Quiescent Smooth Muscle Cells Research Articles

Genetic Regulation of SMC Gene Expression and Splicing Predict Causal CAD Genes.

Coronary artery disease (CAD) is the leading cause of death worldwide. Recent meta-analyses of genome-wide association studies have identified over 175 loci associated with CAD. The majority of these loci are in noncoding regions and are predicted to regulate gene expression. Given that vascular smooth muscle cells (SMCs) play critical roles in the development and progression of CAD, we aimed to identify the subset of the CAD loci associated with the regulation of transcription in distinct SMC phenotypes. We measured gene expression in SMCs isolated from the ascending aortas of 151 heart transplant donors of various genetic ancestries in quiescent or proliferative conditions and calculated the association of their expression and splicing with ~6.3 million imputed single-nucleotide polymorphism markers across the genome. We identified 4910 expression and 4412 splicing quantitative trait loci (sQTLs) representing regions of the genome associated with transcript abundance and splicing. A total of 3660 expression quantitative trait loci (eQTLs) had not been observed in the publicly available Genotype-Tissue Expression dataset. Further, 29 and 880 eQTLs were SMC-specific and sex-biased, respectively. We made these results available for public query on a user-friendly website. To identify the effector transcript(s) regulated by CAD loci, we used 4 distinct colocalization approaches. We identified 84 eQTL and 164 sQTL that colocalized with CAD loci, highlighting the importance of genetic regulation of mRNA splicing as a molecular mechanism for CAD genetic risk. Notably, 20% and 35% of the eQTLs were unique to quiescent or proliferative SMCs, respectively. One CAD locus colocalized with a sex-specific eQTL (TERF2IP), and another locus colocalized with SMC-specific eQTL (ALKBH8). The most significantly associated CAD locus, 9p21, was an sQTL for the long noncoding RNA CDKN2B-AS1, also known as ANRIL, in proliferative SMCs. Collectively, our results provide evidence for the molecular mechanisms of genetic susceptibility to CAD in distinct SMC phenotypes.

Genetic regulation of circular RNA expression in human aortic smooth muscle cells and vascular traits.

Circular RNAs (circRNAs) are a class of non-coding RNAs that have cell-type-specific expression and are relevant in cardiovascular disease. Aortic smooth muscle cells (SMCs) play a crucial role in cardiovascular disease. In this study, we employed a systems genetics approach to identify SMC circRNA transcripts and their relevance in cardiovascular traits across the genome. We quantified circRNA expression across 151 quiescent and proliferative human aortic SMCs from donors of various genetic ancestries. We identified 1,589 expressed circRNAs. Between quiescent and proliferative SMCs, we identified 173 differentially expressed circRNAs. To characterize the genetic regulation of circRNA expression, we associated the genotypes of 6.3 million single nucleotide polymorphisms (SNPs) with circRNA abundance and found 96 circRNAs that were associated with genetic loci. Three SNPs were associated with circRNA expression in proliferative SMCs but not quiescent SMCs. We identified six SNPs that had distinct association directions with circRNA isoforms from the same gene. Lastly, to identify the relevance of circRNAs in cardiovascular disease, we overlapped genetic loci associated with circRNA expression with vascular disease-related genome-wide association studies loci. We identified 14 blood pressure, one myocardial infarction, and three coronary artery disease loci, which were associated with a circRNA transcript but not an mRNA transcript. Overall, our results provide insight into the genetic basis of vascular disease traits mediated by circRNA expression.

Effects of traditional cigarette and electronic cigarette on smooth muscle cell phenotypic modulation

Background and Aims : Cigarette smoke (CS) is a risk factor for cardiovascular disease. Whole traditional cigarette (TC) smoke contains over 7000 chemical components and several are deemed to be toxic. Thus, alternative next-generation tobacco products (E-cigarettes (E-cig)) are being developed as less dangerous. Vascular homeostasis is maintained by differentiated quiescent smooth muscle cells (SMCs) that, following environmental cues, may undergo transcriptional changes affecting contractile proteins and their proliferative phenotype.

