle cell abundance as well as cellular proliferation in ApoE-/- mice. Moreover, these effects were concomitant to changes in VLDL (very low-density lipoprotein)-triglyceride and HDL (high-density lipoprotein)-cholesterol levels. Immunoblotting further revealed reduced vimentin and pCREB (phospho-cyclic AMP response element-binding protein) accompanied with augmented smooth muscle-myosin heavy chain expression in aortic vessels of leptin-treated double knockout versus leptin-treated ApoE-/-; also confirmed in aortic smooth muscle cells from the mice genotypes, incubated ± leptin in vitro. Finally, TSP-1 deletion impeded plaque burden in leptin-treated ApoE-/- on western diet, independent of plasma lipid alterations. The present study provides evidence for a protective effect of TSP-1 deletion on leptin-stimulated atherogenesis. Our findings suggest a regulatory role of TSP-1 on leptin-induced vascular smooth muscle cell phenotypic transition and inflammatory lesion invasion. Collectively, these results underscore TSP-1 as a potential target of leptin-induced vasculopathy.

Phenotype transitions induced by mechanical stimuli in airway smooth muscle are regulated by differential interactions of parvin isoforms with paxillin and Akt.

Mechanical tension and humoral stimuli can induce transitions in airway smooth muscle phenotype between a synthetic inflammatory state that promotes cytokine secretion and a differentiated state that promotes the expression of smooth muscle phenotype-specific proteins. When tissues are maintained under high tension, Akt activation and eotaxin secretion are suppressed, but expression of the differentiation marker protein, smooth muscle myosin heavy chain (SmMHC), is promoted. When tissues are maintained under low tension, Akt activation and eotaxin secretion are stimulated, and the differentiated phenotype is suppressed. We hypothesized that mechanical stimuli are differentially transduced to Akt-mediated signaling pathways that regulate phenotype expression by α-parvin and β-parvin integrin-linked kinase/PINCH/parvin (IPP) signaling complexes within integrin adhesomes. High tension or ACh triggered paxillin phosphorylation and the binding of phospho-paxillin to β-parvin IPP complexes. This inhibited Akt activation and promoted SmMHC expression. Low tension or IL-4 did not elicit paxillin phosphorylation and triggered the binding of unphosphorylated paxillin to α-parvin IPP complexes, which promoted Akt activation and eotaxin secretion and suppressed SmMHC expression. Expression of a nonphosphorylatable paxillin mutant or β-parvin depletion by siRNA promoted the inflammatory phenotype, whereas the depletion of α-parvin promoted the differentiated phenotype. Results demonstrate that phenotype expression is regulated by the differential interaction of phosphorylated and unphosphorylated paxillin with α-parvin and β-parvin IPP complexes and that these complexes have opposite effects on the activation of Akt. Our results describe a novel molecular mechanism for transduction of mechanical and humoral stimuli within integrin signaling complexes to regulate phenotype expression in airway smooth muscle.

A novel CD147 inhibitor, SP-8356, reduces neointimal hyperplasia and arterial stiffness in a rat model of partial carotid artery ligation

BackgroundNeointimal hyperplasia and its related arterial stiffness are the crucial pathophysiological features in atherosclerosis and in-stent restenosis. Cluster of differentiation 147 (CD147), a member of the immunoglobulin super family that induces the expression of matrix metalloproteinase-9 (MMP-9) by dimerization, may play important roles in neointimal hyperplasia and may therefore be an effective target for the treatment of this condition. Here, we investigated whether a novel CD147 inhibitor SP-8356 ((1S,5R)-4-(3,4-dihydroxy-5-methoxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one) reduces neointimal hyperplasia and arterial stiffness in a rat model of partial carotid artery ligation.MethodsNeointimal hyperplasia was induced in Sprague–Dawley rats by partial ligation of the right carotid artery combined with a high fat diet and vitamin D injection. Rats were subdivided into vehicle, SP-8356 (50 mg/kg), and rosuvastatin (10 mg/kg) groups. The drugs were administrated via intraperitoneal injections for 4 weeks. The elasticity of blood vessels was assessed by measuring pulse wave velocity using Doppler ultrasonography before sacrifice. Histomolecular analysis was carried out on harvested carotid arteries.ResultsSP-8356 significantly reduced MMP activity by inhibiting CD147 dimerization. SP-8356 reduced neointimal hyperplasia and prevented the deterioration of vascular elasticity. SP-8356 had a greater inhibitory effect on neointimal hyperplasia than did rosuvastatin. Furthermore, rosuvastatin did not improve vascular elasticity. SP-8356 increased the expression of smooth muscle myosin heavy chain (SM-MHC), but decreased the expression of collagen type III and MMP-9 in the neointimal region. In contrast to SP-8356, rosuvastatin did not alter the expression of SM-MHC or MMP-9.ConclusionsThe ability of SP-8356 to reduce neointimal hyperplasia and improve arterial stiffness in affected carotid artery suggests that SP-8356 could be a promising therapeutic drug for vascular remodeling disorders involving neointimal hyperplasia and arterial stiffness.

