Smooth Muscle Gene Expression Research Articles

MicroRNA-133 Controls Vascular Smooth Muscle Cell Phenotypic Switch In Vitro and Vascular Remodeling In Vivo

MicroRNA (miR)-1 and -133 play a crucial role in skeletal and cardiac muscle biology and pathophysiology. However, their expression and regulation in vascular cell physiology and disease is currently unknown. The aim of the present study was to evaluate the role, if any, of miR-1 and miR-133 in vascular smooth muscle cell (VSMC) phenotypic switch in vitro and in vivo. We demonstrate here that miR-133 is robustly expressed in vascular smooth muscle cells (VSMCs) in vitro and in vivo, whereas miR-1 vascular levels are negligible. miR-133 has a potent inhibitory role on VSMC phenotypic switch in vitro and in vivo, whereas miR-1 does not have any relevant effect per se. miR-133 expression is regulated by extracellular signal-regulated kinase 1/2 activation and is inversely correlated with VSMC growth. Indeed, miR-133 decreases when VSMCs are primed to proliferate in vitro and following vascular injury in vivo, whereas it increases when VSMCs are coaxed back to quiescence in vitro and in vivo. miR-133 loss- and gain-of-function experiments show that miR-133 plays a mechanistic role in VSMC growth. Accordingly, adeno-miR-133 reduces but anti-miR-133 exacerbates VSMC proliferation and migration in vitro and in vivo. miR-133 specifically suppresses the transcription factor Sp-1 expression in vitro and in vivo and through Sp-1 repression regulates smooth muscle gene expression. Our data show that miR-133 is a key regulator of vascular smooth muscle cell phenotypic switch in vitro and in vivo, suggesting its potential therapeutic application for vascular diseases.

Activation Dynamics and Signaling Properties of Notch3 Receptor in the Developing Pulmonary Artery

Notch3 signaling is fundamental for arterial specification of systemic vascular smooth muscle cells (VSMCs). However, the developmental role and signaling properties of the Notch3 receptor in the mouse pulmonary artery remain unknown. Here, we demonstrate that Notch3 is expressed selectively in pulmonary artery VSMCs, is activated from late fetal to early postnatal life, and is required to maintain the morphological characteristics and smooth muscle gene expression profile of the pulmonary artery after birth. Using a conditional knock-out mouse model, we show that Notch3 receptor activation in VSMCs is Jagged1-dependent. In vitro VSMC lentivirus-mediated Jagged1 knockdown, confocal localization analysis, and co-culture experiments revealed that Notch3 activation is cell-autonomous and occurs through the physical engagement of Notch3 and VSMC-derived Jagged1 in the interior of the same cell. Although the current models of mammalian Notch signaling involve a two-cell system composed of a signal-receiving cell that expresses a Notch receptor on its surface and a neighboring signal-sending cell that provides membrane-bound activating ligand, our data suggest that pulmonary artery VSMC Notch3 activation is cell-autonomous. This unique mechanism of Notch activation may play an important role in the maturation of the pulmonary artery during the transition to air breathing.

Generalized neuromuscular hypoplasia, reduced smooth muscle myosin and altered gut motility in the klotho model of premature aging

Gastrointestinal symptoms, particularly constipation, increase with aging, but their underlying mechanisms are poorly understood due to lack of experimental models. Previously we established the progeric klotho mouse as a model of aging-associated anorexia and gastric dysmotility. We also detected reduced fecal output in these animals; therefore, the aim of this study was to investigate in vivo function and cellular make-up of the small intestinal and colonic neuromuscular apparatus. Klotho expression was studied by RT-PCR and immunohistochemistry. Motility was assessed by dye transit and bead expulsion. Smooth muscle and neuron-specific gene expression was studied by Western immunoblotting. Interstitial cells of Cajal (ICC) and precursors were analyzed by flow cytometry, confocal microscopy, and three-dimensional reconstruction. HuC/D(+) myenteric neurons were enumerated by fluorescent microscopy. Klotho protein was detected in neurons, smooth muscle cells, and some ICC classes. Small intestinal transit was slower but whole-gut transit of klotho mice was accelerated due to faster colonic transit and shorter intestinal lengths, apparent only after weaning. Fecal water content remained normal despite reduced output. Smooth muscle myosin expression was reduced. ICC, ICC precursors, as well as nitrergic and cholinergic neurons maintained their normal proportions in the shorter intestines. Progeric klotho mice express less contractile proteins and develop generalized intestinal neuromuscular hypoplasia mainly arising from stunted postweaning growth. As reduced fecal output in these mice occurs in the presence of accelerated colonic and whole-gut transit, it likely reflects reduced food intake rather than intestinal dysmotility.