Abstract 11990: Insulin-Like Growth Factor Binding Protein 2 is a Novel Marker of Early Smooth Muscle Cell Phenotype Modulation in Thoracic Aortic Aneurysm

Introduction: Single cell RNA sequencing (scRNAseq) enables high resolution analysis of vascular smooth muscle cell (SMC) phenotype modulation in thoracic aortic aneurysm. Insulin-like growth factor binding protein-2 ( Igfbp2 ) expression by SMCs is activated early in a murine Marfan syndrome (MFS) aortic aneurysm model. IGFBP2 modulates IGF1 bioavailability and interacts directly with SMCs via integrins. We hypothesize that IGFBP2 is a marker of early SMC modulation in human aortopathy. Methods: Surgically resected aortic aneurysm tissue was collected from patients spanning the clinical spectrum of monogenic aortopathies ( ACTA2, FBN1, TGFBR1 (n=2), TGFB2, or SMAD3 variants). Tissues were digested and processed for scRNAseq. SMC clusters from all patients were jointly analyzed with pseudotemporal trajectory analysis to reconstruct the SMC phenotypic continuum. Primary MFS human (n=5) aortic root SMCs underwent IGFBP2 silencing via siRNA followed by bulk RNAseq. Results: scRNAseq identified two main SMC clusters, comprising 39% of all cells in the dataset, including a quiescent contractile SMCs and a modulated, synthetic subset. Pseudotemporal ordering generated a continuous ‘pseudotime’ scale inversely correlated with contractile gene (e.g., CNN1, MYH11) expression, modeling gradual de-differentiation in vivo . We identified similar markers of late SMC modulation (e.g., TNFRSF11B , MMP2, CYTL1, COL1A1 ) in patients of all genotypes. IGFBP2 expression showed comparatively earlier activation and peak with reduced expression late in pseudotime. Silencing IGFBP2 in human MFS SMCs in vitro significantly depressed contractile gene expression. Conclusions: SMC modulation occurs in consistent pattern across hereditary aortopathy subtypes. IGFBP2 activation occurs early in this trajectory, marking cells in phenotypic transition. IGFBP2 may promote contractile phenotype maintenance, presenting a novel pathway for potential therapies.

CREB depletion in smooth muscle cells promotes medial thickening, adventitial fibrosis and elicits pulmonary hypertension.

Levels of the cAMP-responsive transcription factor, CREB, are reduced in medial smooth muscle cells in remodeled pulmonary arteries from hypertensive calves and rats with chronic hypoxia-induced pulmonary hypertension. Here, we show that chronic hypoxia fails to promote CREB depletion in pulmonary artery smooth muscle cells or elicit significant remodeling of the pulmonary arteries in mice, suggesting that sustained CREB expression prevents hypoxia-induced pulmonary artery remodeling. This hypothesis was tested by generating mice, in which CREB was ablated in smooth muscle cells. Loss of CREB in smooth muscle cells stimulated pulmonary artery thickening, right ventricular hypertrophy, profound adventitial collagen deposition, recruitment of myeloid cells to the adventitia, and elevated right ventricular systolic pressure without exposure to chronic hypoxia. Isolated murine CREB-null smooth muscle cells exhibited serum-independent proliferation and hypertrophy in vitro and medium conditioned by CREB-null smooth muscle cells stimulated proliferation and expression of extracellular matrix proteins by adventitial fibroblasts. We conclude that CREB governs the pathologic switch from homeostatic, quiescent smooth muscle cells to proliferative, synthetic cells that drive arterial remodeling contributing to the development or pulmonary hypertension.

