Expression In Smooth Muscle Cells Research Articles

The role of demineralized bone particle in a PLGA scaffold designed to create a media equivalent for a tissue engineered blood vessel

Poly(lactic-co-glycolic acid) (PLGA) is a copolymer used for scaffolds in tissue engineering due to its biodegradable and biocompatible properties. Demineralized bone particle (DBP) is a natural material containing bone morphogenic proteins (BMPs). The objective of this study was to determine if DBP induced differentiation of mesenchymal stem cells (MSCs) into a smooth muscle cells (SMCs) phenotype and act in a similar manner when treated with TGF-β1 as SMCs under the same conditions. PLGA scaffolds were fabricated with or without DBP using the solvent casting/salt leaching technique. Proliferation of MSCs and SMCs was measured using the MTT assay, while the expression of the smooth muscle-specific markers α-smooth muscle actin (α-SMA), smooth muscle 22 alpha (SM22α), and calponin was measured using RT-PCR and western blotting. Results showed PLGA with DBP scaffolds increased cell proliferation of SMCs (1 and 3 days) and MSCs (3 and 7 days) compared to the PLGA only scaffolds. Smooth muscle-specific genes were significantly increased in SMCs cultured for 7 days on PLGA with DBP scaffolds compared to PLGA only scaffolds and were also significantly increased when used SMC medium containing TGF-β1. Interestingly, the smooth muscle-specific genes were not altered in MSCs by the addition of TGF-β1 to the PLGA with DBP. However, in the 6-well and PLGA only groups, TGF-β1 addition caused increased levels of SM22α and calponin mRNA. Western blotting results were similar to the mRNA levels. These results suggest that the cell growth and gene expression of SMCs were up-regulated by DBP and TGF-β1 in PLGA scaffold; however, they were inadequate for the shift of MSCs into a SMCs phenotype.

Serum Response Factor Is Essential for Prenatal Gastrointestinal Smooth Muscle Development and Maintenance of Differentiated Phenotype.

Background/AimsSmooth muscle cells (SMCs) characteristically express serum response factor (SRF), which regulates their development. The role of SRF in SMC plasticity in the pathophysiological conditions of gastrointestinal (GI) tract is less characterized.MethodsWe generated SMC-specific Srf knockout mice and characterized the prenatally lethal phenotype using ultrasound biomicroscopy and histological analysis. We used small bowel partial obstruction surgeries and primary cell culture using cell-specific enhanced green fluorescent protein (EGFP) mouse lines to study phenotypic and molecular changes of SMCs by immunofluorescence, Western blotting, and quantitative polymerase chain reaction. Finally we examined SRF change in human rectal prolapse tissue by immunofluorescence.ResultsCongenital SMC-specific Srf knockout mice died before birth and displayed severe GI and cardiac defects. Partial obstruction resulted in an overall increase in SRF protein expression. However, individual SMCs appeared to gradually lose SRF in the hypertrophic muscle. Cells expressing low levels of SRF also expressed low levels of platelet-derived growth factor receptor alpha (PDGFRαlow) and Ki67. SMCs grown in culture recaptured the phenotypic switch from differentiated SMCs to proliferative PDGFRαlow cells. The immediate and dramatic reduction of Srf and Myh11 mRNA expression confirmed the phenotypic change. Human rectal prolapse tissue also demonstrated significant loss of SRF expression.ConclusionsSRF expression in SMCs is essential for prenatal development of the GI tract and heart. Following partial obstruction, SMCs down-regulate SRF to transition into proliferative PDGFRαlow cells that may represent a phenotype responsible for their plasticity. These findings demonstrate that SRF also plays a critical role in the remodeling process following GI injury.

THORACIC AORTIC DILATIONS IN PATIENTS WITH BICUSPID AORTIC VALVE DISEASE IS MARKED BY ACCELERATED SMOOTH MUSCLE CELL AGING