The cAMP-responsive Rap1 Guanine Nucleotide Exchange Factor, Epac, Induces Smooth Muscle Relaxation by Down-regulation of RhoA Activity

Agonist activation of the small GTPase, RhoA, and its effector Rho kinase leads to down-regulation of smooth muscle (SM) myosin light chain phosphatase activity, an increase in myosin light chain (RLC(20)) phosphorylation and force. Cyclic nucleotides can reverse this process. We report a new mechanism of cAMP-mediated relaxation through Epac, a GTP exchange factor for the small GTPase Rap1 resulting in an increase in Rap1 activity and suppression of RhoA activity. An Epac-selective cAMP analog, 8-pCPT-2'-O-Me-cAMP ("007"), significantly reduced agonist-induced contractile force, RLC(20), and myosin light chain phosphatase phosphorylation in both intact and permeabilized vascular, gut, and airway SMs independently of PKA and PKG. The vasodilator PGI(2) analog, cicaprost, increased Rap1 activity and decreased RhoA activity in intact SMs. Forskolin, phosphodiesterase inhibitor isobutylmethylxanthine, and isoproterenol also significantly increased Rap1-GTP in rat aortic SM cells. The PKA inhibitor H89 was without effect on the 007-induced increase in Rap1-GTP. Lysophosphatidic acid-induced RhoA activity was reduced by treatment with 007 in WT but not Rap1B null fibroblasts, consistent with Epac signaling through Rap1B to down-regulate RhoA activity. Isoproterenol-induced increase in Rap1 activity was inhibited by silencing Epac1 in rat aortic SM cells. Evidence is presented that cooperative cAMP activation of PKA and Epac contribute to relaxation of SM. Our findings demonstrate a cAMP-mediated signaling mechanism whereby activation of Epac results in a PKA-independent, Rap1-dependent Ca(2+) desensitization of force in SM through down-regulation of RhoA activity. Cyclic AMP inhibition of RhoA is mediated through activation of both Epac and PKA.

NADPH oxidase 4 mediates TGF-β-induced smooth muscle α-actin via p38MAPK and serum response factor

In contrast to other cell types, vascular smooth muscle cells modify their phenotype in response to external signals. NADPH oxidase 4 (Nox4) is critical for maintenance of smooth muscle gene expression; however, the underlying mechanisms are incompletely characterized. Using smooth muscle α-actin (SMA) as a prototypical smooth muscle gene and transforming growth factor-β (TGF-β) as a differentiating agent, we examined Nox4-dependent signaling. TGF-β increases Nox4 expression and activity in human aortic smooth muscle cells (HASMC). Transfection of HASMC with siRNA against Nox4 (siNox4) abolishes TGF-β-induced SMA expression and stress fiber formation. siNox4 also significantly inhibits TGF-β-stimulated p38MAPK phosphorylation, as well as that of its substrate, mitogen-activated protein kinase-activated protein kinase-2. Moreover, the p38MAPK inhibitor SB-203580 nearly completely blocks the SMA increase induced by TGF-β. Inhibition of either p38MAPK or NADPH oxidase-derived reactive oxygen species impairs the TGF-β-induced phosphorylation of Ser103 on serum response factor (SRF) and reduces its transcriptional activity. Binding of SRF to myocardin-related transcription factor (MRTF) is also necessary, because downregulation of MRTF by siRNA abolishes TGF-β-induced SMA expression. Taken together, these data suggest that Nox4 regulates SMA expression via activation of a p38MAPK/SRF/MRTF pathway in response to TGF-β.

