SMC Migration Research Articles

Inhibition of Semicarbazide-sensitive Amine Oxidase Reduces Atherosclerosis in Cholesterol-fed New Zealand White Rabbits

Inflammation, oxidative stress, and the formation of advanced glycated end-products (AGEs) are important components of atherosclerosis. Vascular adhesion protein-1 (VAP-1) participates in inflammation. Its enzymatic activity, semicarbazide-sensitive amine oxidase (SSAO), can catalyze oxidative deamination reactions to produce hydrogen peroxide and aldehydes, leading to the subsequent generation of AGEs. This study aimed to investigate the effect of VAP-1/SSAO inhibition on atherosclerosis. In our study, immunohistochemical staining showed that atherosclerotic plaques displayed higher VAP-1 expression than normal arterial walls in apolipoprotein E-deficient mice, cholesterol-fed New Zealand White rabbits and humans. In cholesterol-fed rabbits, VAP-1 was expressed on endothelial cells and smooth muscle cells in the thickened intima of the aorta. Treatment with PXS-4728A, a selective VAP-1/SSAO inhibitor, in cholesterol-fed rabbits significantly decreased SSAO-specific hydrogen peroxide generation in the aorta and reduced atherosclerotic plaques. VAP-1/SSAO inhibition also lowered blood low-density lipoprotein cholesterol, reduced the expression of adhesion molecules and inflammatory cytokines, suppressed recruitment and activation of macrophages, and decreased migration and proliferation of SMC. In conclusion, VAP-1/SSAO inhibition reduces atherosclerosis and may act through suppression of several important mechanisms for atherosclerosis.

Abstract 619: Regulatory Mechanism of Lmod1 Mediated Predisposition to Coronary Artery Disease

Atherosclerotic coronary artery disease (CAD) continues to be the leading cause of mortality and morbidity worldwide, with an estimated 40% of one’s lifetime risk attributed to genetic factors. Meta-analysis of genome-wide association studies implemented to identify this risk in human populations, has now identified rs2820315 (P=7.7E-10; OR=1.05), in the smooth muscle cell-restricted gene, Leiomodin 1 (LMOD1), as the leading genetic polymorphism associated with CAD. However, the causal mechanism by which this polymorphism is responsible for predisposition to CAD remains to be identified. Expression quantitative trait loci (eQTL) mapping in GTEx and STARNET databases revealed that carriers of the risk allele, rs34091558, which is in tight linkage disequilibrium with rs2820315, display significantly attenuated LMOD1 expression in vascular tissues than carriers of the ancestral allele. Allelic expression imbalance analyses in heterozygous HCASMC donors further demonstrated cis-acting effects of the polymorphism on LMOD1 gene expression. To determine the mechanism responsible for this reduction in LMOD1 expression, we performed position weight matrix (PWM) motif analyses and found that rs34091558 disrupts the binding site of a transcription factor called forkhead box O3 (FOXO3). Subsequent chromatin immunoprecipitation and reporter assays demonstrated reduced FOXO3 binding and transcriptional activity by the risk allele in cultured HCASMCs. Platelet-derived growth factor BB (PDGF-BB) stimulation also significantly reduced LMOD1 expression coincident with FOXO3 knockdown. Finally, both gain and loss-of-function for FOXO3 and LMOD1 in HCASMC delineated a regulatory circuit by which LMOD1 regulates SMC proliferation, migration and contraction, characteristic features of atherosclerotic lesion progression. Taken together, these results provide compelling functional evidence that: 1) rs3091558 is associated with reduced LMOD1 expression, 2) this reduction appears to be mediated through the inhibition of FOXO3 binding and 3) changes in vessel wall processes through LMOD1 dysregulation may partially explain the heritable risk for CAD.

N-oleoylethanolamide suppresses intimal hyperplasia after balloon injury in rats through AMPK/PPARα pathway