Abstract 213: Deletion of Epha2 Reduces Fibroproliferative Remodeling in Atherosclerosis and Vascular Smooth Muscle

Advanced atherosclerosis involves the recruitment of vascular smooth muscle cells, leading to smooth muscle-mediated fibroproliferative remodeling and plaque stiffening. We previously demonstrated the neuronal guidance receptor EphA2 shows enhanced expression in murine and human atherosclerosis, and EphA2 deletion in atherosclerosis-prone ApoE knockout mice attenuates lesion formation and endothelial cell activation in models of atherosclerosis. We now demonstrate deletion of EphA2 attenuates plaque progression to advanced stages associated with diminished smooth muscle content. While contractile smooth muscle cells show minimal EphA2 expression in vitro and in vivo , transition to the synthetic phenotype results in enhanced EphA2 expression and luciferase reporter activity in vitro and in atherosclerotic lesions. Furthermore, forced expression of EphA2 in quiescent smooth muscle cells is sufficient to suppress contractile smooth muscle markers, suggesting a role for EphA2 in smooth muscle phenotypic switch. Depletion of EphA2 results in significant reductions in proliferation within atherosclerotic plaques and in smooth muscle cells with associated reductions in serum-induced ERK and Akt signaling. Conversely, overexpression of EphA2 enhances smooth muscle proliferation in vitro , suggesting EphA2 is sufficient to drive proliferation. EphA2-deficient mice show reductions in plaque fibrosis and matrix remodeling. Consistent with this, depletion of EphA2 in vascular smooth muscle cells exhibit a significant reduction in matrix deposition. Together these data suggest a role for EphA2 in smooth muscle-mediated fibroproliferative remodeling, representing the first link between EphA2 signaling and smooth muscle function in atherosclerosis.

ASSA14-03-20 Cellular repressor of E1A-stimulated genes protects against angiotensin II-induced vascular remodelling via degradation of angiotensin II type 1 receptor

Background Cellular repressor of E1A-stimulated genes (CREG), a new cardiovascular homeostasis regulator, has been proposed to reduce the neointimal formation after vascular injury via the maintenance of the quiescent mature vascular smooth muscle cell (VSMC) phenotype. We hypothesised that CREG is a negative regulator of angiotensin (Ang) II-mediated vascular remodelling. Methods Ten-week-old male heterozygous CREG-deficient (CREG +/- ) mice and their littermate wild-type (WT) mice were used. An osmotic minipump was implanted subcutaneously to infuse a dose of Ang II (1.5 mg/kg · d) or saline (vehicle) for 14 days. Ang II-infused WT mice were then treated with placebo or recombinant human CREG (rhCREG; 300 μg/kg · d). Systolic blood pressure (SBP); extent of vascular remodelling; protein level of collagen type I/III, angiotensin-converting enzyme-2 (ACE2), angiotensin receptor type 1 (AT1R) and Rab7 were evaluated. Primary VSMC culture was performed to assess AT1R and collagen protein expression before and after CREG gene transfection and knockdown. Results CREG levels are high in vascular media under basal conditions but rapidly decrease in response to Ang II. Ang II infusion for 14 days resulted that levels of SBP, intima medial thickness and vascular remodelling of the aorta and mesenteric artery were significantly greater in CREG +/- mice compared with the WT controls. Vascular gene expression level of CREG was lower in Ang II-treated CREG +/- mice than in WT mice. However, daily treatment of Ang II-infused WT mice with rhCREG blunted the increase of SBP. Ang II-mediated vascular remodelling and expression of collagen type I/III were also suppressed by rhCREG. Moreover, rhCREG treatment inhibited Ang II-mediated up-regulation of AT1R expression and down-regulation of ACE2 expression. Ang II-induced vascular remodelling was inhibited by rhCREG in association with reduced plasma Ang II and increased plasma Ang 1–7 levels. In adult VSMCs, CREG over-expression increased the expression of Rab7 by a regulatory mechanism of ubiquitination. Importantly, Ang II-mediated collagen production and AT1R expression was inhibited by CREG over-expression and augmented by CREG knock-down in a rab7-dependent manner. Conclusions Elevated Ang II induced hypertension and vascular remodelling, which were exacerbated by CREG deficiency, whereas rhCREG attenuated Ang II-induced adverse vascular remodelling. Hence, CREG is an important negative regulator of Ang II-induced vascular disease and suppresses adverse vascular remodelling.

Platelet-derived growth factor regulation of type-5 phosphodiesterase in human and rat penile smooth muscle cells.