Sex Steroid Receptors in Male Human Bladder: Expression and Biological Function

In male, lower urinary tract symptoms (LUTS) have been associated, beside benign prostatic hyperplasia, to some unexpected comorbidities (hypogonadism, obesity, metabolic syndrome), which are essentially characterized by an unbalance between circulating androgens/estrogens. Within the bladder, LUTS are linked to RhoA/Rho-kinase (ROCK) pathway overactivity. To investigate the effects of changing sex steroids on bladder smooth muscle. ER α, ER β, GPR30/GPER1 and aromatase mRNA expression was analyzed in male genitourinary tract tissues, and cells isolated from bladder, prostate, and urethra. Estrogen and G1 effect on RhoA/ROCK signaling output like cell migration, gene expression, and cytoskeletal remodeling, and [Ca(2+) ](i) was also studied in hB cells. Contractile studies on bladder strips from castrated male rats supplemented with estradiol and testosterone was also performed. The effects of classical (ER α, ER β) and nonclassical (GPR30/GPER1) estrogen receptor ligands (17 β-estradiol and G1, respectively) and androgens on RhoA/ROCK-.mediated cell functions were studied in hB cells. Contractility studies were also performed in bladder strips from castrated male rats supplemented with testosterone or estradiol. Aromatase and sex steroid receptors, including GPR30, were expressed in human bladder and mediates several biological functions. Both 17 β-estradiol and G1 activated calcium transients and induced RhoA/ROCK signaling (cell migration, cytoskeleton remodeling and smooth muscle gene expression). RhoA/ROCK inhibitors blunted these effects. Estrogen-, but not androgen-supplementation to castrated rats increased sensitivity to the ROCK inhibitor, Y-27632 in isolated bladder strips. In hB cells, testosterone elicited effects similar to estrogen, which were abrogated by blocking its aromatization through letrozole. Our data indicate for the first time that estrogen-more than androgen-receptors up-regulate RhoA/ROCK signaling. Since an altered estrogen/androgen ratio characterizes conditions, such as aging, obesity and metabolic syndrome, often associated to LUTS, we speculate that a relative hyperestrogenism may induce bladder overactivity through the up-regulation of RhoA/ROCK pathway.

Repression of Smooth Muscle Differentiation by a Novel High Mobility Group Box-containing Protein, HMG2L1

The molecular mechanisms regulating smooth muscle-specific gene expression during smooth muscle development are poorly understood. Myocardin is an extraordinarily powerful cofactor of serum response factor (SRF) that stimulates expression of smooth muscle-specific genes. In an effort to search for proteins that regulate myocardin function, we identified a novel HMG box-containing protein HMG2L1 (high mobility group 2 like 1). We found that HMG2L1 expression is correlated with the smooth muscle cell (SMC) synthetic phenotype. Overexpression of HMG2L1 in SMCs down-regulated smooth muscle marker expression. Conversely, depletion of endogenous HMG2L1 in SMCs increases smooth muscle-specific gene expression. Furthermore, we found HMG2L1 specifically abrogates myocardin-induced activation of smooth muscle-specific genes. By GST pulldown assays, the interaction domains between HMG2L1 and myocardin were mapped to the N termini of each of the proteins. Finally, we demonstrated that HMG2L1 abrogates myocardin function through disrupting its binding to SRF and abolishing SRF-myocardin complex binding to the promoters of smooth muscle-specific genes. This study provides the first evidence of this novel HMG2L1 molecule playing an important role in attenuating smooth muscle differentiation.

182 EXPRESSION OF THE PROANGIOGENIC FACTOR CYSTEINE-RICH PROTEIN 61 GENE IN MECHANICALLY STRAINED BLADDER SMOOTH MUSCLE CELLS REQUIRES THE COMBINED ACTIVITIES OF THE MYOCARDIN-RELATED TRANSCRIPTION FACTOR AND HISTONE ACETYL TRANSFERASE