Blebbistatin modulates prostatic cell growth and contrapctility through myosin II signaling.

To investigate the effect of blebbistatin (BLEB, a selective myosin inhibitor) on regulating contractility and growth of prostate cells and to provide insight into possible mechanisms associated with these actions. BLEB was incubated with cell lines of BPH-1 and WPMY-1, and intraprostatically injected into rats. Cell growth was determined by flow cytometry, and in vitro organ bath studies were performed to explore muscle contractility. Smooth muscle (SM) myosin isoform (SM1/2, SM-A/B, and LC17a/b) expression was determined via competitive reverse transcriptase PCR. SM myosin heavy chain (MHC), non-muscle (NM) MHC isoforms (NMMHC-A and NMMHC-B), and proteins related to cell apoptosis were further analyzed via Western blotting. Masson's trichrome staining was applied to tissue sections. BLEB could dose-dependently trigger apoptosis and retard the growth of BPH-1 and WPMY-1. Consistent with in vitro effect, administration of BLEB to the prostate could decrease rat prostatic epithelial and SM cells via increased apoptosis. Western blotting confirmed the effects of BLEB on inducing apoptosis through a mechanism involving MLC20 dephosphorylation with down-regulation of Bcl-2 and up-regulation of BAX and cleaved caspase 3. Meanwhile, NMMHC-A and NMMHC-B, the downstream proteins of MLC20, were found significantly attenuated in BPH-1 and WPMY-1 cells, as well as rat prostate tissues. Additionally, BLEB decreased SM cell number and SM MHC expression, along with attenuated phenylephrine-induced contraction and altered prostate SMM isoform composition with up-regulation of SM-B and down-regulation of LC17a, favoring a faster contraction. Our novel data demonstrate BLEB regulated myosin expression and functional activity. The mechanism involved MLC20 dephosphorylation and altered SMM isoform composition.

Abstract 313: Loss of Contractile Smooth Muscle Cell Phenotype in a Mouse Model of Marfan Syndrome

Objectives: Thoracic aortic aneurysm (TAA) is a chronic vascular disease and the 15th leading cause of death in the US. TAA can result from a genetic disorder such as Marfan syndrome (MFS). MFS patients typically have fibrillin-1 deficiency, causing elastic fiber defects in the aortic media that initiate a series of cellular events. We sought to determine the association of smooth muscle cell (SMC) conversion from contractile to synthetic phenotype with changes in extracellular matrix (ECM), medial cell density, and arterial biomechanics. Methods: A mouse model of MFS (Fbn1 C1039G/+, Het) was used to study progression of TAA and cellular events over a 3-12 month time course. Small animal ultrasound was used to image the ascending aorta and to measure diameters at several positions. Immunohistochemistry (IHC) was performed on proximal aorta sections with smooth muscle-myosin heavy chain (SM-MHC) and SM alpha actin as markers for contractile SMCs. Second harmonic generation (SHG) microscopy was used to quantitate aortic medial collagen. Results: Ultrasound data show that aortic diameter significantly increases with time in Het mice. There is a statistically significant difference between genotypes independent of time. IHC images demonstrate that while SM-MHC expression in wild type (WT) mice remains nearly constant, in Het mice at 3 months the expression is comparable to that of WT mice but decreases at 6 months and starts to recover to the WT level by 12 months. SHG images show a time dependent increase in medial collagen expression in Het mice vs. WT. These results suggest loss of contractile SMCs in the media and remodeling of ECM. Conclusion: Our results to date demonstrate loss of contractile SMCs and increased medial collagen deposition in the proximal aorta during TAA progression. Loss of contractile cells together with changes in ECM are expected to alter aortic biomechanics. To quantify these alterations, we are currently studying active and passive biomechanics of ascending aortas of WT and Het mice.