Relaxation of cavernous smooth muscle cells (SMCs) is a key component in the control of the erectile mechanism. SMCs can switch their phenotype from a contractile differentiated state to a proliferative and dedifferentiated state in response to a change of local environmental stimuli. Proliferation and contraction are both regulated by the intracellular second messengers cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), which are degraded by phosphodiesterases (PDEs). The most abundant PDE present in corpora cavernosa is the electrolytic cGMP-specific phosphodiesterase type 5 (PDE5). We investigated the cellular localization of PDE5 in in vitro cultured corpora cavernosa cells and the effect of mitogenic stimulation on PDE5 expression. Biochemical ad molecular techniques on cultured SMCs from human and rat penis. We studied the ability of the quiescent SMC phenotype vs. the proliferating phenotype in modulation of PDE5 expression. We demonstrated that PDE5 is localized in the cytoplasm, in the perinuclear area, and in discrete cytoplasmic foci. As previously demonstrated in human myometrial cells, the cytoplasmic foci may correspond to centrosomes. In corpora cavernosa, PDE5 protein levels are strongly regulated by the mitotic activity of the SMCs, as they were increased in quiescent cultures. In contrast, treatment with platelet-derived grow factor (PDGF), one of the most powerful mitogenic factors for SMCs, reduces the expression of PDE5 after 24 hours of treatment. We found that PDGF treatment downregulates PDE5 expression in proliferating SMCs, suggesting that PDE5 may represent one of the markers of the contractile phenotype of the SMCs of corpora cavernosa.

Decorin mimic regulates platelet-derived growth factor and interferon-γ stimulation of vascular smooth muscle cells.

Following balloon injury, smooth muscle cells (SMCs) serve as targets for many of the pro-inflammatory and pro-fibrotic factors, including platelet-derived growth factor (PDGF) and interferon-γ (IFN-γ) released from activated inflammatory cells and platelets. Previously, our lab designed a mimic of the proteoglycan decorin, termed DS-SILY20, that suppressed vascular SMC proliferation, migration, and protein synthesis in vitro, and injured vessels treated with DS-SILY20 demonstrated reduced hyperplasia in vivo. Here we characterize the effects of DS-SILY20 on modulating PDGF and IFN-γ stimulation in both proliferative and quiescent human SMCs to further evaluate the potential impact of DS-SILY20-SMC interaction on restenosis. Nanomolar dissociation constants were observed between DS-SILY20 and both PDGF and IFN-γ. PDGF significantly increased migration, proliferation, and protein and cytokine expression, as well as increased ERK-1/2 and p38 MAPK phosphorylation in both quiescent and proliferative cultures. However, DS-SILY20 inhibited these increases, presumably through sequestration of the PDGF. Consistent with the complex responses seen with IFN-γ in SMC physiology in the literature, the response of SMC cultures to IFN-γ was variable and complex. However, where increased activity was seen with IFN-γ, DS-SILY20 attenuated this activity. Overall, the results suggest that DS-SILY20 would be an ideal alternative to traditional therapeutics used and may be an effective therapy for the prevention of intimal hyperplasia after balloon angioplasty.

Bortezomib protects from varicose-like venous remodeling.

Despite the high prevalence of venous diseases that are associated with and based on the structural reorganization of the venous vessel wall, not much is known about their mechanistic causes. In this context, we demonstrated that the quantity of myocardin, a transcriptional regulator of the contractile and quiescent smooth muscle cell phenotype, was diminished in proliferating synthetic venous smooth muscle cells (VSMCs) of human and mouse varicose veins by 51 and 60%, respectively. On the basis of the relevance of proteasomal activity for such phenotypic changes, we hypothesized that the observed VSMC activation is attenuated by the proteasome inhibitor bortezomib. This drug fully abolished VSMC proliferation and loss of myocardin in perfused mouse veins and blocked VSMC invasion in collagen gels by almost 80%. In line with this, topical transdermal treatment with bortezomib diminished VSMC proliferation by 80%, rescued 90% of VSMC myocardin abundance, and inhibited varicose-like venous remodeling by 67 to 72% in a mouse model. Collectively, our data indicate that the proteasome plays a pivotal role in VSMC phenotype changes during venous remodeling processes. Its inhibition protects from varicose-like vein remodeling in mice and may thus serve as a putative therapeutic strategy to treat human varicose veins.