Vascular smooth muscle cell (VSMC) proliferation and migration are crucial events in the pathological course of restenosis after percutaneous coronary intervention (PCI). N-oleoylethanolamide (OEA) is a bioactive lipid amide released upon dietary fat digestion with many reported actions. However, the effect of OEA on restenosis after vascular injury remains unknown. Here, we investigated the effects of OEA on intimal hyperplasia after balloon injury in vivo, its effect on VSMC proliferation and migration induced by platelet-derived growth factor (PDGF) stimulation in vitro, and the underlying mechanism underlying these effects. The results showed that OEA-treated rats displayed a significant reduction in neointima formation after balloon injury. In cultured VSMCs, treatment with OEA decreased cell proliferation and migration induced by PDGF. OEA treatment both in vivo and in vitro led to an increase in adenosine monophosphate-activated protein kinase (AMPK) phosphorylation and peroxisome proliferator-activated receptor alpha (PPARα), and a decrease in proliferating cell nuclear antigen (PCNA) and cyclinD1 expression. Pharmacological inhibition of AMPK and PPARα reversed the suppressive effects of OEA on VSMC proliferation and migration, suggesting that the suppressive effect of OEA on VSMC proliferation and migration is mediated through the activation of AMPK and PPARα. In conclusion, our present study demonstrated that OEA attenuated neointima formation in response to balloon injury by suppressing SMC proliferation and migration through an AMPK and PPARα-dependent mechanism. Our data suggests that OEA may be a potential therapeutic agent for restenosis after PCI.

Abstract 452: Serine Carboxypeptidase 1 Mediates Vascular Remodeling by Increasing Inflammatory Cell Infiltration

In pathological vascular remodeling, contractile vascular smooth muscle cells (VSMCs) switch their phenotype to highly proliferative and migratory states leading to neointimal formation. Inflammatory cell recruitment and infiltration, which is dependent on the increased expression of adhesion molecules on the endothelial cells, is a key event to initiate SMC phenotypic modulation in vascular remodeling. Serine carboxypeptidase 1 (scpep1), a novel protease containing the putative catalytic triad (Ser-Asp-His) common to all members of the serine protease family, has been proved to be involved in vascular remodeling by promoting SMC proliferation and migration in a catalytic triad-dependent manner. To determine whether Scpep1 modulates leukocyte adhesion and infiltration, a flow-induced model of vascular remodeling was conducted in wild-type (WT) or Scpep1 knockout (KO) mice. Scpep1-null mice show a decreased number of infiltrated leukocytes into the intima and media compared to WT mice. Further, mice devoid of Scpep1 show a dramatic reduction of vascular cell adhesion molecule-1 (VCAM-1) and intercellular cell adhesion molecule-1 (ICAM-1) expression in vessels in comparison with that of WT mice. Consistent with our in vivo data, the expression levels of ICAM-1 and VCAM-1 on human umbilical vein endothelial cells (HUVECs) transfected with SiRNA against Scpep1 were significantly decreased after TNF-α treatment. Taken together, these data suggest that Scpep1 may increase leukocyte extravasation by increasing the expression of VCAM-1 and ICAM-1 adhesion molecules.

Myeloperoxidase-oxidized high density lipoprotein impairs atherosclerotic plaque stability by inhibiting smooth muscle cell migration

BackgroundHigh density lipoprotein (HDL) has been proved to be a protective factor for coronary heart disease. Notably, HDL in atherosclerotic plaques can be nitrated (NO2-oxHDL) and chlorinated (Cl-oxHDL) by myeloperoxidase (MPO), likely compromising its cardiovascular protective effects.MethodHere we determined the effects of NO2-oxHDL and Cl-oxHDL on SMC migration using wound healing and transwell assays, proliferation using MTT and BrdU assays, and apoptosis using Annexin-V assay in vitro, as well as on atherosclerotic plaque stability in vivo using a coratid artery collar implantation mice model.ResultsOur results showed that native HDL promoted SMC proliferation and migration, whereas NO2-oxHDL and Cl-oxHDL inhibited SMC migration and reduced capacity of stimulating SMC proliferation as well as migration, respectively. OxHDL had no significant influence on SMC apoptosis. In addition, we found that ERK1/2-phosphorylation was significantly lower when SMCs were incubated with NO2-oxHDL and Cl-oxHDL. Furthermore, transwell experiments showed that differences between native HDL, NO2-oxHDL and Cl-oxHDL was abolished after PD98059 (MAPK kinase inhibitor) treatment. In aortic SMCs from scavenger receptor BI (SR-BI) deficient mice, differences between migration of native HDL, NO2-oxHDL and Cl-oxHDL treated SMCs vanished, indicating SR-BI’s possible role in HDL-associated SMC migration. Importantly, NO2-oxHDL and Cl-oxHDL induced neointima formation and reduced SMC positive staining cells in atherosclerotic plaque, resulting in elevated vulnerable index of atherosclerotic plaque.ConclusionThese findings implicate MPO-catalyzed oxidization of HDL may contribute to atherosclerotic plaque instability by inhibiting SMC proliferation and migration through MAPK-ERK pathway which was dependent on SR-BI.