CD137 induces vascular muscle cells phenotype transformation through activating nuclear factor of activated T-cells 1 signaling

Objective: To investigate whether CD137 induces primary vascular muscle cells (VSMCs) phenotype transformation through activating nuclear factor of activated T-cells 1(NFATc1) signaling. Methods: VSMCs were obtained from aorta of C57BL/6J mice (8 weeks, male) through tissue-piece inoculating. Cells were divided into control group, CD137 agonist group (treated with CD137L recombinant protein) and anti-CD137 group (treated with anti-CD137 antibody). In si-RNA transfection assay, cells were divided into si-control group and si-NFATc1 group which were transfected with control or si-NFATc1 sequence respectively. The levels of NFATc1 and other phenotype related protein such as α-smooth muscle actin (α-SMA), smooth muscle myosin heavy chain (SM-MHC), vimentin were detected by Q-PCR and Western blot. Nuclear protein expression and activity of NFATc1 were detected by immunofluorescence and Western blot. Transwell assay was performed to measure the migration of VSMCs. Results: According to Western blot, the expression of NFATc1 and vimentin was significantly upregulated (5.07±0.36 vs. 1.00±0.00, P<0.05; 3.23±0.27 vs. 1.00±0.00, P<0.05) while α-SMA and SM-MHC expressions was significantly downregulated (0.73±0.15 vs. 1.00±0.00, P<0.05; 0.45±0.05 vs. 1.00±0.00, P<0.05) in CD137 agonist group compare to control group. Compared with CD137 agonist group, the expression of NFATc1 and vimentin was significantly downregulated (1.56±0.27 vs. 5.07±0.36, P<0.05; 1.21±0.17 vs. 3.23±0.27, P<0.05), but the levels of α-SMA and SM-MHC were significantly upregulated (2.01±0.43 vs. 0.73±0.15, P<0.05; 2.85 ±0.32 vs. 0.45±0.05, P<0.05) in anti-CD137 group. Compared with si-con group, the expression of SM-MHC and α-SMA was significantly upregulated while the expression of vimentin was significantly downregulated in si-NFATc1 group. Transwell assay results demonstrated that migration cell numbers was significantly higher in CD137L group compared with control group(3.85±0.31 vs. 1.00±0.00, P<0.05), this effect was significantly attenuated by inhibiting NFATc1. Conclusion: CD137 could induce VSMC phenotype transformation through activating NFATc1 signaling.

Involvement of gap junctions between smooth muscle cells in sustained hypoxic pulmonary vasoconstriction development: a potential role for 15-HETE and 20-HETE.

In response to hypoxia, the pulmonary artery normally constricts to maintain optimal ventilation-perfusion matching in the lung, but chronic hypoxia leads to the development of pulmonary hypertension. The mechanisms of sustained hypoxic pulmonary vasoconstriction (HPV) remain unclear. The aim of this study was to determine the role of gap junctions (GJs) between smooth muscle cells (SMCs) in the sustained HPV development and involvement of arachidonic acid (AA) metabolites in GJ-mediated signaling. Vascular tone was measured in bovine intrapulmonary arteries (BIPAs) using isometric force measurement technique. Expression of contractile proteins was determined by Western blot. AA metabolites in the bath fluid were analyzed by mass spectrometry. Prolonged hypoxia elicited endothelium-independent sustained HPV in BIPAs. Inhibition of GJs by 18β-glycyrrhetinic acid (18β-GA) and heptanol, nonspecific blockers, and Gap-27, a specific blocker, decreased HPV in deendothelized BIPAs. The sustained HPV was not dependent on Ca(2+) entry but decreased by removal of Ca(2+) and by Rho-kinase inhibition with Y-27632. Furthermore, inhibition of GJs decreased smooth muscle myosin heavy chain (SM-MHC) expression and myosin light chain phosphorylation in BIPAs. Interestingly, inhibition of 15- and 20-hydroxyeicosatetraenoic acid (HETE) synthesis decreased HPV in deendothelized BIPAs. 15-HETE- and 20-HETE-stimulated constriction of BIPAs was inhibited by 18β-GA and Gap-27. Application of 15-HETE and 20-HETE to BIPAs increased SM-MHC expression, which was also suppressed by 18β-GA and by inhibitors of lipoxygenase and cytochrome P450 monooxygenases. More interestingly, 15,20-dihydroxyeicosatetraenoic acid and 20-OH-prostaglandin E2, novel derivatives of 20-HETE, were detected in tissue bath fluid and synthesis of these derivatives was almost completely abolished by 18β-GA. Taken together, our novel findings show that GJs between SMCs are involved in the sustained HPV in BIPAs, and 15-HETE and 20-HETE, through GJs, appear to mediate SM-MHC expression and contribute to the sustained HPV development.

