Expression In Smooth Muscle Cells Research Articles

Oxidized low-density lipoprotein induced hepatoma-derived growth factor upregulation mediates foam cell formation of cultured rat aortic vascular smooth muscle cells

Vascular smooth muscle cells (SMCs) are important vascular components that are essential for the regulation of vascular functions during vascular atherosclerogenesis and vascular injury. Oxidized low-density lipoprotein (oxLDL) is known to induce SMC activation and foam cell transformation. This study characterized the role of hepatoma-derived growth factor (HDGF) in oxLDL-induced foam cell formation in cultured primary rat aortic SMCs. OxLDL exposure significantly increased HDGF expression and extracellular release. It also upregulated atherogenic regulators in SMCs, including TLR4, MyD88, LOX-1, and CD36. Exogenous HDGF stimulation not only increased the expression of cognate receptor nucleolin, but also the innate immunity regulators TLR4/MyD88 and lipid metabolism regulators, including LOX-1 and CD36. Oil red O staining showed that HDGF did not initiate, but enhanced oxLDL-driven foam cell formation in SMCs. Further signaling characterization demonstrated that oxLDL evoked activation of PI3K/Akt and p38 MAPK signaling pathways, both of which were involved in the upregulation of HDGF, LOX-1, and CD36 induced by oxLDL. Gene knockdown experiments using LOX-1 targeted siRNA demonstrated that LOX-1 expression was critical for oxLDL-induced HDGF upregulation, while HDGF gene depletion completely abolished oxLDL-triggered TLR4, LOX-1, and CD36 overexpression and foam cell formation in SMCs. These findings strongly suggest that oxLDL-induced HDGF upregulation participates in subsequent LOX-1 and CD36 expression in aortic SMCs and mechanistically contributes to the formation of SMC-derived foam cells. The oxLDL/LOX-1/HDGF axis may serve as a target for anti-atherogenesis therapy.

Roles of vascular endothelial and smooth muscle cells in the vasculoprotective effect of insulin in a mouse model of restenosis.

Background:Insulin exerts vasculoprotective effects on endothelial cells (ECs) and growth-promoting effects on vascular smooth muscle cells (SMCs) in vitro, and suppresses neointimal growth in vivo. Here we determined the role of ECs and SMCs in the effect of insulin on neointimal growth.Methods:Mice with transgene CreERT2 under the control of EC-specific Tie2 (Tie2-Cre) or SMC-specific smooth muscle myosin heavy chain promoter/enhancer (SMMHC-Cre) or littermate controls were crossbred with mice carrying a loxP-flanked insulin receptor (IR) gene. After CreERT2-loxP-mediated recombination was induced by tamoxifen injection, mice received insulin pellet or sham (control) implantation, and underwent femoral artery wire injury. Femoral arteries were collected for morphological analysis 28 days after wire injury.Results:Tamoxifen-treated Tie2-Cre+ mice showed lower IR expression in ECs, but not in SMCs, than Tie2-Cre− mice. Insulin treatment reduced neointimal area after arterial injury in Tie2-Cre− mice, but had no effect in Tie2-Cre+ mice. Tamoxifen-treated SMMHC-Cre+ mice showed lower IR expression in SMCs, but not in ECs, than SMMHC-Cre− mice. Insulin treatment reduced neointimal area in SMMHC-Cre− mice, whereas unexpectedly, it failed to inhibit neointima formation in SMMHC-Cre+ mice.Conclusion:Insulin action in both ECs and SMCs is required for the “anti-restenotic” effect of insulin in vivo.

No direct involvement of Phactr-1 in non-atherosclerotic arteriopathies: Results from 3 different Phactr-1 transgenic knockout mice

Genome-wide association studies have revealed robust associations of common genetic polymorphisms in the Phactr1 locus (6p24) (encoding phosphatase and actin regulator 1) with cervico-cerebral artery dissection (CCeAD), spontaneous coronary artery dissection (SCAD) and fibromuscular dysplasia (FMD). These common elements of genetic predisposition provide a proof of concept for important shared mechanisms at the molecular level between CCeAD, SCAD and FMD vascular diseases. Deciphering genetic, molecular and physiological importance of PHACTR1. Using various tissue-specific Cre-driver mouse lines, Phactr1 was deleted either in endothelial cells (EC) using 2 tissue-specific Cre-driver (one of them was embryonic) and smooth muscle cells (SMC) with a third tissue-specific Cre-driver to assess its role in the pathogenesis of CCeAD, SCAD or FMD. To test the efficacy of the Phactr1 deletion after cre-induction, we confirmed firstly, a decrease in Phactr1 transcription and Phactr1 expression in EC and SMC isolated from Phactr1 iPDGFB and Phactr1 iSMA mice. Irrespective to the tissue or the duration of the deletion, mice did not spontaneously display pathological phenotype or vascular impairment: mouse survival, growth, blood pressure, large vessel morphology or actin organization were not different in KO mice than their comparatives littermates. Challenging vascular function and repair either by angiotensin-II-induced hypertension or limb ischemia did not lead to vascular morphology or function impairment in Phactr1-deleted mice. Similarly, there was no more consequences of Phactr1 deletion during embryogenesis in ECs. Loss of function of PHACTR-1 in the cell types mostly involved in vascular physiology does not appear to induce a pathological vascular phenotype.