Individuals with a bicuspid aortic valve (BAV) are at increased risk for ascending aortic dilation and dissection. Loss of aortic medial smooth muscle cells (SMCs) and disruption of the extracellular matrix are well-recognized pathologies, but the underlying cellular mechanisms remain elusive. We tested the hypothesis that the dilated aorta in patients with BAV was marked by accelerated cellular aging. Samples of human ascending aorta were obtained from individuals with BAV undergoing thoracic aorta replacement (n=37, age 54.7±2.2, aortic diameter 4.8±0.9 cm) or patients with a tricuspid aortic valve and non-dilated aorta undergoing heart transplantation or coronary bypass procedures (n=8, age 55.3±8.1, aortic diameter 3.1±0.3 cm). Assessment of fresh aortic samples for senescence-associated β-galactosidase revealed evidence for rare medial cell senescence that was 4.2-fold more prevalent in dilated aortas (0.83±0.10%) than in non-dilated aortas (0.20±0.10%, p=0.048). Expression of p16 was abundantly detected in medial SMCs within dilated aortas (24.0±1.5%) and 3-fold more abundant than in non-dilated aortas (8.2±1.5%, p<0.0001). Interestingly, medial p16 expression did not correlate with telomere length of medial cells, as determined by quantitative and standardized PCR (p=0.35). Immunostaining for γH2A.X (phosphorylated Ser139) interestingly revealed discrete nuclear DNA double-strand breakage signals in 25.7±3.8% of medial cells in dilated aortas from patients with BAV, which was 2.3-fold higher than that found in non-dilated aortas (11.0±4.9, p=0.03). Medial cell outgrowths from BAV patients showed increased senescence-associated ß-galactosidase activity compared to control patient SMC outgrowths (38.9±2.9 vs. 19.5±1.9, p=0.0005). Furthermore, incubation of early passage SMCs with 50nM of angiotensin II for 3 days caused a 1.4-fold increase in senescence-associated ß-galactosidase expressing SMCs from BAV patients (p=0.0006), but elicited no change in senescence-associated ß-galactosidase expression in SMCs from control patients (p=0.83). These findings identify a previously unrecognized phenomenon of accelerated SMC aging in the aortas of patients with BAV, with cellular senescence and unresolved DNA breaks. Accelerated cell aging could thus be a driver of aortic wall degeneration in these patients and a potential therapeutic target.

Modulation of cGMP/cAMP crosstalk on the level of PDE3 in animals lacking NO-sensitive guanylyl cyclase

NO-sensitive guanylyl cyclase (NO-GC) is accepted to be the major receptor for the signaling molecule NO. Deletion of NO-GC in mice leads to disturbed NO/cGMP signaling. As a result, these mice show abolished NO-dependent relaxation of smooth muscle-containing tissues in the cardiovascular and gastrointestinal systems. Mice with general deletion (GCKO) suffer from increased blood pressure, reduced bleeding time, erectile dysfunction and die prematurely due to gastrointestinal dysmotility. Mice lacking NO-GC specifically in smooth muscle cells (SMC) show an elevated blood pressure similar to that seen in GCKO animals. Crosstalk between cGMP and cAMP signaling has been reported to occur in many different cells. Phosphodiesterase 3 (PDE3) is thought to be one of the prominent mediators of this crosstalk. Through its inhibitory action on PDE3, cGMP may regulate cAMP levels and cAMP-mediated signaling. Here, we reasoned that, in animals deficient in NO-GC, lack of NO-induced cGMP synthesis might affect cGMP/cAMP crosstalk on the level of PDE3. Smooth muscle relaxation induced by forskolin to stimulate adenylyl cyclase was similar in WT, GCKO and SMC-GCKO aortae. RIA-based detection of basal cAMP levels in aortic tissue from these mice did not reveal differences. Similarly, FRET measurements showed that PGE1-induced cAMP synthesis in isolated smooth muscle cells was unaffected by NO-GC deletion. However, aortae from GCKO and SMC-GCKO animals were more sensitive to milrinone, a specific inhibitor of PDE3, as the concentration curve for relaxation was shifted to the left. Analysis of PDE3 expression in aorta showed an approximately 50% reduction in PDE3 protein and specific enzyme activity. Similar reduction was detected in platelets but not in heart tissue of GCKO animals. Induction of SMC-specific NO-GC deletion by tamoxifen led to a parallel decline of NO-GC and PDE3 expression. Both proteins were strongly reduced already 5 days after the last tamoxifen injection. In contrast, systolic blood pressure in these animals started to rise only after 10 days and was maximal after 50 days. These data indicate that cGMP, not the increase in blood pressure, is responsible for the expression regulation of PDE3. We conclude that cGMP modulates PDE3 expression in SMC in order to keep up functional cAMP signaling.

XBP 1-Deficiency Abrogates Neointimal Lesion of Injured Vessels Via Cross Talk With the PDGF Signaling.

Smooth muscle cell (SMC) migration and proliferation play an essential role in neointimal formation after vascular injury. In this study, we intended to investigate whether the X-box-binding protein 1 (XBP1) was involved in these processes. In vivo studies on femoral artery injury models revealed that vascular injury triggered an immediate upregulation of XBP1 expression and splicing in vascular SMCs and that XBP1 deficiency in SMCs significantly abrogated neointimal formation in the injured vessels. In vitro studies indicated that platelet-derived growth factor-BB triggered XBP1 splicing in SMCs via the interaction between platelet-derived growth factor receptor β and the inositol-requiring enzyme 1α. The spliced XBP1 (XBP1s) increased SMC migration via PI3K/Akt activation and proliferation via downregulating calponin h1 (CNN1). XBP1s directed the transcription of mir-1274B that targeted CNN1 mRNA degradation. Proteomic analysis of culture media revealed that XBP1s decreased transforming growth factor (TGF)-β family proteins secretion via transcriptional suppression. TGF-β3 but not TGF-β1 or TGF-β2 attenuated XBP1s-induced CNN1 decrease and SMC proliferation. This study demonstrates for the first time that XBP1 is crucial for SMC proliferation via modulating the platelet-derived growth factor/TGF-β pathways, leading to neointimal formation.