Potentially positive ageing-related variations of medial smooth muscle cells in the saphenous veins used as aortocoronary bypass grafts.

Currently, elderly people constitute a large proportion of patients undergoing coronary artery bypass grafting (CABG). Activated smooth muscle cells in the tunica media of saphenous vein (SV) grafts are thought to play a key role in the formation of neointima and development of occluding atherosclerotic plaques. The aim of this study was to identify ageing-related variations in the expression of the smooth muscle cells pro-teins that may impact on patency rate of the grafts and the CABG outcomes. The study involved 216 consecutive patients with the mean of 62.7 ± 8.4 years who underwent isolated CABG with at least one SV aortocoronary bypass graft. Expression of a-smooth muscle actin (a-SM actin), smooth muscle-myosin heavy chain (SM-MHC), calponin (CALP), cytokeratin 8 (CK-8), metalloproteinase-2 (MMP-2) and tissue inhibitors of metalloproteinases-2 and -3 (TIMP-2, TIMP-3) in the SV wall was assessed by immunohistochemistry and correlated with the age of patients. Calponin and a-SM actin were expressed in all studied SV transplants. SM-MHC immunoreactivity was observed in SV segments in 68.5% of patients, whereas MMP-2a and TIMPs expression was found in 75% of cases. In more than 50% of analyzed SV transplants, no expression of cytokeratin-8 was found. Moderate correlations between preexisting expressions of either cytoskeletal or hemostatic proteins in the tunica media of the SV grafts and the age of CABG patients were demonstrated. They were positive for SM-MHC (r = 0.494), CALP (r = 0.548), TIMP-2 (r = 0.413) and TIMP-3 (r = 0.406) whereas negative for CK-8 (r = -0.528) and MMP-2 (r = -0.417). Age-dependent decreases in the expression of MMP-2 and CK-8 accompanied by increases in expression of SM-MHC, TIMP-2 and TIMP-3 may promote SV graft patency and, thus, suggest a rationale for common use of SV grafts in the elderly.

Bronchial but not Tracheal Smooth Muscle is Hypercontractile in an Equine Model of Severe Asthma

Asthma is an inflammatory disease of the airways characterized by airway hyperresponsiveness, an exaggerated bronchoconstrictive response of airway smooth muscle (ASM) to stimuli. However, whether asthmatic ASM is mechanically different from non-asthmatic ASM is still unclear. Most studies so far have been performed on the trachealis muscle but it might not be representative of smooth muscle in the more peripheral airways. Thus, we addressed this question in an equine model of asthma (horse with heaves) and compared the trachealis (TSM) and peripheral bronchial (BSM) airway smooth muscle mechanics.Muscle strips from de-epithelialized TSM and BSM (less than 10 mm in diameter) were mounted horizontally in an experimental chamber between a length controller and a force transducer (Aurora Scientific Inc.). Maximal velocity of shortening (Vmax), methacholine dose response and stress were measured.We found no difference in Vmax between TSM from control horses and horses with heaves (0.124±0.01vs. 0.119±0.01; control vs. heaves; P=0.8), whereas the BSM exhibited a significantly higher Vmax (0.114±0.01 vs. 0.239±0.02; P=0.03). Western blot analysis showed a significant increase of the (+)insert fast smooth muscle myosin heavy chain (SMMHC) isoform in BSM of heaves-affected horses (0.78±0.04 vs. 1.0±0.05, P=0.0092), while no such difference was found in TSM (0.91±0.05 vs.0.97±0.08;P=0.54). The expression of calponin was significantly increased in TSM of heaves-affected horses (0.92±0.15 vs. 2.5±0.57, P=0.037; but this was not observed in BSM (0.37±0.14 vs. 0.49±0.18; P=0.62). There were no differences detected in the expression of MLCK, total SMMHC and transgelin. Peripheral airways may have a greater exposure to inflammatory cells than the trachealis which may explain their differences in mechanics. Further studies await to determine the mechanism underlying in observed effect.Supported by: CIHR(MOP102718), NIH(RO1HL103405), RFBR(12-04-0076)