C-Kit expression in smooth muscle cells reduces atherosclerosis burden in hyperlipidemic mice

Background and aimsIncreased receptor tyrosine kinase (RTK) activity has been historically linked to atherosclerosis. Paradoxically, we recently found that global deficiency in c-Kit function increased atherosclerosis in hyperlipidemic mice. This study aimed to investigate if such unusual atheroprotective phenotype depends upon c-Kit's function in smooth muscle cells (SMC). MethodsWe studied atherosclerosis in a SMC-specific conditional knockout mice (KitSMC) and control littermate. Tamoxifen (TAM) and vehicle treated mice were fed high fat diet for 16 weeks before atherosclerosis assessment in the whole aorta using oil red staining. Smooth muscle cells were traced within the aortic sinus of conditional c-Kit tracing mice (KitSMC eYFP) and their control littermates (KitWT eYFP) by immunofluorescent confocal microscopy. We then performed RNA sequencing on primary SMC from c-Kit deficient and control mice, and identified significantly altered genes and pathways as a result of c-Kit deficiency in SMC. ResultsAtherosclerosis significantly increased in KitSMC mice with respect to control groups. In addition, the loss of c-Kit in SMC increased plaque size and necrotic core area in the aortic sinus of hyperlipidemic mice. Smooth muscle cells from KitSMC eYFP mice were more prone to migrate and express foam cell markers (e.g., Mac2 and MCAM) than those from control littermate animals. RNAseq analysis showed a significant upregulation in genes associated with cell proliferation, migration, lipid metabolism, and inflammation secondary to the loss of Kit function in primary SMCs. ConclusionsLoss of c-Kit increases SMC migration, proliferation, and expression of foam cell markers in atherosclerotic plaques from hyperlipidemic mice.

Deleterious impact of nerve growth factor precursor (proNGF) on bladder urothelial and smooth muscle cells

The nerve growth factor precursor (proNGF) activates p75NTR receptor and promotes cell death in different tissues, yet this pathophysiological effect is not fully described in the bladder. The aim of this study was to identify the biological effect of proNGF/p75NTR activation on urothelial and smooth muscle (SM) cells of rodents' bladder. Cell viability was assessed by MTT assay which showed a significant reduction in urothelial viability after 24 h of incubation with proNGF in culture medium [5 or 10 nM], an effect not seen in SM cells. Western blot analysis on cellular protein extracts showed increased expression of the transmembrane TNF-α and activation of RhoA in urothelial cells exposed to proNGF with no evidence of a nuclear translocation of NF-κB assessed by western blotting on nuclear extracts and immunofluorescence. The activation of p75NTR-death domain related pathways in urothelial cells such as TNF-α or RhoA had a downstream effect on NO release and the junctional protein occludin, as estimated respectively by colorimetric and western blotting. On the other hand, proNGF did not induce TNF-α or RhoA expression in SM cells, but induced a significant NF-κB nuclear translocation. ProNGF had a different impact on SM as evidenced by a significant dose- and time-dependent increase in SM proliferation and migration examined by MTT test and cell migration assay. Together, our results indicate that activation of proNGF/p75NTR axis induces degenerative changes to the urothelial layer impacting its barrier and signaling integrity, while promoting adaptive proliferative changes in detrusor SM cells that can interfere with the contractile phenotype essential for proper bladder function.

AAV-mediated AP-1 decoy oligonucleotide expression inhibits aortic elastolysis in a mouse model of Marfan syndrome.