Late-outgrowth endothelial progenitors from patients with coronary artery disease: Endothelialization of confluent stromal cell layers

Patients with coronary artery disease (CAD) are the primary candidates to receive small-diameter tissue-engineered blood vessels (TEBVs). Peripheral blood derived endothelial progenitor cells (EPCs) from CAD patients (CAD EPCs) represent a minimally invasive source of autologous cells for TEBV endothelialization. We have previously shown that human CAD EPCs are highly proliferative and express many of the hallmarks of mature and healthy endothelial cells; however, their behavior on stromal cells that comprise the media of TEBVs has not yet been evaluated. Primary CAD EPCs or control human aortic endothelial cells (HAECs) were seeded over confluent, quiescent layers of human smooth muscle cells (SMCs) using a direct co-culture model. The percent coverage, adhesion strength, alignment under flow and generation of flow-induced nitric oxide of the seeded CAD EPCs were compared to that of HAECs. The integrin-binding profile of CAD EPCs was also evaluated over a layer of confluent, quiescent SMCs. Direct comparison of our CAD EPC results to analogous co-culture studies with cord blood EPCs show that both types of blood-derived EPCs are viable options for endothelialization of TEBVs.

TGFβ1-induced Baf60c regulates both smooth muscle cell commitment and quiescence.

Smooth muscle cells (SMCs) play critical roles in a number of diseases; however, the molecular mechanism underlying their development is unclear. Although the role of TGFβ1 signaling in SMC development is well established, the downstream molecular signals are not fully understood. We used several rat multipotent adult progenitor cell ((r)MAPC) lines that express levels of Oct4 mRNA similar to hypoblast stem cells (HypoSC), and can differentiate robustly to mesodermal and endodermal cell types. TGFβ1 alone, or with PDGF-BB, induces differentiation of rMAPCs to SMCs, which expressed structural SMC proteins, including α-smooth muscle actin (αSMA), and contribute to the SMC coat of blood vessels in vivo. A genome-wide time-course transcriptome analysis revealed that transcripts of Baf60c, part of the SWI/SNF actin binding chromatin remodeling complex D-3 (SMARCD3/BAF60c), were significantly induced during MAPC-SMC differentiation. We demonstrated that BAF60c is a necessary co-regulator of TGFβ1 mediated induction of SMC genes. Knock-down of Baf60c decreased SMC gene expression in rMAPCs whereas ectopic expression of Baf60c was sufficient to commit rMAPCs to SMCs in the absence of exogenous cytokines. TGFβ1 activates Baf60c via the direct binding of SMAD2/3 complexes to the Baf60c promoter region. Chromatin- and co-immunoprecipitation studies demonstrated that regulation of SMC genes by BAF60c is mediated via interaction with SRF binding CArG box-containing promoter elements in SMC genes. We noted that compared with TGFβ1, Baf60c overexpression in rMAPC yielded SMC with a more immature phenotype. Similarly, Baf60c induced an immature phenotype in rat aortic SMCs marked by increased cell proliferation and decreased contractile marker expression. Thus, Baf60c is important for TGFβ-mediated commitment of primitive stem cells (rMAPCs) to SMCs and is associated with induction of a proliferative state of quiescent SMCs. The MAPC-SMC differentiation system may be useful for identification of additional critical (co-)regulators of SMC development.

Genome-Wide Microarray Analyses Identify the Protein C Receptor as a Novel Calcineurin/Nuclear Factor of Activated T Cells–Dependent Gene in Vascular Smooth Muscle Cell Phenotypic Modulation