MiRNA-181a/b Regulates Phenotypes of Vessel Smooth Muscle Cells Through Serum Response Factor.

The phenotypic modulation of vessel smooth muscle cells (VSMCs) plays a crucial role in the physiological and pathological conditions of vasculature in response to local environmental changes. The phenotypic transition of VSMCs is largely modulated by the serum response factor (SRF). miR-181a and miR-181b are members of the well-studied miR-181 family and both have complementary sequence in the 3' untranslated region (UTR) of SRF gene. In this article, evidence insinuates that miR-181a/b was involved in VSMCs differentiation through upregulating synthetic marker genes and downregulating contractile ones, respectively. We also confirmed the roles of the miR-181a/b in promoting SMC proliferation, migration, and synthetic phenotype transformation. In addition, miR-181a/b was indicated as directly targeting at 3' UTR of SRF by dual-luciferase assay and Western blot assay. In a word, miR-181a/b is one of the factors involved in VSMC differentiation toward a synthetic phenotype through targeting at SRF. These findings may provide a potential therapeutic approach that miR-181a/b took part in regulating the vessel disorders.

Factor VII‐activating protease deficiency promotes neointima formation by enhancing leukocyte accumulation

Background Factor VII-activating protease (FSAP) is a multifunctional circulating plasma serine protease involved in thrombosis and vascular remodeling processes. The Marburg I single-nucleotide polymorphism (MI-SNP) in the FSAP-coding gene is characterized by low proteolytic activity, and is associated with increased rates of stroke and carotid stenosis in humans. Objectives To determine whether neointima formation after vascular injury is increased in FSAP-/- mice. Methods and Results The neointimal lesion size and the proliferation of vascular smooth muscle cells (VSMCs) were significantly enhanced in FSAP-/- mice as compared with C57BL/6 control mice after wire-induced injury of the femoral artery. Accumulation of leukocytes and macrophages was increased within the lesions of FSAP-/- mice at day 3 and day 14. Quantitative zymography demonstrated enhanced activity of gelatinases/matrix metalloproteinase (MMP)-2 and MMP-9 within the neointimal lesions of FSAP-/- mice, and immunohistochemistry showed particular costaining of MMP-9 with accumulating leukocytes. Using intravital microscopy, we observed that FSAP deficiency promoted the intravascular adherence and the subsequent transmigration of leukocytes in vivo in response to chemokine ligand 2 (CCL2). CCL2 expression was increased in FSAP-/- monocytes but not in the vessel wall. There was no difference in the expression of platelet-derived growth factor (PDGF-BB). Conclusions FSAP deficiency causes an increase in CCL2 expression and CCL2-mediated infiltration of leukocytes into the injured vessel, thereby promoting SMC proliferation and migration by the activation of leukocyte-derived gelatinases. These results provide a possible explanation for the observed association of the loss-of-function MI-SNP with vascular proliferative diseases.

Abstract 510: Perivascular-Cell Derived Oct4 is Essential for Angiogenesis Following Hindlimb Ischemia

Ischemic vascular diseases such as coronary, carotid, and peripheral artery disease (PAD) require vascular growth and remodeling in order to restore blood flow to ischemic tissue. Human therapeutic studies aimed at promoting neovascularization have been largely unsuccessful in part because these approaches have focused on stimulating endothelial cell (EC) growth without coordinate investment of smooth muscle cells and pericytes (SMC-P) needed for functional blood vessels that are stable and able to regulate perfusion. Thus, there is a critical need to identify mechanisms that mediate perivascular cell coverage during vascular network remodeling, a process that requires perivascular cell detachment, migration, and investment of endothelial tubes. Previous work from the Owens lab demonstrated that the stem cell pluripotency gene Oct4, previously thought to be permanently epigenetically silenced in all somatic cells, is reactivated in SMC within atherosclerotic lesions. SMC specific KO of Oct4 results in marked reductions in the number of SMC within lesions due to failure of cells to migrate out of the media. That is, Oct4 reactivation appears to be critical for SMC migration, at least in the context of atherosclerosis. Therefore, the present study tested the hypothesis that SMC-P reactivation of Oct4 is essential for neovascularization following hindlimb ischemia (HLI), a mouse model of PAD. To test this hypothesis, we used a unique mouse line enabling SMC-P specific tamoxifen-inducible lineage tracing (termed Myh11 eYFP), allowing their tracking following phenotypic switching and loss of identifying genes, combined with SMC-P specific knockout of Oct4. We subjected both Oct4WT and Oct4KO Myh11 eYFP mice to HLI and monitored perfusion recovery via Laser Doppler at days 0, 3, 7, 14, and 21 post-HLI. We found that SMC-P specific KO of Oct4 results in significantly impaired perfusion recovery at days 14 and 21 post-HLI. We harvested hindlimb muscle at day 21 post-HLI and observed that Oct4 KO mice have significantly impaired angiogenesis, but intact arteriogenesis, compared to WT controls. Future studies will investigate Oct4 downstream targets necessary for angiogenesis following HLI.