Identification and characterization of intimal smooth muscle cell with innate immune capacity in atherosclerosis

Smooth muscle cells (SMC) contribute to the development and stability of atherosclerotic lesions. The phenotype transition and modulation of SMC is involved in many atherosclerotic pathological process. To date, more and more evidences indicated that innate immune response play important roles in atherosclerosis. As the first line of host defense and innate immunity, pathogen recognition receptors (PPR) have been shown to be involved in many mechanistic pathways in atherosclerosis. To investigate the role of innate immunity in SMC phenotype modulation in atherosclerosis, we analyzed PPR expression profile between different phenotypes of SMC and immune cells. Our data at the first time identified the high level of PPR expression in intimal SMC and characterized the comprehensive innate immune signatures. With comparison to other phenotypes of SMC and immune cells, we found intimal SMC possess a pro-inflammatory macrophage-like phenotype. To investigate the functional features and regulatory mechanism underlying the distinct phenotype, we used specific ligands for PRRs activation and showed that the innate immune capacity acquired by intimal SMC contribute to chemokine and cytokine secretion, migration promotion and extracellular matrix remodelling. Last but not least, we found the macrophage-like phenotype in intimal SMC and the pro-inflammatory capacity is associated with the elevated level of key transcription factors STAT1a, IRF8, IRF9, KLF4 and HIF2a. In conclusion, we propose a critical role of intimal SMC with innate immune capacity in the development of atherosclerosis. Targeting the phenotypic modulation of intimal SMC might be a new strategy for further clinical treatment.

Abstract 676: Microrna-30 and Cthrc1: Translating Aging into Vascular Stiffness and Abdominal Aortic Aneurysm

Abdominal aortic aneurysm (AAA) is a major source of vascular morbidity and mortality, with increasing age representing one of the strongest risk factors. While increases in aortic extra-cellular matrix deposition may be protective, changes in vessel architecture with age result in heterogeneous stiffness and vascular calcification, which appear to pre-dispose to AAA development. MicroRNAs (miRs) are key regulators of vascular homeostasis and pathobiology. Array profiling and qRT-PCR of aneurysmal aortic tissue in a murine AAA model (elastase-infused C57/B6) showed significant down-regulation of the miR-30 family, which is believed to have a role in vascular calcification. Increased age augmented this response, particularly for miR-30a, 30b and 30c. RNASeq profiling of a related AAA model (Ang-II in ApoE-/- KO) showed similar aortic down-regulation. Further, of differentially down-regulated miRs, miR-30 had the highest inverse correlation with mRNA gene targets. One predicted target - Cthrc1 (collagen triple helix repeat containing-1) - was the most consistently and significantly up-regulated gene across all time points in the AAA models. The protein resides within vascular smooth muscle cells (SMCs) and fibroblasts, responds to injury, and may regulate collagen expression and deposition. Immunofluorescence staining of AAA vs. normal aortas revealed increased expression of CTHRC1. We further found that miR-30 family members bind to the CTHRC1 3’ UTR and regulate gene expression in vitro, and that antagomir suppression of miR-30 upregulates CTHRC1 in SMCs. We also demonstrated that signaling pathways known to increase in activity with aging within the aorta, and which are associated with vascular calcification and fibrosis (e.g. IL6-based inflammatory signaling, TGF-β signaling, and BMP-2 signaling) down-regulate miR-30 family expression in SMCs, and inversely up-regulate CTHRC1 expression. Forced overexpression of miR-30 in SMC in vitro down-regulates RUNX2, a key promoter of vascular stiffness and calcification. Taken together, these results suggest a significant role for miR-30 in pathways related to matrix deposition and calcification, regulating aortic pathobiology and age-related susceptibility to AAA.

Estrogen Regulates the Expression of Small-Conductance Ca2+-Activated K+ Channels in Colonic Smooth Muscle Cells