You have accessJournal of UrologyBladder and Urethra: Anatomy, Physiology and Pharmacology I1 Apr 2010182 EXPRESSION OF THE PROANGIOGENIC FACTOR CYSTEINE-RICH PROTEIN 61 GENE IN MECHANICALLY STRAINED BLADDER SMOOTH MUSCLE CELLS REQUIRES THE COMBINED ACTIVITIES OF THE MYOCARDIN-RELATED TRANSCRIPTION FACTOR AND HISTONE ACETYL TRANSFERASE Mary Hanna, Haibo Liu, and Brahim Chaqour Mary HannaMary Hanna More articles by this author , Haibo LiuHaibo Liu More articles by this author , and Brahim ChaqourBrahim Chaqour More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.238AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The cellular components of the urinary bladder elicit adaptive responses to pressure overload acutely by remodeling their cytoskeletal structures and chronically through hypertrophic growth of smooth muscle cells (SMCs). The long term responses are associated with the activation of angiogenesis to improve tissue perfusion which, otherwise, becomes impaired as a result of a mismatch between the smooth muscle mass and capillary density, leading to hypoxia/ischemia, altered muscle contractility and organ failure. Neoangiogenesis is promoted by mechano-responsive angiogenic factors such as cysteine-rich protein 61 (Cyr61) which enhances sprouting of new blood vessels and protects against oxidative stress. In this study, we examined the expression and regulation of the Cyr61 gene by mechanical forces in bladder SMCs. METHODS Cultured human bladder SMCs were subjected to cyclic mechanical stretch using a mechanical device designed to impart a biaxial strain to the cells. Partial urethral obstruction of the bladder was created in female Sprague Dawley rats by loosely tying the urethra. The expression and activation of the Cyr61 gene was examined by real-time PCR, transfection and chromatin immunoprecipitation (ChIP) assays. RESULTS Mechanical strain-dependent induction of the Cyr61 gene involves signaling cascades through RhoA-mediated actin remodeling and the p38 stress-activated protein kinase (SAPK). Actin signaling controls serum response factor (SRF) activity via SRF interaction with the myocardin-related transcriptional activator (MRTF)-A and its tethering to a single CArG-box sequence within the Cyr61 promoter. Such activity was abolished in mechanically stimulated mouse MRTF-A null cells or upon inhibition of cyclic AMP responsive element (CREB)-binding protein (CBP) histone acetyl transferase (HAT). Mechanical strain induced CBP-mediated acetylation of histones 3 and 4 at the SRF binding site. Inhibition of p38 SAPK reduced CBP HAT activity and its recruitment to the SRF-MRTF-A complex. Similarly, mechanical overload-induced Cyr61 gene expression in obstructed bladders was associated with nuclear localization of MRTF-A and enrichment of the Cyr61 promoter with both MRTF-A and acetylated histone H3. CONCLUSIONS Signal-controlled activation of SRF, MRTF-A and CBP provides a novel connection between mechanical forces and angiogenic gene expression in bladder smooth muscle. Brooklyn, NY© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e72-e73 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Mary Hanna More articles by this author Haibo Liu More articles by this author Brahim Chaqour More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

887 THE POTENTIAL ROLE OF SIALORPHIN IN MEDIATING UROGENITAL SYMPTOMS IN DIABETES

You have accessJournal of UrologySexual Function/Dysfunction/Andrology: Basic Research II1 Apr 2010887 THE POTENTIAL ROLE OF SIALORPHIN IN MEDIATING UROGENITAL SYMPTOMS IN DIABETES Giulia Calenda, Nirmala Kanika, Sylvia Suadicani, Moses Tar, Dwaraka Kuppam, David Spray, Arnold Melman, and Kelvin Davies Giulia CalendaGiulia Calenda More articles by this author , Nirmala KanikaNirmala Kanika More articles by this author , Sylvia SuadicaniSylvia Suadicani More articles by this author , Moses TarMoses Tar More articles by this author , Dwaraka KuppamDwaraka Kuppam More articles by this author , David SprayDavid Spray More articles by this author , Arnold MelmanArnold Melman More articles by this author , and Kelvin DaviesKelvin Davies More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.1643AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Diabetes is associated with the development of urogenital complications such as erectile dysfunction (ED) and lower urinary tract symptoms (LUTS). It remains to be determined if ED and LUTS have an underlying common molecular mediator. We have previously demonstrated that diabetes results in down regulation of the Vcsa1 gene expression in corporal smooth muscle. Sialorphin, the protein product of Vcsa1, acts as a potent endogenous inhibitor of neutral endopeptidase (NEP) and has been shown to play an important role in erectile physiology by modulating corporal smooth muscle tone. Because sialorphin is released into the circulation it could also act on non-corporal smooth muscle tissue, such as the bladder. The work described here investigated if diabetes results in lowered circulating sialorphin levels, and if changes in sialorphin levels could have a similar impact on the biochemistry of distinct urogenital tissues. METHODS Diabetes was induced in Sprague-Dawley rat using streptozotocin (STZ). The levels of sialorphin in the serum of age matched controls (AMC), STZ-diabetic and STZ-diabetic rats treated with insulin were determined by radioimmunoassay (RIA). The effect of sialorphin on intracellular calcium ([Ca2+]i) was determined in cultured bladder and corporal smooth muscle cells from diabetic and non-diabetic rats using calcium imaging. RESULTS Circulating levels of sialorphin are decreased in diabetic rats by approximately 35±3.6%. In insulin treated STZ-diabetic animals circulating levels of sialorphin were not significantly different from AMC (87±3.4% of AMC). In vitro we demonstrate that sialorphin potentiates the Ca2+- releasing effect of bradykinin in both bladder (by about 73±8.9%) and corporal (by about 40±6.3%) smooth muscle cells, probably through the inhibition of NEP-mediated degradation of bradykinin. CONCLUSIONS In this study we show that diabetes is associated with a decrease of circulating sialorphin in rats. In addition sialorphin acts to modulate [Ca2+]i in both corporal and bladder smooth muscle cells, probably though its actions as a very effective NEP inhibitor. Intracellular calcium is an important mediator of smooth muscle tone, which in turn is an important determinant of both erectile and bladder function. Our work suggests that the down regulation of circulating sialorphin levels which occur with diabetes acts as a common factor for the development of LUTS in diabetes. Bronx, NY© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e347 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Giulia Calenda More articles by this author Nirmala Kanika More articles by this author Sylvia Suadicani More articles by this author Moses Tar More articles by this author Dwaraka Kuppam More articles by this author David Spray More articles by this author Arnold Melman More articles by this author Kelvin Davies More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