Abstract 14743: Intracellular Adenosine Suppresses Vsmc Phenotypic Switch Through Klf4 Gene Methylation

Background and objective: It is well known that adenosine affects the growth of vascular smooth muscle cells via adenosine receptors. Adenosine kinase (ADK) is an intracellular enzyme regulating the levels of adenosine in intracellular and further extracellular compartments. The effect of ADK on VSMC phenotypic switch is unknown. In this study, we evaluate phenotypic changes of VSCMs both in vitro and in vivo following deletion of ADK in VSMCs. Methods and results: The expression of ADK in vascular wall was significant upregulated in mouse carotid artery ligation model. Heterogeneous deletion of ADK or specific ADK deletion in VSMCs led to remarkably decreased neointima in ligated mouse carotid arteries (Fig. A&B). This was accompanied with increased expression of VSMC differentiation markers and transcriptional factor KLF4 (Fig. D). In an in vitro setup, treatment of VSMCs with ADK shRNA or ADK inhibitors suppressed serum or PDGF-induced SMC proliferation and migration (Fig. C). This effect remained intact in the presence of adenosine receptor antagonists to A1, A2A, A2B and A3. Deletion of ADK in VSMCs increased the levels of intracellular adenosine, decreased the methylation levels of genes and histones, and augmented KLF4 expression. It is being investigated that upregulation of KLF4 was resulted from its gene hypomethylation in response to elevated intracellular adenosine following ADK deletion/knockdown or inhibition. Conclusions: Our data demonstrate that ADK deletion/inhibition suppresses neointima formation in injured arteries through a novel epigenetic pathway but not classic adenosine receptors, and ADK is a promising therapeutic target in the treatment of arterial diseases such as atherosclerosis and arterial neointima formation following arterial angioplasty.

MiR-138 promotes smooth muscle cells proliferation and migration in db/db mice through down-regulation of SIRT1

BackgroundDiabetic vascular smooth muscle cells (VSMCs) exhibit significantly increased rates of proliferation and migration, which was the most common pathological change in atherosclerosis. In addition, the study about the role for miRNAs in the regulation of VSMC proliferation is just beginning to emerge and additional miRNAs involved in VSMC proliferation modulation should be identified. MethodsThe expression of miR-138 and SIRT1 were examined in SMCs separated from db/db mice and in SMC lines C-12511 exposed to high glucose with qRT-PCR and western blot. The regulation of miR-138 on the expression of SMCs was detected with luciferase report assay. VSMCs proliferation and migration assays were performed to examine the effect of miR-138 inhibitor on VSMCs proliferation and migration. ResultsWe discovered that higher mRNA level of miR-138 and reduced expression of SIRT1 were observed in SMCs separated from db/db mice and in SMC lines C-12511. Moreover, luciferase report assay showed that the activity of SIRT1 3′-UTR was highly increased by miR-138 inhibitor and reduced by miR-138 mimic. In addition, we examined that the up-regulation of NF-κB induced by high glucose in SMCs was reversed by resveratrol and miR-138 inhibitor. MTT and migration assays showed that miR-138 inhibitor attenuated the proliferation and migration of smooth muscle cells. ConclusionIn this study, we revealed that miR-138 might promote proliferation and migration of SMC in db/db mice through suppressing the expression of SIRT1.

Abstract 281: Local MicroRNA Modulation Using a Novel Anti-mir-21-eluting Stent Effectively Prevents In-stent Restenosis