Marfan syndrome is one of the most common inherited disorders of connective tissue caused by fibrillin-1 mutations, characterized by enhanced transcription factor AP-1 DNA binding activity and subsequently abnormally increased expression and activity of matrix-metalloproteinases (MMPs). We aimed to establish a novel adeno-associated virus (AAV)-based strategy for long-term expression of an AP-1 neutralizing RNA hairpin (hp) decoy oligonucleotide (dON) in the aorta to prevent aortic elastolysis in a murine model of Marfan syndrome. Using fibrillin-1 hypomorphic mice (mgR/mgR), aortic grafts from young (9 weeks old) donor mgR/mgR mice were transduced ex vivo with AAV vectors and implanted as infrarenal aortic interposition grafts in mgR/mgR mice. Grafts were explanted after 30 days. For in vitro studies, isolated primary aortic smooth muscle cells (SMCs) from mgR/mgR mice were used. Elastica-van-Giesson staining visualized elastolysis, reactive oxygen species (ROS) production was assessed using dihydroethidine staining. RNA F.I.S.H. verified AP-1 hp dON generation in the ex vivo transduced aortic tissue. MMP expression and activity were assessed by western blotting and immunoprecipitation combined with zymography.Transduction resulted in stable therapeutic dON expression in endothelial and SMCs. MMP expression and activity, ROS formation as well as expression of monocyte chemoattractant protein-1 were significantly reduced. Monocyte graft infiltration declined and the integrity of the elastin architecture was maintained. RNAseq analysis confirmed the beneficial effect of AP-1 neutralization on the pro-inflammatory environment in SMCs. This novel approach protects from deterioration of aortic stability by sustained delivery of nucleic acids-based therapeutics and further elucidated how to interfere with the mechanism of elastolysis.

Surfactant Protein A, a Novel Regulator for Smooth Muscle Phenotypic Modulation and Vascular Remodeling-Brief Report.

The objective of this study is to determine the role of SPA (surfactant protein A) in vascular smooth muscle cell (SMC) phenotypic modulation and vascular remodeling. Approach and Results: PDGF-BB (Platelet-derived growth factor-BB) and serum induced SPA expression while downregulating SMC marker gene expression in SMCs. SPA deficiency increased the contractile protein expression. Mechanistically, SPA deficiency enhanced the expression of myocardin and TGF (transforming growth factor)-β, the key regulators for contractile SMC phenotype. In vivo, SPA was induced in medial and neointimal SMCs following mechanical injury in both rat and mouse carotid arteries. SPA knockout in mice dramatically attenuated the wire injury-induced intimal hyperplasia while restoring SMC contractile protein expression in medial SMCs. These data indicate that SPA plays an important role in SMC phenotype modulation and vascular remodeling in vivo. SPA is a novel protein factor modulating SMC phenotype. Blocking the abnormal elevation of SPA may be a potential strategy to inhibit the development of proliferative vascular diseases.

Lack of PI 3-kinase isoform p110alpha in smooth muscle cells impairs aortic wall homoeostasis and thus promotes aortic aneurysm formation

Abstract Background In smooth muscle cells (SMCs), the PI 3-kinase isoform p110α mediates receptor tyrosine kinase dependent proliferation, chemotaxis and cell survival. Since mice, harbouring a smooth muscle specific p110α deficiency (SM-p110α−/−), display reduced vascular wall thickness, we hypothesized that SM-p110α−/− mice might be prone to aortic aneurysm (AA) formation. The pathogenesis of AA is characterized by increased dedifferentiation of SMCs, extracellular matrix (ECM) degeneration and inflammation in the aortic wall. Herein, we investigated how p110α-dependent signal transduction in SMCs affects these processes. Methods and results We examined AA formation in SM-p110α−/− mice and wild-type littermates using the “porcine pancreatic elastase” (PPE) AA model. PPE was infused into the infrarenal aorta to induce AA formation. Ultrasound examination of the aorta revealed an enlarged aortic diameter in all PPE-treated mice. The aortic diameter in SM-p110α−/− mice (0.46±0.12 mm) was significantly increased compared to wild-type animals (0.18±0.03 mm, p<0.01). These data indicate a protective function of p110α in AA formation. Immunocytochemical examination of the tunica media of PPE-perfused SM-p110α−/− mice revealed significantly increased infiltration of CD45+ leukocytes. In particular, the number of MOMA-2+ monocytes / macrophages in the vessel wall was significantly increased indicating elevated inflammation of the aortic wall during AA progression in comparison to wild-type control mice. Ultrastructural analysis of aortic wall morphology in SM-p110α−/− mice using transmission electron microscopy (TEM) showed a deranged tunica media and increased apoptotic cell death. In addition, the media thickness in the abdominal aorta was significantly reduced in SM-p110α−/− mice (29.0±3.1 μm vs. 42.5±4.1 μm). Western blots demonstrated a reduced elastin and fibrillin expression in SMCs from SM-p110α−/− mice. p110α−/− SMCs showed significantly reduced expression of differentiation markers SM-α-actin and SM-MHC. In addition, aortic p110α-deficient SMCs were significantly impaired in their ability to proliferate and migrate. These findings indicate that p110α−/− SMCs are neither differentiated nor dedifferentiated and have therefore largely lost their plasticity. Consequently, p110α deficiency significantly diminished responsiveness of aortic rings to vasodilator acetylcholine and NO-donor nitroglycerin, further indicating impaired contractility of SMCs. Mechanistically, we demonstrated that PDGF and insulin induced phosphorylation and inactivation of key regulators of SMC differentiation and dedifferentiation, Foxo4 and GSK3b, respectively, were abrogated in p110α−/− SMCs. Conclusion These data show that deficiency of p110α in SMCs promotes the formation and progression of AA. Causative are impaired SMC plasticity and ECM homeostasis as well as inflammatory processes in the vascular wall. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft (DFG)