Aim: This study aimed to determine the effects of small-conductance Ca²⁺-activated K⁺ (SK) channels in colonic relaxation and the regulation of SK channels by estrogen. Methods: The contractile activity of muscle strips from male rats was estimated, and drugs including vehicle (DMSO), 17β-estradiol (E2), or apamin (SK blocker) were added, respectively. In a further experiment, muscle strips were preincubated with apamin before exposure to E2. The levels of the SK2 and SK3 protein expression in the colonic smooth muscle cells (SMCs) were detected. SMCs were treated with ICI 182780 (estrogen receptor [ER] antagonist) plus E2, BSA-E2, PPT (ERα agonist), or DPN (ERβ agonist). SK3 mRNA and protein expression levels were detected. Results: The muscle strips responded to E2 with a decrease and to apamin with a transient increase in tension. Preincubation with apamin partially prevented E2-induced relaxation. Two SK channel subtypes, SK2 and SK3, were coexpressed with α-actin in colonic SMCs. The quantitative ratio of the SK transcriptional expression in colonic SMCs was SK3 > SK2. The SK3 expression was upregulated by E2, and was downregulated by ICI 182780, but was not influenced by BSA-E2. Furthermore, the effect of PPT on the expression of SK3 was almost the same as that of E2, while DPN did not influence the protein expression of SK3. Conclusion: These findings indicate that SK3 is involved in the E2-induced relaxing effect on rat colonic smooth muscle. Furthermore, E2 upregulates the expression of SK3 in rat SMCs, and that this effect is mediated via the ERα receptor.

Receptor-interacting protein kinase 3 contributes to abdominal aortic aneurysms via smooth muscle cell necrosis and inflammation.

Depletion of medial smooth muscle cell (SMC) is a major pathological characteristic of abdominal aortic aneurysm (AAA), although the mechanism by which these cells are eliminated remains incompletely understood. We reasoned that necroptosis, a recently described form of necrosis mediated by receptor-interacting protein kinase 3 (RIP3), may contribute to AAA pathology through the induction of SMC death and the significant production of inflammatory cytokines. To test the hypothesis that RIP3-mediated necroptosis is actively involved in aneurysm pathogenesis. RIP3 and RIP1 levels were found to be elevated in human AAAs, most noticeably in SMCs. Elevations of RIP3 and SMC necrosis were also observed in the elastase-induced mouse model of AAAs. Deletion of one or both copies of Rip3 prevented AAA formation. By transplanting Rip3(+/-) aortae to Rip3(+/+) mice, we demonstrated that reduced Rip3 expression in arterial wall was the primary cause of aneurysm resistance. In vitro, adenoviral overexpression of RIP3 was sufficient to trigger SMC necroptosis. Protein kinase C-delta contributed to tumor necrosis factor-α-induced SMC necroptosis by regulating Rip3 expression. Furthermore, Rip3 deficiency impaired tumor necrosis factor-α-induced inflammatory gene expression in aortic SMCs, which was at least in part because of attenuation of p65 Ser536 phosphorylation. In vivo, the lack of RIP3 diminished activation of p65 in SMCs, implicating a necrosis independent function of RIP3 in aneurysms. Enhanced RIP3 signaling in aneurysmal tissues contributes to AAA progression by causing SMC necroptosis, as well as stimulating vascular inflammation, and therefore may serve as a novel therapeutic target for AAA treatment.

Enhanced caveolin-1 expression in smooth muscle cells: Possible prelude to neointima formation.

To study the genesis of neointima formation in pulmonary hypertension (PH), we investigated the role of caveolin-1 and related proteins. Male Sprague Dawley rats were given monocrotaline (M, 40 mg/kg) or subjected to hypobaric hypoxia (H) to induce PH. Another group was given M and subjected to H to accelerate the disease process (M + H). Right ventricular systolic pressure, right ventricular hypertrophy, lung histology for medial hypertrophy and the presence of neointimal lesions were examined at 2 and 4 wk. The expression of caveolin-1 and its regulatory protein peroxisome proliferator-activated receptor (PPAR) γ, caveolin-2, proliferative and anti-apoptotic factors (PY-STAT3, p-Erk, Bcl-xL), endothelial nitric oxide synthase (eNOS) and heat shock protein (HSP) 90 in the lungs were analyzed, and the results from M + H group were compared with the controls, M and H groups. Double immunofluorescence technique was used to identify the localization of caveolin-1 in pulmonary arteries in rat lungs and in human PH lung tissue. In the M + H group, PH was more severe compared with M or H group. In the 4 wk M+H group, several arteries with reduced caveolin-1 expression in endothelial layer coupled with an increased expression in smooth muscle cells (SMC), exhibited neointimal lesions. Neointima was present only in the arteries exhibiting enhanced caveolin-1 expression in SMC. Lung tissue obtained from patients with PH also revealed neointimal lesions only in the arteries exhibiting endothelial caveolin-1 loss accompanied by an increased caveolin-1 expression in SMC. Reduction in eNOS and HSP90 expression was present in the M groups (2 and 4 wk), but not in the M + H groups. In both M groups and in the M + H group at 2 wk, endothelial caveolin-1 loss was accompanied by an increase in PPARγ expression. In the M + H group at 4 wk, increase in caveolin-1 expression was accompanied by a reduction in the PPARγ expression. In the H group, there was neither a loss of endothelial caveolin-1, eNOS or HSP90, nor an increase in SMC caveolin-1 expression; or any alteration in PPARγ expression. Proliferative pathways were activated in all experimental groups. Enhanced caveolin-1 expression in SMC follows extensive endothelial caveolin-1 loss with subsequent neointima formation. Increased caveolin-1 expression in SMC, thus, may be a prelude to neointima formation.