Calcineurin (Cn) and the nuclear factor of activated T cells (NFAT) family of transcription factors are critical in vascular smooth muscle cell (SMC) development and pathology. Here, we used a genomics approach to identify and validate NFAT gene targets activated during platelet-derived growth factor-BB (PDGF-BB)-induced SMC phenotypic modulation. Genome-wide expression arrays were used to identify genes both (1) differentially activated in response to PDGF-BB and (2) whose differential expression was reduced by both the Cn inhibitor cyclosporin A and the NFAT inhibitor A-285222. The 20 most pharmacologically sensitive genes were validated by quantitative reverse transcription-polymerase chain reaction analysis of PDGF-BB-stimulated SMCs in the presence of Cn/NFAT inhibitors, including the VIVIT peptide. In all experiments, protein C receptor (PROCR) gene activation was reduced. We showed that PROCR expression was virtually absent in untreated, quiescent SMCs. PDGF-BB stimulation, however, induced significant PROCR promoter activation and downstream protein expression in a Cn/NFAT-dependent manner. Mutation of a species-conserved, NFAT binding motif significantly attenuated PDGF-BB-induced PROCR promoter activity, thereby distinguishing NFAT as the first PROCR transcriptional activator to date. Moreover, SMC PROCR expression was upregulated in the neointima as early as 7 days following acute vascular injury in rat carotid arteries. We hereby report PROCR as a novel, NFAT-dependent gene that may be implicated in vascular restenosis and consequent inward remodeling.

Glutathione peroxidase-deficient smooth muscle cells cause paracrine activation of normal smooth muscle cells via cyclophilin A

Background/aimsReduced activity of the antioxidant glutathione peroxidase-1 (GPx1) correlates with increased risk of cardiovascular events in patients with coronary artery disease. However, it remains unclear whether this imbalance in antioxidant capacity directly contributes to activation of vascular cells. In response to oxidative stress, smooth muscle cells (SMCs) secrete the pro-inflammatory immunomodulator cyclophilin A (CyPA). We hypothesized that reduction in vascular cell GPx1 activity causes secretion of CyPA and paracrine-mediated activation of NF-κB and proliferation of SMCs. Methods/resultsUsing a murine model of GPx1 deficiency (GPx1+/−), we found elevated levels of hydrogen peroxide levels and increased secretion of CyPA in both arterial segments and cultured SMCs as compared to wild type (WT). Conditioned media from GPx1+/− SMCs caused increased NF-κB activation of quiescent WT SMCs, and this was inhibited by the antioxidant N-acetyl-l-cysteine or by cyclosporine A (CsA). In co-culture experiments, SMCs derived from GPx1+/− aorta caused increased proliferation of WT SMCs, which was also inhibited by CsA. ConclusionsReduction in vascular cell GPx1 activity and the associated increase in oxidative stress cause CyPA-mediated paracrine activation of SMCs. These findings identify a novel mechanism by which an imbalance in antioxidant capacity may contribute to vascular disease.

IGF-1 receptor transactivation mediates Src-dependent cortactin phosphorylation in response to angiotensin IIThis article is one of a selection of papers published in a special issue celebrating the 125th anniversary of the Faculty of Medicine at the University of Manitoba.

Release of angiotensin II (Ang II) after vascular injury promotes tissue repair by stimulating phenotypic modulation of smooth muscle cells, which enables cell proliferation and migration. This process requires cytoskeleton remodeling, which involves cortactin, a scaffold protein that is phosphorylated by Src kinase in response to Ang II. Since insulin-like growth factor (IGF)-1 receptor transactivation mediates intracellular signals originating from the Ang II type 1 (AT1) receptor in a Src kinase-dependent manner, we examined whether IGF-1 receptor transactivation was also required for cortactin phosphorylation. Treatment of quiescent smooth muscle cells with Ang II resulted in both cortactin phosphorylation and its translocation to the plasma membrane. Both events were prevented by 1-(1,1-dimethylethyl)-1-(4-methylphenyl)-1H-pyrazolo(3,4-d)pyrimidin-4-amine (PP1), a Src kinase inhibitor, and by AG1024, an inhibitor of the IGF-1 receptor tyrosine kinase. Additionally, PP1 and AG1024 blocked the association of cortactin with actin-related protein (Arp) 3, an actin nucleation factor. These results indicate that Src kinase and the IGF-1 receptor kinase are necessary for activating cortactin. Phosphorylation of Src kinase in Ang II-treated cells was subsequently examined and was shown to be prevented by AG1024. Furthermore, Src kinase phosphorylation was blocked by inhibitors of protein kinase C (PKC), but not by inhibitors of phosphatidylinositol (PI) 3-kinase. These data establish that IGF-1 receptor transactivation is required for Src kinase-mediated cortactin phosphorylation and cytoskeletal reorganization in response to Ang II.