The major limitation of vascular angioplasty remains the occurrence of in-stent restenosis (ISR). The central role of miRNAs in the pathophysiology of the proliferative response in cardiovascular disease offers attractive concepts for possible therapeutic interventions, with systemic miRNA modulation however potentially causing substantial off-target effects. Aim of the current study was to test the feasibility of local miRNA modulation using drug eluted stent (DES) technology. Utilizing human ISR tissue samples, as well as a humanized rodent model of balloon-injured human internal mammary arteries with subsequent stent implantation in RNU rats, we were able to identify several miRNAs dys-regulated, including the well-characterized miR-21. We inhibited miR-21 (with a FAM-tagged-LNA-anti-miR-21), using either systemic or local delivery via DES. Systemic suppression of miR-21 showed a dose-dependent reduction of luminal obliteration, while not impeding vascular re-endothelialization. However, miR-21 expression in liver, heart, lung, and kidney appeared significantly reduced, with increased levels of serum creatinine, pointing towards the expected off-target effects of systemic miRNA modulation. Local mir-21 suppression, using the anti-miR-21-DES, effectively reduced myointimal hyperplasia and ISR compared to bare metal stents, while not exerting detectable off-target effects on any other organ we investigated. Additional in vitro experiments in cultured human coronary artery smooth muscle and endothelial cells further confirmed the ability of anti_miR-21 to limit SMC proliferation and migration by repressing mRNA and protein levels of its established target PTEN, while not affecting the proliferative response in the ECs. This is the first experimental study to demonstrate the efficacy, feasibility and suitability of an anti-miRNA-eluting stent (anti-miR-21-DES) for the reduction of ISR through dominant inhibition of SMC proliferation.

8. Thrombomodulin Demonstrates Critical Beneficial Direct Effects On Smooth Muscle Cell Physiology

Introduction and Objectives: Following vascular injury, critical endothelial cell (EC) function is lost. Thrombomodulin (TM), an endothelial anticoagulant, is one lost regulator. TM has known effects through inhibition of thrombin, but here we sought to examine direct, independent effects of TM on SMC physiology. We hypothesized that exogenous TM would induce a favorable SMC phenotype and would inhibit SMC migration. Methods: Primary human saphenous vein SMC were explanted and used in early passage (1-4). SMCmigration was measured using: (1) a 4-hour Boyden-chamber chemotaxis assay, and (2) a 24-hour ElectricCell-substrate Impedance Sensing (ECIS) wound assay.Migration experiments were conducted with serum-starved SMC exposed to increasing doses of soluble human TM and results quantified by cell counts. Thrombin served as positive control and serum-free media as negative control. Dose-dose comparisons were performed using a t-test (a< .05). Multiphoton laser scanning microscopy was used to assess the effect of exogenous TM on SMC phenotype. Results: SMC demonstrated low, baseline migration in serum-free conditions. Thrombin significantly stimulated SMCmigration as expected. TM, independent of thrombin, significantly inhibited SMC migration in a dose-dependent fashion (Figure 1). 70% reduction was observed at 5mg/mL (56±1.7 vs. 18±3.5 cells/5hpf, P1⁄4 .0005). SMC exposed to TM demonstrated a spindle-shapedmorphology with organized stress fibers consistent with a differentiated, contractile phenotype, whereas, control thrombin stimulation lead to a dedifferentiated, synthetic phenotype (Figure 2). Conclusions: Thrombomodulin demonstrated direct effects on SMC physiology, independent from thrombin, includingmaintenance of a differentiated, contractile phenotype and inhibition of migration. These findings provide new knowledge in understanding the biology of vascular injury, and supports a strategy focusedon restoring key endothelial function to prevent intimal hyperplasia.

Regulation of mitochondrial morphology by positive feedback interaction between PKCδ and Drp1 in vascular smooth muscle cell.

Dynamin-related protein-1 (Drp1) plays a critical role in mitochondrial fission which allows cell proliferation and Mdivi-1, a specific small molecule Drp1 inhibitor, is revealed to attenuate proliferation. However, few molecular mechanisms-related to Drp1 under stimulus for restenosis or atherosclerosis have been investigated in vascular smooth muscle cells (vSMCs). Therefore, we hypothesized that Drp1 inhibition can prevent vascular restenosis and investigated its regulatory mechanism. Angiotensin II (Ang II) or hydrogen peroxide (H2 O2 )-induced proliferation and migration in SMCs were attenuated by down-regulation of Drp1 Ser 616 phosphorylation, which was demonstrated by in vitro assays for migration and proliferation. Excessive amounts of ROS production and changes in mitochondrial membrane potential were prevented by Drp1 inhibition under Ang II and H2 O2 . Under the Ang II stimulation, activated Drp1 interacted with PKCδ and then activated MEK1/2-ERK1/2 signaling cascade and MMP2, but not MMP9. Furthermore, in ex vivo aortic ring assay, inhibition of the Drp1 had significant anti-proliferative and -migration effects for vSMCs. A formation of vascular neointima in response to a rat carotid artery balloon injury was prevented by Drp1 inhibition, which shows a beneficial effect of Drp1 regulation in the pathologic vascular condition. Drp1-mediated SMC proliferation and migration can be prevented by mitochondrial division inhibitor (Mdivi-1) in in vitro, ex vivo and in vivo, and these results suggest the possibility that Drp1 can be a new therapeutic target for restenosis or atherosclerosis.