NFAT5 positively regulates SMαA gene expression in vascular smooth muscle

Nuclear factor of activated T‐cells 5 (NFAT5) is a tonicity‐responsive transcription factor; however, NFAT5 function in vascular SMCs is unknown. The SM α‐actin (SMαA) gene contains an NFAT5 cis element overlapping the first intronic CArG. We hypothesized that NFAT5 positively regulates SMαA gene expression through binding to this consensus sequence. NFAT5 overexpression increased SMαA promoter‐reporter activity, which was attenuated by mutating the NFAT5 site. siRNA knockdown of endogenous NFAT5 inhibited an Ang II‐mediated upregulation of SMαA promoter activity. Conversely, platelet‐derived growth factor‐BB decreased SMαA expression concomitant with a 70% decrease in NFAT5 binding to the SMαA first intron. In the A404 SM precursor cell line, treatment with retinoic acid to induce SMαA mRNA was preceded by increased NFAT5 expression. Microarray analysis of NFAT5 null mouse embryonic fibroblasts indicated a 64% decrease in SMαA mRNA compared to WT. Additionally, aortas from heterozygous NFAT5 mice have a 41% decrease in SMαA protein at 5 wks of age, returning to WT levels by 20 wks. In the rat carotid artery balloon injury model, NFAT5 protein was upregulated at 7 and 21 days, corresponding with an increase in SMαA protein back to basal levels and resolution to the contractile phenotype. In conclusion, we have identified NFAT5 as a novel positive regulator of SMαA gene expression in vascular SMCs. (NIH HL81682)

Modulation of Myocardin Function by the Ubiquitin E3 Ligase UBR5

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

MicroRNAs and Heart Failure Diagnosis

See related article, pages 1035–1039 Heart disease is the greatest noninfectious health hazard ever to confront the human race. It is the leading cause of death in industrialized nations, and, accordingly, it is responsible for a huge economic burden to society. It is estimated that 5 million Americans have heart failure (HF), a syndrome with mortality of approximately 50% at 5 years.1 Now, evidence indicates that the epidemic of HF is extending to the developing world. The road to advances in the treatment of HF is littered with failures, and many new means of diagnosing and tracking disease progression have disappointed. Therefore, there is an urgent need to identify novel mechanisms, markers, and therapeutic targets in HF pathogenesis. MicroRNAs (miRNAs or miRs) are among the most exciting areas in medicine and science today. MicroRNAs are endogenous, highly conserved, ≈22-nucleotide-long noncoding RNAs that regulate the stability and subsequent translation of nascent mRNA transcripts. Generally, miRs inhibit protein translation and/or promote mRNA degradation by base-pairing with 3′ untranslated regions within a transcript. MicroRNAs were first discovered in 1993 in Caenorhabditis elegans and are known to be critical to early vertebrate development and in an increasing number of basic molecular processes. Over the last few years, miRs have emerged as important mechanisms in human disease, including cardiovascular disease, diabetes, and cancer.2–6 Now, there is burgeoning interest in testing whether miRs could serve as markers for disease progression or prognostication. Indeed, in the cancer literature, strategies are emerging to use miR profiles for diagnosis and risk stratification. Currently, miR profiling is being evaluated in prostate cancer, head and neck squamous cell carcinomas, breast cancer, acute lymphoblastic leukemia, malignant mesothelioma, and other tumors.2,7–10 Most clinical studies to date on miRs have relied on tissue-based measurement of miR abundances, but miRs are …