Inducible nitric oxide synthase contributes to MAPK‐phosphatase induction and abundance of cyclooxygenase‐2 expression in an inflammatory setting

Cyclooxygenase (COX)‐2 and inducible nitric oxide synthase (iNOS) are responsive to inflammatory stimuli and have been localized to vascular smooth muscle cells (SMCs) in pathophysiological settings, e.g. atherosclerosis. Furthermore, the production of, and subsequent impact of COX products appear to be correlative with the status of NO synthesis and bioavailability. This study examined the role of inflammation‐driven NO synthesis on COX‐2 expression in rat aortic SMCs. Concurrent stimulation of quiescent SMCs with lipopolysaccharide (LPS) and interferon (IFN)‐γ induced iNOS and COX‐2 expression maximally at 2‐4hr. Inhibition of NO synthesis (LNMMA) significantly reduced nitrite (140±8 vs. 19±7 nmol/mg protein) and enhanced LPS+IFN‐γ‐mediated COX‐2 mRNA (156‐ vs. 43‐fold increase) and protein expression (5‐fold) at 30 and 60min. COX‐2 induction was associated with activation of the ERK, p38 and JNK pathways. Importantly, LNMMA enhanced ERK and p38 phosphorylation, and suppressed MAPK‐phosphatase (MKP)‐1 induction. Consistent with these data, exposure of SMCs from iNOS‐/‐ mice to LPS+IFN‐γ produced a more robust induction (p<0.05) of COX‐2 and a dramatically attenuated upregulation of MKP‐1 vs. WT. These findings provide pharmacological and genetic evidence implicating an inhibitory influence of iNOS‐derived species on phosphorylation cascades associated with COX‐2‐induction in SMCs.

The new immunosuppressive agent FK778 attenuates neointima formation in an experimental venous bypass graft model

Neointima formation as a result of smooth muscle cell (SMC) proliferation still contributes significantly to venous bypass graft stenosis and failure. The new immunosuppressive agent FK778 has recently been shown to exert anti-proliferative effects. We examined the inhibitory potential of FK778 on venous SMC proliferation and neointima formation in an experimental venous bypass graft model. Quiescent venous SMCs were incubated for 48 h with 10% FCS and different concentrations of FK778. SMC proliferation was measured by Ki67 immunostaining. Uridine was added to reverse FK778 induced pyrimidine synthesis blockade. The effect of FK778 treatment on neointima formation in vivo was assessed in an autologous epigastric vein-to femoral artery interposition graft model in rats. In vitro, FK778 inhibited venous SMC proliferation in a dose dependent manner. This effect was reversed by addition of uridine. In vivo, 3-week oral treatment with FK778 (15 mg/kg/d) significantly reduced neointima formation and stenosis in venous bypass grafts. We show that the immunosuppressive agent FK778 can prevent neointima formation in experimental venous bypass grafts by inhibiting venous SMC proliferation. FK778 might provide a new pharmacological therapy to limit vein graft stenosis and bypass graft failure.

Lysyl Oxidase Oxidizes Cell Membrane Proteins and Enhances the Chemotactic Response of Vascular Smooth Muscle Cells