High Throughput Screen Identifies the DNMT1 (DNA Methyltransferase-1) Inhibitor, 5-Azacytidine, as a Potent Inducer of PTEN (Phosphatase and Tensin Homolog): Central Role for PTEN in 5-Azacytidine Protection Against Pathological Vascular Remodeling.

Our recent work demonstrates that PTEN (phosphatase and tensin homolog) is an important regulator of smooth muscle cell (SMC) phenotype. SMC-specific PTEN deletion promotes spontaneous vascular remodeling and PTEN loss correlates with increased atherosclerotic lesion severity in human coronary arteries. In mice, PTEN overexpression reduces plaque area and preserves SMC contractile protein expression in atherosclerosis and blunts Ang II (angiotensin II)-induced pathological vascular remodeling, suggesting that pharmacological PTEN upregulation could be a novel therapeutic approach to treat vascular disease. Approach and Results: To identify novel PTEN activators, we conducted a high-throughput screen using a fluorescence based PTEN promoter-reporter assay. After screening ≈3400 compounds, 11 hit compounds were chosen based on level of activity and mechanism of action. Following in vitro confirmation, we focused on 5-azacytidine, a DNMT1 (DNA methyltransferase-1) inhibitor, for further analysis. In addition to PTEN upregulation, 5-azacytidine treatment increased expression of genes associated with a differentiated SMC phenotype. 5-Azacytidine treatment also maintained contractile gene expression and reduced inflammatory cytokine expression after PDGF (platelet-derived growth factor) stimulation, suggesting 5-azacytidine blocks PDGF-induced SMC de-differentiation. However, these protective effects were lost in PTEN-deficient SMCs. These findings were confirmed in vivo using carotid ligation in SMC-specific PTEN knockout mice treated with 5-azacytidine. In wild type controls, 5-azacytidine reduced neointimal formation and inflammation while maintaining contractile protein expression. In contrast, 5-azacytidine was ineffective in PTEN knockout mice, indicating that the protective effects of 5-azacytidine are mediated through SMC PTEN upregulation. Our data indicates 5-azacytidine upregulates PTEN expression in SMCs, promoting maintenance of SMC differentiation and reducing pathological vascular remodeling in a PTEN-dependent manner.

453-P: Cell-Specific Role of Insulin in an In Vivo Model of Restenosis

The effects of insulin on the vasculature remains a hotly debated and controversial topic. Its role on endothelial cells (ECs) is thought to be vasculoprotective, whereas its effects on vascular smooth muscle cells (SMCs) are mainly mitogenic according to in vitro data. We have previously demonstrated a suppressive effect of insulin treatment on neointimal growth in rodent models of restenosis under conditions of normal insulin sensitivity that was abolished in insulin resistant conditions. The objective of this study was to determine the endothelial-and SMC-specific effects of insulin treatment on neointimal growth in a murine model of restenosis. Mice were generated by crossing loxP-flanked insulin receptor (IR) mice with mice overexpressing tamoxifen-inducible Cre recombinase under the control of Tie2 (endothelial-specific receptor tyrosine kinase) or SMMHC (smooth muscle myosin heavy chain) promoters. Mice were fed with standard chow and implanted with insulin pellet (0.1 U/day) or sham (control) three days prior to undergoing femoral artery wire injury. Lastly, femoral arteries were collected for morphological analysis 28 days after wire injury. Tamoxifen-induced Tie2-Cre+ mice showed decreased IR expression in ECs compared with Tie2-Cre-, with no change in IR expression in SMCs. Neointimal area in Tie2-Cre- mice was reduced following insulin treatment, whereas it was unaffected in Tie2-Cre+ mice. Similarly, tamoxifen-induced SMMHC-Cre+ mice showed a reduction in IR expression in SMCs, but not in the ECs, compared to SMMHC-Cre- mice. In SMMHC-Cre- mice, neointimal area was decreased with insulin treatment; however, insulin surprisingly failed to have the same effects in SMMHC-Cre+ mice. These data demonstrate that insulin action in both ECs and SMCs is required for the antirestenotic effect of insulin in insulin sensitive conditions. Disclosure M. Gonzalez: None. Y. Mori: Research Support; Self; Taisho Pharmaceutical Co., Ltd. J. Guo: None. L.S. Dingwell: None. A. Giacca: None.