Marker profile for the evaluation of human umbilical artery smooth muscle cell quality obtained by different isolation and culture methods.

Even though umbilical cord arteries are a common source of vascular smooth muscle cells, the lack of reliable marker profiles have not facilitated the isolation of human umbilical artery smooth muscle cells (HUASMC). For accurate characterization of HUASMC and cells in their environment, the expression of smooth muscle and mesenchymal markers was analyzed in umbilical cord tissue sections. The resulting marker profile was then used to evaluate the quality of HUASMC isolation and culture methods. HUASMC and perivascular-Wharton's jelly stromal cells (pv-WJSC) showed positive staining for α-smooth muscle actin (α-SMA), smooth muscle myosin heavy chain (SM-MHC), desmin, vimentin and CD90. Anti-CD10 stained only pv-WJSC. Consequently, HUASMC could be characterized as α-SMA+, SM-MHC+, CD10- cells, which are additionally negative for endothelial markers (CD31 and CD34). Enzymatic isolation provided primary HUASMC batches with 90-99% purity, yet, under standard culture conditions, contaminant CD10+cells rapidly constituted more than 80% of the total cell population. Contamination was mainly due to the poor adhesion of HUASMC to cell culture plates, regardless of the different protein coatings (fibronectin, collagen I or gelatin). HUASMC showed strong attachment and long-term viability only in 3D matrices. The explant isolation method achieved cultures with only 13-40% purity with considerable contamination by CD10+cells. CD10+cells showed spindle-like morphology and up-regulated expression of α-SMA and SM-MHC upon culture in smooth muscle differentiation medium. Considering the high contamination risk of HUASMC cultures by CD10+neighboring cells and their phenotypic similarities, precise characterization is mandatory to avoid misleading results.

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.

Inhaled corticosteroids modulate the (+)insert smooth muscle myosin heavy chain in the equine asthmatic airways.

Overexpression of the (+)insert smooth muscle myosin heavy chain (SMMHC) isoform could contribute to airway bronchospasm by increasing the velocity of contraction. Whether the (+)insert isoform is present in the small airways and its expression is reversible in asthma are unknown. To determine the anatomical location and the expression kinetics of the (+)insert SMMHC isoform in airways of horses with heaves and to evaluate its modulation in response to disease status. We evaluated the (+)insert SMMHC isoform in the airways of horses with heaves during disease exacerbation and remission, and in controls. The expression kinetics of the SMMHC (+)insert was then assessed at multiple time points in two studies: first, in horses with heaves treated for a 1-year period with antigen avoidance alone, inhaled corticosteroids alone or both; second, in horses with heaves before and after a 30-day natural antigen exposure. Gene expression analysis was assessed by quantitative PCR and protein expression was confirmed by targeted mass spectrometry. The (+)insert SMMHC isoform was significantly increased in central and peripheral airways, but not in the trachea of heaves-affected horses in clinical exacerbation when compared horses with heaves in remission and controls. Both corticosteroid administration and antigen avoidance led to a significant reduction of the (+)insert expression in the airways. The (+)insert SMMHC isoform was not significantly increased in airways after 1 month of antigenic re-exposure. The (+)insert SMMHC expression is increased throughout the bronchial tree in horses with heaves and reversible by corticosteroids administration and antigen avoidance.