Temporal and Embryonic Lineage-Dependent Regulation of Human Vascular SMC Development by NOTCH3

Vascular smooth muscle cells (SMCs), which arise from multiple embryonic progenitors, have unique lineage-specific properties and this diversity may contribute to spatial patterns of vascular diseases. We developed in vitro methods to generate distinct vascular SMC subtypes from human pluripotent stem cells, allowing us to explore their intrinsic differences and the mechanisms involved in SMC development. Since Notch signaling is thought to be one of the several key regulators of SMC differentiation and function, we profiled the expression of Notch receptors, ligands, and downstream elements during the development of origin-specific SMC subtypes. NOTCH3 expression in our in vitro model varied in a lineage- and developmental stage-specific manner so that the highest expression in mature SMCs was in those derived from paraxial mesoderm (PM). This pattern was consistent with the high expression level of NOTCH3 observed in the 8–9 week human fetal descending aorta, which is populated by SMCs of PM origin. Silencing NOTCH3 in mature SMCs in vitro reduced SMC markers in cells of PM origin preferentially. Conversely, during early development, NOTCH3 was highly expressed in vitro in SMCs of neuroectoderm (NE) origin. Inhibition of NOTCH3 in early development resulted in a significant downregulation of specific SMC markers exclusively in the NE lineage. Corresponding to this prediction, the Notch3-null mouse showed reduced expression of Acta2 in the neural crest-derived SMCs of the aortic arch. Thus, Notch3 signaling emerges as one of the key regulators of vascular SMC differentiation and maturation in vitro and in vivo in a lineage- and temporal-dependent manner.

Pioglitazone inhibits high glucose-induced expression of receptor for advanced glycation end products in coronary artery smooth muscle cells.

Receptor for advanced glycation end products (RAGE) is critical in inflammatory diseases, including diabetes and atherosclerosis. The mechanism underlying the effect of peroxisome proliferator-activated receptor γ (PPARγ) agonist pioglitazone (PIO) on RAGE expression in coronary artery smooth muscle cells (SMCs) stimulated by high glucose concentrations remains to be elucidated. In the present study, the effect and mechanism of action of PIO on RAGE expression in SMCs was investigated following treatment with high glucose concentrations. Rat coronary artery SMCs were pretreated with PIO alone, PIO and GW9662 (a PPARγ antagonist), diphenyleneiodonium (DPI; a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor) or the antioxidant pyrrolidine dithiocarbamate (PDTC; a nuclear factor-κB (NF-κB) inhibitor), followed by treatment with high glucose. RAGE mRNA and protein expression, reactive oxygen species (ROS) production and NF-κB nuclear translocation were investigated. Glucose induced RAGE expression in a dose-dependent manner, with maximal effect at a concentration of 25 mmol/l following treatment for 48 h. PIO, DPI and PDTC reduced high glucose-induced increases in RAGE protein and mRNA expression. PIO prominently downregulated RAGE expression and inhibited high glucose-induced increases in ROS production and NF-κB activation (P<0.05). Pretreatment with PIO and GW9662 did not exhibit this inhibitory effect. High glucose may stimulate RAGE expression in coronary artery SMCs through NADPH oxidase-mediated ROS generation and NF-κB activation. PIO downregulated RAGE expression and inhibited ROS production and NF-κB activation via PPARγ activation, which may prevent the inflammatory effect of AGE/RAGE system in diabetes.

Differential effects of tumor necrosis factor-α on matrix metalloproteinase-2 expression in human myometrial and uterine leiomyoma smooth muscle cells.