Meprin‐α (Mep1A) enhances TNF‐α secretion by mast cells and aggravates abdominal aortic aneurysms

Abdominal aorticaneurysm (AAA) rupture is mainly due to elastic lamina degradation. As a metalloendopeptidase, meprin-α (Mep1A) critically modulates the activity of proteins and inflammatory cytokines in various diseases. Here, we sought to investigate the functional role of Mep1A in AAA formation and rupture. AAA tissues were detected by using real-time PCR (RT-PCR), western blotting (WB), and immunohistochemistry. Further mechanistic studies used RT-PCR, WB, and enzyme-linked immunosorbent assays. Mep1A mediated AAA formation by regulating the mast cell (MC) secretion of TNF-α, which promoted matrix metalloproteinase (MMP) expression and apoptosis in smooth muscle cells (SMCs). Importantly, increased Mep1A expression was found in human AAA tissues and in angiotensin II-induced mouse AAA tissues. Mep1A deficiency reduced AAA formation and increased the survival rate of AAA mice. Pathological analysis showed that Mep1A deletion decreased elastic lamina degradation and SMC apoptosis in AAA tissues. Furthermore, Mep1A was expressed mainly in MCs, wherein it mediated TNF-α expression. Mep1A inhibitor actinonin significantly inhibited TNF-α secretion in MCs. TNF-α secreted by MCs enhanced MMP2 expression in SMCs and promoted SMC apoptosis. Taken together, these data suggest that Mep1A may be vital in AAA pathophysiology by regulating TNF-α production by MCs. Knocking out Mep1A significantly decreased AAA diameter and improved AAA stability in mice. Therefore, Mep1A is a potential new therapeutic target in the development of AAA.

Regulation of KCNMA1 transcription by Nrf2 in coronary arterial smooth muscle cells

The large conductance Ca2+-activated K+ (BK) channels, composed of the pore-forming α subunits (BK-α, encoded by KCNMA1 gene) and the regulatory β1 subunits (BK-β1, encoded by KCNMB1 gene), play a unique role in the regulation of coronary vascular tone and myocardial perfusion by linking intracellular Ca2+ homeostasis with excitation-contraction coupling in coronary arterial smooth muscle cells (SMCs). The nuclear factor erythroid 2-related factor 2 (Nrf2) belongs to a member of basic leucine zipper transcription factor family that regulates the expression of antioxidant and detoxification enzymes by binding to the antioxidant response elements (AREs) of these target genes. We have previously reported that vascular BK-β1 protein expression was tightly regulated by Nrf2. However, the molecular mechanism underlying the regulation of BK channel expression by Nrf2, particularly at transcription level, is unknown. In this study, we hypothesized that KCNMA1 and KCNMB1 are the target genes of Nrf2 transcriptional regulation. We found that BK channel protein expression and current density were diminished in freshly isolated coronary arterial SMCs of Nrf2 knockout (KO) mice. However, BK-α mRNA expression was reduced, but not that of BK-β1 mRNA expression, in the arteries of Nrf2 KO mice. Promoter-Nrf2 luciferase reporter assay confirmed that Nrf2 binds to the ARE of KCNMA1 promoter, but not that of KCNMB1. Adenoviral expression and pharmacological activation of Nrf2 increased BK-α and BK-β1 protein levels and enhanced BK channel activity in coronary arterial SMCs. Hence, our results indicate that Nrf2 is a key determinant of BK channel expression and function in vascular SMCs. Nrf2 facilitates BK-α expression through a direct increase in gene transcription, whereas that on BK-β1 is through a different mechanism.

Transcriptional and posttranscriptional regulation of the SMC-selective blood pressure-associated gene, ARHGAP42.