Cell Division Cycle 7 Mediates Transforming Growth Factor-β-induced Smooth Muscle Maturation through Activation of Myocardin Gene Transcription

Smooth muscle (SM) development consists of several processes, including cell fate determination, differentiation, and maturation. The molecular mechanisms controlling SM early differentiation have been studied extensively. However, little is known about the mechanism underlying SM maturation. Cell division cycle 7 (Cdc7) has been shown to regulate cell fate determination in the initial phase of transforming growth factor-β (TGF-β)-induced SM differentiation. Our present study indicates that Cdc7 also regulates SM maturation. Knockdown of Cdc7 suppresses TGF-β-induced expression of SM myosin heavy chain, a late marker of SM differentiation. Cdc7 overexpression, on the other hand, enhances SM myosin heavy chain expression. Interestingly, Cdc7 activates the mRNA expression and promoter activity of myocardin (Myocd), a master regulator of SM differentiation, whose transcription is blocked in the initial phase of the differentiation because TGF-β does not induce Myocd mRNA until after the early SM markers are induced. These data suggest that Cdc7 mediates TGF-β-induced SM maturation via activation of Myocd transcription. Mechanistically, Cdc7 physically and functionally interacts with Nkx2.5 to regulate Myocd promoter activity. Cdc7 appears to enhance Nkx2.5 binding to Myocd promoter, leading to Myocd activation. Taken together, our studies demonstrate that Cdc7 regulates the initial and late phase of SM differentiation through distinct mechanisms.

Abstract 543: ADAR1-mediated RNA Editing, a Novel Mechanism Controlling Phenotypic Modulation of Vascular Smooth Muscle Cells

Vascular smooth muscle (SMC) phenotypic modulation, the transition from a contractile to a proliferative phenotype accompanied by neointima formation following vascular injury, plays a critical role in the development and progression of several proliferative cardiovascular diseases such as atherosclerosis, hypertension, restenosis after angioplasty or bypass, diabetic vascular complications, and transplantation arteriopathy. A hallmark feature of the phenotypic modulation is the down-regulation of SMC contractile genes. Platelet-derived growth factor-BB (PDGF-BB), a well-known stimulator of SMC phenotypic modulation, down-regulates SMC gene expression via posttranscriptional regulation of the related genes. The post-transcriptional mechanisms involved in SMC phenotypic gene expression, however, remain largely unknown. We found that the down-regulation of SMC contractile genes is caused by abnormal RNA editing of their precursor mRNAs (pre-mRNAs). This abnormal pre-mRNA editing is facilitated by adenosine deaminase acting on RNA (ADAR), which converts adenosines to inosines (A→I editing). PDGF-BB induces ADAR1 while down-regulating the expression of SMC myosin heavy chain (SMMHC) and calponin (CNN). Knockdown of ADAR1 by shRNA restores PDGF-BB-blocked SMMHC and CNN expression, demonstrating that ADAR1 plays an essential role in SMC phenotype modulation. In vivo animal studies show that SMMHC and CNN pre-mRNA is accumulated while their mature mRNA is decreased in balloon-injured rat carotid arteries. Moreover, ADAR1 is highly induced in media layer SMCs initially, and neointima SMCs subsequently following the injury. Of importance, knockdown of ADAR1 dramatically inhibits injury-induced neointima formation, demonstrating a critical role of ADAR1 in vascular remodeling in vivo . Taken together, our study unraveled a novel molecular mechanism, i.e., pre-mRNA editing, governing SMC phenotypic modulation.

Abstract 299: Cell Division Cycle 7 Mediates Transforming Growth Factor-ß-induced Smooth Muscle Maturation

Cell division cycle 7 (Cdc7) has been shown to regulate cell fate determination in the initial phase of transforming growth factor-β (TGF-β)-induced smooth muscle cell (SMC) differentiation. It is unknown, however, if Cdc7 is involved in SMC maturation or the late phase differentiation. In this study, we found that Cdc7 regulates the late phase SMC differentiation through a mechanism different from the cell fate determination. Knockdown of Cdc7 suppresses TGF-β-induced expression of smooth muscle myosin heavy chain (SMMHC), a late marker of SMC differentiation in addition to the early markers. Cdc7 overexpression, on the other hand, enhances SMMHC expression. Interestingly, Cdc7 activates the mRNA expression and gene transcription of myocardin (Myocd), a master regulator for SMC differentiation, whose transcription is blocked in the initial phase of the differentiation because TGF-β does not induce Myocd mRNA until 16 h after the stimulation. These data suggest that Cdc7 mediates TGF-β-induced late SMC differentiation via activating Myocd. Mechanistically, Cdc7 physically and functionally interacts with Nkx2.5 to regulate Myocd promoter activity. Cdc7 appears to enhance Nkx2.5 binding to Myocd promoter, leading to Myocd activation. Taken together, our studies demonstrate that Cdc7 regulates the initial and late phase of SMC differentiation through distinct mechanisms.