Does tumor necrosis factor-α (TNF-α) differentially regulate matrix metalloproteinase-2 (MMP-2) expression in leiomyomas compared with normal myometrium? TNF-α up-regulates MMP-2 expression and stimulates cell migration through the activation of extracellular signal-regulated kinase (ERK) signaling pathway in leiomyoma smooth muscle cells (SMCs), but not in normal myometrial SMCs. Uterine leiomyoma, the benign smooth muscle cell tumor, is the single most common indication for hysterectomy. High expression of MMPs or TNF-α has been reported in uterine leiomyomas; however, the molecular mechanism underlying these observations remains unknown. Samples were obtained between 2009 and 2013 from 12 women of reproductive age at the proliferative phase of the menstrual cycle by hysterectomy. Leiomyomas and matched normal myometrium from each woman were analyzed in vitro. Western blot, RT-qPCR and a wound-healing assay were used to investigate the effects of TNF-α on MMP-2 expression and intracellular signal transduction in cultured SMCs from leiomyomas and matched myometrium. Western blot and RT-qPCR analyses using tissues from clinical patients showed that the levels of MMP-2 protein (P = 0.008) and mRNA (P = 0.009) were significantly higher in uterine leiomyomas compared with their matched myometrium. Treatment with TNF-α significantly up-regulated the protein (P = 0.039) and mRNA (P = 0.037) levels of MMP-2 in cultured leiomyoma SMCs but not in matched myometrial SMCs. The extracellular signal-regulated kinase (ERK) and nuclear factor-kappa B (NF-κB) pathways were activated by TNF-α in leiomyoma SMCs. Specific inhibitors of the ERK or NF-κB pathway (PD98059 or Bay11-7082) suppressed TNF-α-induced MMP-2 expression in leiomyoma SMCs. The wound-healing assay revealed that TNF-α promoted the migration of cultured leiomyoma SMCs (P = 0.036); however, PD98059 compromised the cell migration triggered by TNF-α. This study is descriptive and although we observed clear differential regulation of MMP-2 by TNF-α at mRNA and protein levels in leiomyoma, future studies are needed to identify why the difference in TNF-α response exists between human leiomyoma tissue and normal myometrium. Including some of the experiments such as transfection studies for TNF-α and MMP-2 promoter mapping could have added more insight as to why this difference exists. In addition, further studies in vivo are needed to verify the results obtained from primary cultured SMCs. Considering the positive effect of TNF-α on leiomyoma SMC migration, strategies targeting TNF-α, in parallel with the production of more specific inhibitors of MMPs, may provide alternative therapeutic approaches for the treatment of leiomyoma. This work was partially supported by grants from the Program for New Century Excellent Talents in University (NCET-12-0282), National Natural Science Foundation of China (81371620) and Tianjin Natural Science Foundation (12JCZDJC24900). The authors have no conflicts of interest to declare.

Perlecan Heparan Sulfate Is Required for the Inhibition of Smooth Muscle Cell Proliferation by All-trans-Retinoic Acid.

Smooth muscle cell (SMC) proliferation is a key process in stabilization of atherosclerotic plaques, and during restenosis after interventions. A clearer understanding of SMC growth regulation is therefore needed to design specific anti-proliferative therapies. Retinoic acid has been shown to inhibit proliferation of SMCs both in vitro and in vivo and to affect the expression of extracellular matrix molecules. To explore the mechanisms behind the growth inhibitory activity of retinoic acid, we hypothesized that retinoids may induce the expression of perlecan, a large heparan sulfate proteoglycan with anti-proliferative properties. Perlecan expression and accumulation was induced in murine SMC cultures by all-trans-retinoic acid (AtRA). Moreover, the growth inhibitory effect of AtRA on wild-type cells was greatly diminished in SMCs from transgenic mice expressing heparan sulfate-deficient perlecan, indicating that the inhibition is perlecan heparan sulfate-dependent. In addition, AtRA influenced activation and phosphorylation of PTEN and Akt differently in wild-type and mutant SMCs, consistent with previous studies of perlecan-dependent SMC growth inhibition. We demonstrate that AtRA regulates perlecan expression in SMCs and that the inhibition of SMC proliferation by AtRA is, at least in part, secondary to an increased expression of perlecan and dependent upon its heparan sulfate-chains.

BRG1 overexpression in smooth muscle cells promotes the development of thoracic aortic dissection.

BackgroundHere we investigated Brahma-related gene 1 (BRG1) expression in aortic smooth muscle cells (SMCs) and its role in the regulation of the pathological changes in aortic SMCs of thoracic arotic dissection (TAD).MethodsBRG1, matrix metalloproteinase 2 (MMP2), and MMP9 mRNA and protein expression in human aortic specimens were examined by qPCR and western blot, respectively. The percentage of apoptotic and contractile SMCs in aortic specimens were determined by TUNEL assay and α-SMA immunohistochemical staining, respectively. The role of BRG1 in MMP2 and MMP9 expression, cell apoptosis, and phenotype transition in aortic SMCs were investigated using a human aortic SMC line via adenovirus mediated gene transfer. MMPs mRNA and protein levels were analyzed by qPCR and western blot, respectively. The percentage of apoptotic and contractile cells were determined through flow cytometry analysis.ResultsThe expression level of BRG1 in the aortic walls (adventitia-removed) was significantly higher in the TAD than the normal group. BRG1 expression was positively correlated to expression of MMP2 and MMP9 and SMC apoptosis, but was negatively correlated to the percentage of contractile aortic SMCs in TAD specimens. In human aortic SMC line, BRG1 transfection led to significant upregulation of MMP2 and MMP9 expression and a concomitant increase in SMC apoptosis as well as a decrease in the percentage of contractile phenotype of cells.ConclusionsBRG1 is significantly upregulated in the aortic SMCs of TAD, and its overexpression might promote the development of TAD by increasing MMP2 and MMP9 expression, inducing SMC apoptosis and the transition from contractile to synthetic phenotype.