Lysyl oxidase (LOX) is a potent chemokine inducing the migration of varied cell types. Here we demonstrate that inhibition of LOX activity by beta-aminopropionitrile (BAPN) in cultured rat aortic smooth muscle cells (SMCs) reduced the chemotactic response and sensitivity of these cells toward LOX and toward PDGF-BB. The chemotactic activity of PDGF-BB was significantly enhanced in the presence of a non-chemotactic concentration of LOX. We considered the possibility that extracellular LOX may oxidize cell surface proteins, including the PDGF receptor-beta (PDGFR-beta), to affect PDGF-BB-induced chemotaxis. Plasma membranes purified from control SMC contained oxidized PDGFR-beta. The oxidation of this receptor and other membrane proteins was largely prevented in cells preincubated with BAPN. Addition of purified LOX to these cells restored the profile of oxidized proteins toward that of control cells. The high affinity and capacity for the binding of PDGF-BB by cells containing oxidized PDGFR-beta was diminished by approximately 2-fold when compared with cells in which oxidation by LOX was prevented by BAPN. Phosphorylated members of the PDGFR-beta-dependent signal transduction pathway, including PDGFR-beta, SHP2, AKT1, and ERK1/ERK2 (p44/42 MAPK), turned over faster in BAPN-treated than in control SMCs. LOX knock-out mouse embryonic fibroblasts mirrored the effect obtained with SMCs treated with BAPN. These novel findings suggest that LOX activity is essential to generate optimal chemotactic sensitivity of cells to chemoattractants by oxidizing specific cell surface proteins, such as PDGFR-beta.

Fluvastatin Synergistically Improves the Antiproliferative Effect of Everolimus on Rat Smooth Muscle Cells by Altering p27Kip1/Cyclin E Expression

Multiple intracellular signaling pathways stimulate quiescent smooth muscle cells (SMCs) to exit from G(0) and re-enter the cell cycle. Thus, a combination of two drugs with different mechanisms of action may represent a suitable approach to control SMC proliferation, a prominent feature of in-stent restenosis. In the present study, we investigated the effect of everolimus, a mammalian target of rapamycin inhibitor, in combination with fluvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, on proliferation of rat SMCs. The antiproliferative action of everolimus was amplified by 2.5-fold by the addition of subliminal concentrations of fluvastatin (5 x 10(-7) M), lowering the IC(50) value from 2.5 x 10(-9) to 1.0 x 10(-9) M. The increased antiproliferative effect of everolimus by fluvastatin was prevented in the presence of mevalonate, farnesol, or geranylgeraniol, suggesting the involvement of prenylated proteins. Cell cycle analysis and [3H]thymidine incorporation assay demonstrated that the two drugs synergistically interfered with the progression of G(1) phase. In particular, the drug combination significantly up-regulated p27(Kip1) levels by 47.0%, suppressed cyclin E by 43.0%, and it reduced retinoblastoma (Rb) hyperphosphorylation by 79.0%, compared with everolimus alone. Retroviral overexpression of cyclin E conferred a significant resistance of rat SMCs to the antiproliferative action of the drug combination, measured by cell counting, [3H]thymidine incorporation, and cell cycle analysis, with higher levels of hyperphosphorylated form of Rb. Taken together, these results demonstrated that everolimus acts synergistically with fluvastatin to inhibit SMC proliferation by altering the expression of cyclin E and p27(kip1), which affects Rb phosphorylation and leads to G(1) phase arrest.

CREG promotes a mature smooth muscle cell phenotype and reduces neointimal formation in balloon-injured rat carotid artery

We previously showed that cellular repressor of E1A-stimulated genes (CREG) is up-regulated during serum starvation-induced vascular smooth muscle cell (SMC) differentiation. The aim of this study was to determine the role of CREG in maintaining the quiescent, differentiated phenotype of SMCs both in culture and in balloon-injured rat carotid artery. In cultured SMCs recombinant virus-mediated CREG expression enhanced cellular differentiation, inhibited proliferation, and reduced synthesis of extracellular matrix component fibronectin. In contrast, CREG knockdown via retroviral transfer of short hairpin RNAs abrogated serum starvation-induced SMC differentiation and growth arrest. Both immunostaining and Western analysis demonstrated marked down-regulation of CREG in the vascular media after balloon injury to the rat carotid artery. Retrovirus-mediated CREG transfer to the injured artery inhibited SMC dedifferentiation and proliferation, and reduced neointimal hyperplasia. These results suggest that CREG participates in the maintenance of quiescent mature SMC phenotype in the arterial media by promoting SMC differentiation and growth arrest and that CREG gene transfer has therapeutic potential for vascular diseases associated with neointimal hyperplasia.