We previously showed that ARHGAP42 is a smooth muscle cell (SMC)-selective, RhoA-specific GTPase activating protein that regulates blood pressure and that a minor allele single nucleotide variation within a DNAse hypersensitive regulatory element in intron1 (Int1DHS) increased ARHGAP42 expression by promoting serum response factor binding. The goal of the current study was to identify additional transcriptional and posttranscriptional mechanisms that control ARHGAP42 expression. Using deletion/mutation, gel shift, and chromatin immunoprecipitation experiments, we showed that recombination signal binding protein for immunoglobulin κ-J region (RBPJ) and TEA domain family member 1 (TEAD1) binding to a conserved core region was required for full IntDHS transcriptional activity. Importantly, overexpression of the notch intracellular domain (NICD) or plating SMCs on recombinant jagged-1 increased IntDHS activity and endogenous ARHGAP42 expression while siRNA-mediated knockdown of TEAD1 inhibited ARHGAP42 mRNA levels. Re-chromatin immunoprecipitation experiments indicated that RBPJ and TEAD1 were bound to the Int1DHS enhancer at the same time, and coimmunoprecipitation assays indicated that these factors interacted physically. Our results also suggest TEAD1 and RBPJ bound cooperatively to the Int1DHS and that the presence of TEAD1 promoted the recruitment of NICD by RBPJ. Finally, we showed that ARHGAP42 expression was inhibited by micro-RNA 505 (miR505) which interacted with the ARHGAP42 3'-untranslated region (UTR) to facilitate its degradation and by AK124326, a long noncoding RNA that overlaps with the ARHGAP42 transcription start site on the opposite DNA strand. Since siRNA-mediated depletion of AK124326 was associated with increased H3K9 acetylation and RNA Pol-II binding at the ARHGAP42 gene, it is likely that AK124326 inhibits ARHGAP42 transcription.NEW & NOTEWORTHY First, RBPJ and TEAD1 converge at an intronic enhancer to regulate ARHGAP42 expression in SMCs. Second, TEAD1 and RBPJ interact physically and bind cooperatively to the ARHGAP42 enhancer. Third, miR505 interacts with the ARHGAP42 3'-UTR to facilitate its degradation. Finally, LncRNA, AK124326, inhibits ARHGAP42 transcription.

Coronary Disease-Associated Gene TCF21 Inhibits Smooth Muscle Cell Differentiation by Blocking the Myocardin-Serum Response Factor Pathway.

The gene encoding TCF21 (transcription factor 21) has been linked to coronary artery disease risk by human genome-wide association studies in multiple racial ethnic groups. In murine models, Tcf21 is required for phenotypic modulation of smooth muscle cells (SMCs) in atherosclerotic tissues and promotes a fibroblast phenotype in these cells. In humans, TCF21 expression inhibits risk for coronary artery disease. The molecular mechanism by which TCF21 regulates SMC phenotype is not known. To better understand how TCF21 affects the SMC phenotype, we sought to investigate the possible mechanisms by which it regulates the lineage determining MYOCD (myocardin)-SRF (serum response factor) pathway. Modulation of TCF21 expression in human coronary artery SMC revealed that TCF21 suppresses a broad range of SMC markers, as well as key SMC transcription factors MYOCD and SRF, at the RNA and protein level. We conducted chromatin immunoprecipitation-sequencing to map SRF-binding sites in human coronary artery SMC, showing that binding is colocalized in the genome with TCF21, including at a novel enhancer in the SRF gene, and at the MYOCD gene promoter. In vitro genome editing indicated that the SRF enhancer CArG box regulates transcription of the SRF gene, and mutation of this conserved motif in the orthologous mouse SRF enhancer revealed decreased SRF expression in aorta and heart tissues. Direct TCF21 binding and transcriptional inhibition at colocalized sites were established by reporter gene transfection assays. Chromatin immunoprecipitation and protein coimmunoprecipitation studies provided evidence that TCF21 blocks MYOCD and SRF association by direct TCF21-MYOCD interaction. These data indicate that TCF21 antagonizes the MYOCD-SRF pathway through multiple mechanisms, further establishing a role for this coronary artery disease-associated gene in fundamental SMC processes and indicating the importance of smooth muscle response to vascular stress and phenotypic modulation of this cell type in coronary artery disease risk.

Uremic serum-induced calcification of human aortic smooth muscle cells is a regulated process involving Klotho and RUNX2.

Vascular calcification (VC) is common in subjects with chronic kidney disease (CKD) and is associated with increased cardiovascular risk. It is an active process involving transdifferentiation of arterial smooth muscle cells (SMCs) into osteogenic phenotype. We investigated the ability of serum from CKD subjects to induce calcification in human SMCs in vitro (calcific potential of sera: CP), and associated changes in expression of Runt-related transcription factor 2 (RUNX2), SM22α, and Klotho. Sera from subjects with CKD (18 stage 3, 17 stage 4/5, and 29 stage 5D) and 20 controls were added to human cultured SMCs and CP quantified. The CP of CKD sera was greater (P<0.01) than that of controls, though not influenced by CKD stage. Modification of diet in renal disease estimated glomerular filtration rate (MDRD-4 eGFR) (P<0.001), serum phosphate (P=0.042), receptor activator of nuclear factor κappa-B ligand (RANKL) (P=0.001), parathyroid hormone (PTH) (P=0.014), and high-density lipoprotein (HDL)/cholesterol ratio (P=0.026) were independent predictors of CP accounting for 45% of variation. Adding calcification buffer (CB: calcium chloride [7 mM] and β-glycerophosphate [7 mM]) increased the CP of control sera to approximate that of CKD sera. CP of CKD sera was unchanged. CKD sera increased RUNX2 expression (P<0.01) in human SMCs and decreased SM22α expression (P<0.05). Co-incubating control but not CKD serum with CB further increased RUNX2 expression (P<0.01). Both SM22α and Klotho expression decreased significantly (P<0.01) in the presence of CKD serum, and were virtually abolished with stage 5D sera. These findings support active regulation by CKD serum of in vitro VC by induction of RUNX2 and suppression of SM22α and Klotho.