OP1-6 - Induction of antioxidant genes by sulforaphane and klotho in human aortic smooth muscle cells

Vascular smooth muscle cell (SMC) dysfunction due to enhanced oxidative stress contributes to age-related cardiovascular diseases such as atherosclerosis and calcification. Klotho, a protein with multiple pleiotropic effects associated with anti-ageing, can extend lifespan when overexpressed while its deficiencies result in rapid aging. It exists in membrane and secreted forms with distinct pleiotropic functions, with the secreted form regulating ion channels, suppressing growth factor signaling and oxidative stress while the transmembrane protein forms a co-receptor for FGF23, although the mechanisms involved in its actions remain to be fully elucidated. Sulforaphane (SFN) is an isothiocyanate present in cruciferous vegetables that can induce antioxidant defence enzymes such as heme oxygenase-1 (HO-1) and peroxiredoxin-1 (Prx-1). The present study investigates whether SFN or klotho increases antioxidant defences in cultured human aortic SMC. Cells were treated (0-24h) with either SFN (0-5µM) or klotho (0-1nM) and HO-1, Prx-1 expression determined by western blot analyses and glutathione (GSH) levels measured using the o-phthalaldehyde fluorescence assay. SFN significantly (p<0.05, n=3) enhanced both HO-1 and Prx-1 protein expression and GSH levels in SMC at 12 and 24h while klotho treatments significantly (p<0.05, n=3) augmented HO-1 and Prx-1 expression only at 24h but significantly increased GSH levels after both 8h and 24h. Interestingly preliminary data suggest that treatment of SMC with SFN for 12 or 24h enhances the expression of klotho. Taken together, these findings demonstrate that both SFN and klotho can enhance antioxidant defences which may protect against vascular SMC dysfunction in age-related cardiovascular diseases.Supported by the British Heart Foundation, Heart Research UK and the 'Marco Polo' Program, University of Bologna.

Abstract 052: Alpha1a-Adrenoceptor Genetic Variant-Triggered Hyperproliferation in Cardiovascular Cells is Mediated by Novel Interacting Protein Spinophilin

Objectives: Alpha1-Adrenergic Receptors (α1ARs), members of the G protein-coupled receptor (GPCR) superfamily, play a major role in regulating cardiovascular (CV) function. Recently, we discovered that naturally occurring human α1aAR-G247R (247R) genetic variant, identified in the 3rd intracellular loop (3iL) of the receptor in highly hypertensive patient, triggers constitutive hyperproliferation in fibroblasts, cardiomyoblasts and smooth muscle cells (SMC). Specific proteins mediating this signaling remain unknown. Spinophilin (SPL) is a ubiquitously expressed protein controlling GPCR signaling by binding its 3rd intracellular loop (3iL). We hypothesize that SPL mediates α1aAR signaling and examined whether SPL directly interacts with α1aAR-WT (WT) and 247R. Methods: Cells were co-transfected with HA-α1ARs and full length Myc-SPL or its fragments to determine SPL domains responsible for binding to α1ARs. Cell lysates were co-immunoprecipitated with HA-tag antibodies. SPL levels were analyzed by Western blotting. SPL knockdown experiments were performed by transiently transfecting cells with SPL or scrambled siRNA, cell proliferation was determined by cell counting. Results: We demonstrate a distinct interaction of SPL with WT and 247R, WT interaction being the strongest. Different domains of SPL differentially interact with WT or 247R. SPL 1-480aa fragment interacts stronger with WT indicating interaction with 3iL, while SPL 480-817 fragment interacts stronger with 247R. Endogenous SPL levels are increased in 247R cells compared to WT or control cells. Inhibition of SPL expression in SMC with siRNA reduces 247R-triggered hyperproliferation to near normal levels and has no effect in WT cells. Conclusions: We identified SPL as a novel interacting protein involved in mediating intracellular signaling of α1aAR and its genetic variant in CV cells. Different domains of SPL differentially bind to WT or 247R indicating that SPL has a distinct role in regulation of their signaling pathways. Our findings also reveal that SPL is critical for 247R-triggered EGFR transactivation pathways. Thus SPL could be considered as a potentially novel target in α1aAR-mediated cardiovascular disorders.