Phosphorylation of Myocardin by Extracellular Signal-regulated Kinase

The contractile phenotype of smooth muscle (SM) cells is controlled by serum response factor (SRF), which drives the expression of SM-specific genes including SM alpha-actin, SM22, and others. Myocardin is a cardiac and SM-restricted coactivator of SRF that is necessary for SM gene transcription. Growth factors inducing proliferation of SM cells inhibit SM gene transcription, in a manner dependent on the activation of extracellular signal-regulated kinases ERK1/2. In this study, we found that ERK1/2 phosphorylates mouse myocardin (isoform B) at four sites (Ser(812), Ser(859), Ser(866), and Thr(893)), all of which are located within the transactivation domain of myocardin. The single mutation of each site either to alanine or to aspartate has no effect on the ability of myocardin to activate SRF. However, the phosphomimetic mutation of all four sites to aspartate (4xD) significantly impairs activation of SRF by myocardin, whereas the phosphodeficient mutation of all four sites to alanine (4xA) has no effect. This translates to a reduced ability of the 4xD (but not of 4xA) mutant of myocardin to stimulate expression of SM alpha-actin and SM22, as assessed by corresponding promoter, mRNA, or protein assays. Furthermore, we found that phosphorylation of myocardin at these sites impairs its interaction with acetyltransferase, cAMP response element-binding protein-binding protein, which is known to promote the transcriptional activity of myocardin. In conclusion, we describe a novel mode of modulation of SM gene transcription by ERK1/2 through a direct phosphorylation of myocardin.

Nitric oxide stimulates myoglobin gene and protein expression in vascular smooth muscle

Mb (myoglobin) is a haemoprotein present in cardiac, skeletal and smooth muscle and is primarily responsible for the storage and 'facilitated transfer' of molecular oxygen from the cell membrane to mitochondria. Also, Mb plays a role in regulating *NO (nitric oxide) homoeostasis through (i) binding *NO (Mb-NO complex); (ii) oxidation of *NO to nitrate; and (iii) formation of vasoactive S-nitroso-Mb [Rayner, B.S., Wu, B.-J., Raftery, M., Stocker, R. and Witting, P.K. (2005) J. Biol. Chem. 280, 9985-9993]. Pathological *NO concentrations affect mitochondrial function and decrease cell viability through inducing apoptosis. Treatment of cultured rat VSMCs (vascular smooth muscle cells) with cumulative doses (0.1, 1 or 10 microM) of *NO from the donors diethylamineNONOate or spermineNONOate (N-[2-aminoethyl]-N-[2-hydroxy-3-nitrosohydrazine]-1,2-ethelenediamine) yielded a time-dependent increase in Mb gene expression. Concomitant transcriptional activation increased the concentration of Mb within cultured rat or primary human VSMCs as judged by Western blot analysis and indirect immunofluorescence microscopy. Cell viability did not decrease in these cells at the *NO doses tested. Importantly, sub-culturing isolated rat aortic segments for 7 days in the presence of L-arginine at 37 degrees C stimulated *NO production with a parallel increase in Mb in the underlying VSMCs. Overall, exposure of VSMCs (either in cell culture or intact vessels) to pathological *NO promotes an up-regulation of the Mb gene and protein, suggesting a feedback relationship between *NO and Mb that regulates the concentration of the potent cell signalling molecule in the vessel wall, similar to the role haemoglobin plays in the vessel lumen.

Relationship of lipoprotein-associated phospholipase A2 and oxidized low density lipoprotein in carotid atherosclerosis

Plasma levels of lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) and oxidized low density lipoprotein (oxLDL) have been identified as risk factors for cardiovascular disease. Lp-PLA(2) is the sole enzyme responsible for the hydrolysis of oxidized phospholipids on LDL particles in atherosclerotic plaques. We have studied the relationship between Lp-PLA(2) and oxLDL in carotid endarterectomy (CEA) tissues and in matched plasmas. In extracts from CEA anatomical segments, the levels of oxLDL were significantly associated with the levels of Lp-PLA(2) protein (r = 0.497) and activity (r = 0.615). OxLDL and Lp-PLA(2) mass/activity were most abundant in the carotid bifurcation and internal segments where plaque was most abundant. In extracts from CEA atheroma, the levels of oxLDL and Lp-PLA(2) were significantly correlated (r = 0.634). In matched plasma and atheroma extracts, the levels of Lp-PLA(2) were negatively correlated (r = - 0.578). The ratio of Lp-PLA(2) to oxLDL was higher in atheromatous tissue (277:1) than in normal tissue (135:1) and plasma (13:1). Immunohistochemical experiments indicated that in plaques, oxLDL and Lp-PLA(2) existed in overlapping but distinctly different distribution. Fluorescence microscopy showed both oxLDL and Lp-PLA(2) epitopes on the same LDL particle in plasma but not in plaque. These results suggest that the relationship between Lp-PLA(2) and oxLDL in the atherosclerotic plaque is different from that in the plasma compartment.