P1937Deficiency of PI 3-kinase isoform p110alpha in smooth muscle cells impairs vascular integrity and promotes aortic aneurysm formation

Abstract Background The pathobiology of aortic aneurysms is characterized by vascular inflammation, extracellular matrix degeneration, and particularly by loss and dedifferentiation of vascular smooth muscle cells (SMCs). In SMCs, the PI 3-kinase isoform p110α mediates receptor tyrosine kinase dependent proliferation, chemotaxis, and survival. Smooth muscle specific p110α deficient mice (SM-p110α−/− mice) display reduced medial wall thickness, substantially reduced neointima formation and media hypertrophy after balloon injury of the carotid artery. Objective We hypothesized that loss of p110α signaling impairs vascular integrity and promotes development and progression of abdominal aortic aneurysms (AAA). We aimed to elucidate the impact of p110α deficiency on vascular integrity, SMC phenotypic modulation, vascular inflammation, and AAA formation. Methods and results Ultra-structural characterization of aortic wall morphology in abdominal aortas from SM-p110α−/− mice by transmission electron microscopy (TEM) revealed disarranged structure of tunica media as indicated by disorganized elastic fibers, detached SMCs, and elastic fiber breaks. Western blots showed reduced elastin and fibrillin expression in SMCs from p110α−/− mice. Media thickness was significantly reduced in abdominal aortas from SM-p110α−/− mice compared to wild type (WT) controls (29.0±3.1 vs. 42.5±4.1 μm). Lack of p110α decreased expression of differentiation markers SM-α-actin and SM-MHC. p110α deficiency significantly diminished responsiveness of aortic rings to vasodilator acetylcholine. These data indicate loss of differentiation and impaired contractility of p110α−/− SMCs. We subjected SM-p110α−/− mice and WT littermate controls to the porcine pancreatic elastase (PPE) model of AAA. PPE was infused into the infrarenal aorta, respectively, to induce AAA formation. Ultrasonic examination of abdominal aortas demonstrated an enlarged aortic diameter in PPE challenged mice. AAA formation was significantly (p&lt;0.01) enhanced in SM-p110α−/− (0.46±0.12 mm, n=8) compared to SM-p110α+/+ mice (0.18±0.03 mm, n=4). These data indicate a protective function of p110α in AAA formation. Immunocytochemistry of the aortic medial compartment from PPE-perfused SM-p110α−/− mice revealed significantly increased MOMA-2+ monocyte/macrophage content indicating augmented aortic inflammation during AAA formation compared to WT controls. Furthermore, SMCs from SM-p110α−/− mice expressed reduced amounts of anti-inflammatory angiopoietin1 compared to p110α+/+ SMCs. Moreover, frequent apoptotic/necrotic SMCs were found in the aortic media of SM-p110α−/− mice by TEM, potentially contributing to vascular inflammation in a critical fashion. Conclusion These data indicate that p110α signaling critically contributes to vascular integrity via maintaining SMC plasticity, elastic fiber homeostasis, and anti-inflammatory processes. Consequently, lack of proper p110α signaling promotes progression of AAA formation.

PKCδ Mediates Mineralocorticoid Receptor Activation by Angiotensin II to Modulate Smooth Muscle Cell Function

Angiotensin II (AngII) and the mineralocorticoid receptor (MR) ligand aldosterone both contribute to cardiovascular disorders, including hypertension and adverse vascular remodeling. We previously demonstrated that AngII activates MR-mediated gene transcription in human vascular smooth muscle cells (SMCs), yet the mechanism and the impact on SMC function are unknown. Using an MR-responsive element-driven transcriptional reporter assay, we confirm that AngII induces MR transcriptional activity in vascular SMCs and endothelial cells, but not in Cos1 or human embryonic kidney-293 cells. AngII activation of MR was blocked by the MR antagonist spironolactone or eplerenone and the protein kinase C-δ (PKCδ) inhibitor rottlerin, implicating both in the mechanism. Similarly, small interfering RNA knockdown of PKCδ in SMCs prevented AngII-mediated MR activation, whereas knocking down of MR blocked both aldosterone- and AngII-induced MR function. Coimmunoprecipitation studies reveal that endogenous MR and PKCδ form a complex in SMCs that is enhanced by AngII treatment in association with increased serine phosphorylation of the MR N terminus. AngII increased mRNA expression of the SMC-MR target gene, FKBP51, via an MR-responsive element in intron 5 of the FKBP51 gene. The impact of AngII on FKBP51 reporter activity and gene expression in SMCs was inhibited by spironolactone and rottlerin. Finally, the AngII-induced increase in SMC number was also blocked by the MR antagonist spironolactone and the PKCδ inhibitor rottlerin. These data demonstrate that AngII activates MR transcriptional regulatory activity, target gene regulation, and SMC proliferation in a PKCδ-dependent manner. This new mechanism may contribute to synergy between MR and AngII in driving SMC dysfunction and to the cardiovascular benefits of MR and AngII receptor blockade in humans.