Differentiated markers in undifferentiated cells: Expression of smooth muscle contractile proteins in multipotent bone marrow mesenchymal stem cells

In studying the differentiation of stem cells along smooth muscle lineage, smooth muscle cell (SMC) contractile proteins serve as markers for the relative state of maturation. Yet, recent evidence suggests that some SMC markers are probably expressed in multipotent mesenchymal stem cells (MSCs). Such a paradox necessitates investigations to re-examine their role as differentiated markers in MSCs. We tried to detect the expression of four widely used SMC markers including α-smooth muscle actin (α-SMA), h1-calponin, desmin and smooth muscle myosin heavy chain (SM-MHC), as well as the other isoforms of calponin family in resting MSCs. Then we used three different conditions to initiate MSCs differentiation along SMC lineage, and examined the alternation of SMC markers expression at both the transcript level and protein level. Desmin and h1-calponin are expressed in MSCs, in the presence or absence of SMC induction conditions. Moreover, MSCs are shown to express all known isoforms of calponin. Double-staining reveals that h1-calponin +/α-SMA - cells constitute the majority of resting MSCs. Under differentiated conditions, expression of SM-MHC was initiated and expression of α-SMA was promoted. The expression of SM-MHC and upregulation of α-SMA are relatively reliable indications of a mature smooth muscle phenotype in MSCs. Given that the cells are particularly rich in calponins expression, we postulate possible roles of these proteins in regulating cellular function by taking part in actin cytoskeleton and signaling. These findings imply that an extensive study of the cell physiology of MSCs should focus on the functional roles for these proteins, rather than simply regard them as differentiated markers.

High pulsatility flow stimulates smooth muscle cell hypertrophy and contractile protein expression.

Proximal arterial stiffening is an important predictor of events in systemic and pulmonary hypertension, partly through its contribution to downstream vascular abnormalities. However, much remains undetermined regarding the mechanisms involved in the vascular changes induced by arterial stiffening. We therefore addressed the hypothesis that high pulsatility flow, caused by proximal arterial stiffening, induces downstream pulmonary artery endothelial cell (EC) dysfunction that in turn leads to phenotypic change of smooth muscle cells (SMCs). To test the hypothesis, we employed a model pulmonary circulation in which upstream compliance regulates the pulsatility of flow waves imposed onto a downstream vascular mimetic coculture composed of pulmonary ECs and SMCs. The effects of high pulsatility flow on SMCs were determined both in the presence and absence of ECs. In the presence of ECs, high pulsatility flow increased SMC size and expression of the contractile proteins, smooth muscle α-actin (SMA) and smooth muscle myosin heavy chain (SM-MHC), without affecting proliferation. In the absence of ECs, high pulsatility flow decreased SMC expression of SMA and SM-MHC, without affecting SMC size or proliferation. To identify the molecular signals involved in the EC-mediated SMC responses, mRNA and/or protein expression of vasoconstrictors [angiotensin-converting enzyme (ACE) and endothelin (ET)-1], vasodilator (eNOS), and growth factor (TGF-β1) in EC were examined. Results showed high pulsatility flow decreased eNOS and increased ACE, ET-1, and TGF-β1 expression. ACE inhibition with ramiprilat, ET-1 receptor inhibition with bosentan, and treatment with the vasodilator bradykinin prevented flow-induced, EC-dependent SMC changes. In conclusion, high pulsatility flow stimulated SMC hypertrophy and contractile protein expression by altering EC production of vasoactive mediators and cytokines, supporting the idea of a coupling between proximal vascular stiffening, flow pulsatility, and downstream vascular function.