Effects of endothelial growth media on proepicardial cell gene expression and morphogenesis in 3D collagen matrices

Proepicardial cells (PE) contribute to embryonic coronary vessel and epicardial development. Cells from the PE region can differentiate into coronary vascular smooth muscle cells and fibroblasts in vitro, but the endothelial specification capability of these cells is controversial. We sought to examine the effects of endothelial cell growth media on gene expression and the morphogenic properties of proepicardial cells in three-dimensional (3D) matrices. A primary culture of avian PE cells was subjected to molecular characterization with selected endothelial specific markers. Morphogenic properties of PE cells were assessed by in vitro assays of coronary vasculogenesis and invasion, which utilized highly defined, serum free, three-dimensional matrix conditions. PE cells maintained mixed cell population properties in the culture based on morphogenic features, immunohistochemistry, and the gene expression data. When suspended in a 3D vasculogenesis in vitro assay, PE cells formed intracellular vacuoles and assembled into multicellular tubes. Further, ultrastructural analysis revealed the presence of pinocytic vacuoles, intercellular junctions, and endothelial specific Weibel Palade bodies. In the invasion assay, PE cells spontaneously invaded control matrices. This invasion was markedly enhanced by lysophosphatidic acid (94±9.6 vs. 285.6±54.9, p<0.05) and was completely blocked with synthetic broad-spectrum metalloproteinase inhibitor GM6001. Isolated PE cells grown in endothelial cell media represent mixed-cell population, characterized by both smooth muscle and endothelial gene expression. When placed in 3D in vitro assays, PE cells manifest morphogenic properties, including multicellular tube assembly and invasion.

Cyclic GMP‐dependent protein kinase (PKG) regulates smooth muscle gene expression : Possible involvement of Elk‐1 sumoylation

Although the regulation of smooth muscle cell (SMC) gene expression by cGMP‐dependent protein kinase (PKG) is now recognized, the mechanisms underling these effects are not fully understood. In this study, we report that PKG expression increased the level of a high molecular weight Elk‐1 as determined by western blot using a phospho‐Elk‐1 antibody. PKG expression also enhanced myocardin‐induced SM22 promoter activity in cultured SMC. With this background, we examined the hypothesis that PKG may modulate Elk‐1 activity through posttranslational modification by sumoylation, thus contributing to the expression of SMC specific genes. Using immunoprecipitation with sumo‐1 antibody and subsequent immunoblot with phospho‐Elk‐1 antibody, we identified the higher molecular weight species as sumoylated Elk‐1. PKG expression in SMC increased Elk‐1 sumoylation two‐fold compared to the control cells. The sumoylation of Elk‐1 was confirmed by the transfection of dominant negative form of the sumo‐conjugating enzyme, DN‐Ubc9. Furthermore, siRNA‐mediated suppression of PKG confirmed the role of the kinase in the up‐regulation of sumoylated phospho‐Elk‐1. Overexpression of Elk‐1, sumo‐1, and PKG in COS‐7 cells confirmed again that PKG increased the sumoylation of Elk‐1. Transfection of combined vectors demonstrated that PKG inhibited Elk‐1 binding and activity on the myocardin‐induced SMC specific gene promoter activity as measured by ChIP assay and reporter gene expression, respectively. Taken together, these data suggest that PKG increased sumoylation of Elk‐1, thereby increasing myocardin‐SRF activity on SMC‐specific gene expression.