CGMP-dependent protein kinase I gamma encodes a nuclear localization signal that regulates nuclear compartmentation and function

cGMP-dependent protein kinase I (PKGI) plays an important role in regulating how cGMP specifies vascular smooth muscle cell (SMC) phenotype. Although studies indicate that PKGI nuclear localization controls how cGMP regulates gene expression in SMC, information about the mechanisms that regulate PKGI nuclear compartmentation and its role in directly regulating cell phenotype is limited. Here we characterize a nuclear localization signal sequence (NLS) in PKGIγ, a proteolytically cleaved PKGI kinase fragment that translocates to the nucleus of SMC. Immuno-localization studies using cells expressing native and NLS-mutant PKGIγ, and treated with a small molecule nuclear transport inhibitor, indicated that PKGIγ encodes a constitutively active NLS that requires importin α and β for regulation of its compartmentation. Moreover, studies utilizing a genetically encoded nuclear phospho-CREB biosensor probe and fluorescence lifetime imaging microscopy demonstrated that this NLS controls PKGIγ nuclear function. In addition, although cytosolic PKGIγ-activity was observed to stimulate MAPK/ERK-mediated nuclear CREB signaling in SMC, NLS-mediated PKGIγ nuclear activity alone was determined to increase the expression of differentiation marker proteins in these cells. These results indicate that NLS-mediated nuclear PKGIγ localization plays an important role in how PKGI regulates vascular SMC phenotype.

Differential expression of caveolin-1 in human myometrial and uterine leiomyoma smooth muscle

Uterine leiomyomas, the most common neoplasms of the female genital tract, are benign tumors of the uterus arising from the smooth muscle cells (SMCs) of the myometrium with an involvement of estrogen. Caveolin-1 (Cav-1), a major protein component in caveolae membrane lipid rafts, is down-regulated in several estrogen-related cancer cells, and overexpression of Cav-1 inhibits proliferation of cancer cells and vascular SMCs as well. Therefore, we hypothesize that Cav-1 is down-regulated in human uterine leiomyoma. Western blot using tissues from clinical patients showed that Cav-1 expression was significantly lower or undetectable in uterine leiomyoma compared with their matched myometrium (P < .001). This finding was confirmed by immunohistochemistry and confocal microscopy. The cav-1 mRNA level in uterine leiomyomas was also significantly lower as detected by reverse transcription-quantitative polymerase chain reaction analysis (P = .001). To further study the underlying mechanism, we performed primary cell culture, and found that the expression of Cav-1 remained low in cultured leiomyoma SMCs (P = .009). Serum withdrawal did not change Cav-1 expression in leiomyoma SMCs, but increased expression in myometrial SMCs (P = .006). 17-β estradiol inhibited the expression of Cav-1 protein (P = .047) and mRNA (P = .007) in leiomyoma SMCs, whereas it stimulated expression in myometrial SMCs (P = .043). 17-β estradiol, although activating the mitogen-activated protein kinase pathway in both SMCs, did not stimulate their proliferation. We conclude that human uterine leiomyomas in vitro express low levels of Cav-1, which may result from estrogen inhibition. This effect of estrogen may contribute to the pathogenesis of uterine leiomyoma. Further studies in vivo are needed to verify these results.

Abstract 84: Deletion of the Type II TGF-ß Receptor in Smooth Muscle Causes Severe Aortopathy in Mice

Introduction: Prenatal deletion of the type II TGF-beta receptor (TBRII) prevents normal vascular morphogenesis, impairs smooth muscle cell (SMC) differentiation and matrix accumulation, and results in embryonic death in mid-gestation (~embryonic day 13-14). However, the role of TBRII in adult SMC is unknown. Clarification of the postnatal role of TBRII has important clinical implications because TBRII deletion should ablate TGF-beta signaling and blockade of TGF-beta signaling is currently envisioned as a treatment for human aortopathies. Hypothesis: We hypothesized that postnatal deletion of TBRII in SMC would impair aortic homeostasis, resulting in aortopathy. Methods: We bred mice with a tamoxifen-inducible SMC-specific Cre allele and a floxed Tgfbr2 allele. Mice (6-19 wk old) were injected with tamoxifen or vehicle, and euthanized 4-14 wk later. Aortic mRNA and protein were extracted and Tgfbr2 mRNA transcripts and TBRII protein measured. Aortas were examined grossly after a thoracoabdominal incision, and a subset was examined histologically using both H&amp;E and VVG stains. Results: There were no sudden deaths. Tamoxifen-treated doubly transgenic mice had a ~85% decrease in aortic Tgfbr2 transcripts and decreased TBRII protein on western blot. Tamoxifen-treated doubly transgenic mice exhibited several aortic pathologies, none of which were present either in vehicle-treated transgenic or tamoxifen-treated nontransgenic mice: aortic intramural hematoma (19 of 69), rare frank aortic dissection with blood in a large false lumen, penetrating aortic ulcers (7 of 9), medial thickening (46% increase; P &lt; 0.001), aortic dilation, and adventitial thickening. Most of these pathologies were confined to the ascending aorta and arch. In descending thoracic aortas we observed occasional dilation (grossly) and consistent adventitial thickening (on histologic sections). No definite pathology was observed in abdominal aortas. Conclusions: TBRII expression in SMC plays a critical role in maintaining postnatal aortic homeostasis. Because loss of TBRII disrupts TGF-beta signaling, these results suggest that blockade of TGF-beta signaling in humans may cause aortic disease rather than prevent it.