Regulatory T cells protected against abdominal aortic aneurysm by suppression of the COX-2 expression.

CD4+CD25+ regulatory T cells (Tregs) have been shown to protect against the development of abdominal aortic aneurysm (AAA). Cyclooxygenase‐2 (COX‐2), a pro‐inflammatory protein, can convert arachidonic acid into prostaglandins (PGs). The present study was aimed to investigate the effect of Tregs on COX‐2 expression in angiotension II (Ang II)‐induced AAA in ApoE−/− mice. Tregs were injected via tail vein in every 2 weeks. Ang II was continuously infused by a micropump for 28 days to induce AAA. In vivo, compared with the control group, adoptive transfer of Tregs significantly reduced the incidence of AAA, maximal diameter, and the mRNA and protein expression of COX‐2 in mice. Immunofluorescence showed that Tregs treatment reduced COX‐2 expression both in smooth muscle cells (SMCs) and macrophages in AAA. In vitro, the Western blot analysis showed that Tregs reduced Ang II‐induced COX‐2 expression in macrophages and SMCs. Meanwhile, ELISA showed that Tregs reduced Ang II‐induced prostaglandin E2 (PGE2) secretion. Moreover, Tregs increased SMC viability and induced transition of macrophages phenotype from M1 to M2. In conclusion, Tregs treatment dramatically decreased the expression of COX‐2 in vivo and in vitro, suggesting that Tregs could protect against AAA through inhibition of COX‐2. The study may shed light on the immune treatment of AAA.

Medial artery calcification increases neointimal hyperplasia after balloon injury

Arterial calcification predicts accelerated restenosis after angioplasty and stenting. We studied the effects of calcification on neointimal hyperplasia after balloon injury in the rat carotid. Arterial calcification was induced by subcutaneous injection of vitamin D3 or by adventitial application of calcium chloride. After balloon catheter injury, neointimal hyperplasia was significantly increased in rats with medial calcification compared with controls. Neointimal cell proliferation in calcified arteries as assessed by proliferating cell nuclear antigen (PCNA) staining was also higher. In calcified arteries, bone morphogenetic protein 2 (BMP-2)levels were increased at the time of injury suggesting a possible explanation for the altered responses. In vascular smooth muscle cells (SMCs) grown under calcifying conditions , stimulation with BMP-2 significantly increased cell proliferation, however, this did not occur in those grown under non-calcifying conditions. These data suggest that neointimal hyperplasia is accelerated in calcified arteries and that this may be due in part to increased BMP-2 expression in medial SMCs. Treatments aimed at inhibiting restenosis in calcified arteries may differ from those that work in uncalcified vessels.

Promoters to Study Vascular Smooth Muscle.

Smooth muscle cells (SMCs) are a critical component of blood vessel walls that provide structural support, regulate vascular tone, and allow for vascular remodeling. These cells also exhibit a remarkable plasticity that contributes to vascular growth and repair but also to cardiovascular pathologies, including atherosclerosis, intimal hyperplasia and restenosis, aneurysm, and transplant vasculopathy. Mouse models have been an important tool for the study of SMC functions. The development of smooth muscle-expressing Cre-driver lines has allowed for exciting discoveries, including recent advances revealing the diversity of phenotypes derived from mature SMC transdifferentiation in vivo using inducible CreER T2 lines. We review SMC-targeting Cre lines driven by the Myh11, Tagln, and Acta2 promoters, including important technical considerations associated with these models. Limitations that can complicate study of the vasculature include expression in visceral SMCs leading to confounding phenotypes, and expression in multiple nonsmooth muscle cell types, such as Acta2-Cre expression in myofibroblasts. Notably, the frequently employed Tagln/ SM22α- Cre driver expresses in the embryonic heart but can also confer expression in nonmuscular cells including perivascular adipocytes and their precursors, myeloid cells, and platelets, with important implications for interpretation of cardiovascular phenotypes. With new Cre-driver lines under development and the increasing use of fate mapping methods, we are entering an exciting new era in SMC research.