NOTCH3 Expression Is Induced in Mural Cells Through an Autoregulatory Loop That Requires Endothelial-Expressed JAGGED1

Endothelial cells and mural cells (smooth muscle cells, pericytes, or fibroblasts) are known to communicate with one another. Their interactions not only serve to support fully functional blood vessels but also can regulate vessel assembly and differentiation or maturation. In an effort to better understand the molecular components of this heterotypic interaction, we used a 3D model of angiogenesis and screened for genes, which were modulated by coculturing of these 2 different cell types. In doing so, we discovered that NOTCH3 is one gene whose expression is robustly induced in mural cells by coculturing with endothelial cells. Knockdown by small interfering RNA revealed that NOTCH3 is necessary for endothelial-dependent mural cell differentiation, whereas overexpression of NOTCH3 is sufficient to promote smooth muscle gene expression. Moreover, NOTCH3 contributes to the proangiogenic abilities of mural cells cocultured with endothelial cells. Interestingly, we found that the expression of NOTCH3 is dependent on Notch signaling, because the gamma-secretase inhibitor DAPT blocked its upregulation. Furthermore, in mural cells, a dominant-negative Mastermind-like1 construct inhibited NOTCH3 expression, and endothelial-expressed JAGGED1 was required for its induction. Additionally, we demonstrated that NOTCH3 could promote its own expression and that of JAGGED1 in mural cells. Taken together, these data provide a mechanism by which endothelial cells induce the differentiation of mural cells through activation and induction of NOTCH3. These findings also suggest that NOTCH3 has the capacity to maintain a differentiated phenotype through a positive-feedback loop that includes both autoregulation and JAGGED1 expression.

Functional Characterization and Transcriptome Analysis of Embryonic Stem Cell–Derived Contractile Smooth Muscle Cells

Complete transcriptome profiling of contractile smooth muscle cells (SMCs) differentiated from embryonic stem cells is crucial for the characterization of smooth muscle gene expression signatures and will contribute to defining biological and physiological processes in these cells. We have generated a transgenic embryonic stem cell line expressing both the puromycin acetyl transferase and enhanced green fluorescent protein cassettes under the control of the Acta2 promoter. Applying a specific monolayer culture protocol using retinoic acid, a puromycin-resistant and enhanced green fluorescent protein-positive Acta2(+) SMC population of 95% purity was isolated. Acta2(+) SMCs were characterized by semiquantitative and quantitative RT-PCR profiling of SMC markers and by microarray expression profiling, as well as by immunostaining for SMC-specific cytoskeletal proteins. Patch-clamp electrophysiological characterization of these cells identified SMC-specific channels such as the ATP-sensitive potassium channel and the Ca(2+)-activated potassium channel. Culturing of Acta2(+) SMCs in serum-containing medium resulted in a significant number of hypertrophic and binucleated cells failing to complete cell division. Functional characterization of the cells has been proved by stimulation of the cells with vasoactive agents, such as angiotensin II and endothelin. We concluded that our embryonic stem cell-derived SMC population possesses the contractile and hypertrophic phenotype of SMCs incapable of proliferation. This is the first study describing the complete transcriptome of ES-derived SMCs allowing identification of specific biological and physiological processes in the contractile phenotype SMCs and will contribute to the understanding of these processes in early SMCs derived from embryonic stem cells.

Mechanistic Systems Biology of Inflammatory Gene Expression in Airway Smooth Muscle as Tool for Asthma Drug Development

There is compelling evidence that airway smooth muscle cells may function as inflammatory cells in the airway system by producing multiple inflammatory cytokines in response to a large array of external stimuli such as acetylcholine, bradykinin, inflammatory cytokines, and toll-like receptor activators. However, how multiple extracellular stimuli interact in the regulation of inflammatory gene expression in an airway smooth muscle cell remains poorly understood. This review addresses the mechanistic systems biology of inflammatory gene expression in airway smooth muscle by discussing: a) redundancy underlying multiple stimulus-product relations in receptor-mediated inflammatory gene expression, and their regulation by convergent activation of Erk1/2 mitogen-activated protein kinase (MAPK), b) Erk1/2 MAPK-dependent induction of phosphatase expression as a negative feedback mechanism in the robust maintenance of inflammatory gene expression, and c) cyclooxygenase 2-dependent regulation of the differential temporal dynamics of early and late inflammatory gene expression. It is becoming recognized that a single-target approach is unlikely to be effective for the treatment of inflammatory airway diseases because airway inflammation is a result of complex interactions among multiple inflammatory mediators and cells types in the airway system. Understanding the mechanistic systems biology of inflammatory gene expression in airway smooth muscle and other cell types in the airway system may lead to the development of multi-target drug regimens for the treatment of inflammatory airway diseases such as asthma.