Pulmonary artery smooth muscle cell endothelin-1 expression modulates the pulmonary vascular response to chronic hypoxia.

Endothelin-1 (ET-1) increases pulmonary vascular tone through direct effects on pulmonary artery smooth muscle cells (PASMC) via membrane-bound ET-1 receptors. Circulating ET-1 contributes to vascular remodeling by promoting SMC proliferation and migration and inhibiting SMC apoptosis. Although endothelial cells (EC) are the primary source of ET-1, whether ET-1 produced by SMC modulates pulmonary vascular tone is unknown. Using transgenic mice created by crossbreeding SM22α-Cre mice with ET-1(flox/flox) mice to selectively delete ET-1 in SMC, we tested the hypothesis that PASMC ET-1 gene expression modulates the pulmonary vascular response to hypoxia. ET-1 gene deletion and selective activity of SM22α promoter-driven Cre recombinase were confirmed. Functional assays were performed under normoxic (21% O2) or hypoxic (5% O2) conditions using murine PASMC obtained from ET-1(+/+) and ET-1(-/-) mic and in human PASMC (hPASMC) after silencing of ET-1 using siRNA. Under baseline conditions, there was no difference in right ventricular systolic pressure (RVSP) between SM22α-ET-1(-/-) and SM22α-ET-1(+/+) (control) littermates. After exposure to hypoxia (10% O2, 21-24 days), RVSP was and vascular remodeling were less in SM22α-ET-1(-/-) mice compared with control littermates (P < 0.01). Loss of ET-1 decreased PASMC proliferation and migration and increased apoptosis under normoxic and hypoxic conditions. Exposure to selective ET-1 receptor antagonists had no effect on either the hypoxia-induced hPASMC proliferative or migratory response. SMC-specific ET-1 deletion attenuates hypoxia-induced increases in pulmonary vascular tone and structural remodeling. The observation that loss of ET-1 inhibited SMC proliferation, survival, and migration represents evidence that ET-1 derived from SMC plays a previously undescribed role in modulating the response of the pulmonary circulation to hypoxia. Thus PASMC ET-1 may modulate vascular tone independently of ET-1 produced by EC.

Integration of proteomic and transcriptomic profiles identifies a novel PDGF-MYC network in human smooth muscle cells.

BackgroundPlatelet-derived growth factor-BB (PDGF-BB) has been implicated in the proliferation, migration and synthetic activities of smooth muscle cells that characterize physiologic and pathologic tissue remodeling in hollow organs. However, neither the molecular basis of PDGFR-regulated signaling webs, nor the extent to which specific components within these networks could be exploited for therapeutic benefit has been fully elucidated.ResultsExpression profiling and quantitative proteomics analysis of PDGF-treated primary human bladder smooth muscle cells identified 1,695 genes and 241 proteins as differentially expressed versus non-treated cells. Analysis of gene expression data revealed MYC, JUN, EGR1, MYB, RUNX1, as the transcription factors most significantly networked with up-regulated genes. Forty targets were significantly altered at both the mRNA and protein levels. Proliferation, migration and angiogenesis were the biological processes most significantly associated with this signature, and MYC was the most highly networked master regulator. Alterations in master regulators and gene targets were validated in PDGF-stimulated smooth muscle cells in vitro and in a model of bladder injury in vivo. Pharmacologic inhibition of MYC and JUN confirmed their role in SMC proliferation and migration. Network analysis identified the diaphanous-related formin 3 as a novel PDGF target regulated by MYC and JUN, which was necessary for PDGF-stimulated lamellipodium formation.ConclusionsThese findings provide the first systems-level analysis of the PDGF-regulated transcriptome and proteome in normal smooth muscle cells. The analyses revealed an extensive cohort of PDGF-dependent biological processes and connected key transcriptional effectors to their regulation, significantly expanding current knowledge of PDGF-stimulated signaling cascades. These observations also implicate MYC as a novel target for pharmacological intervention in fibroproliferative expansion of smooth muscle, and potentially in cancers in which PDGFR-dependent signaling or MYC activation promote tumor progression.

Anti-atherosclerotic effect of geniposidic acid in a rabbit model and related cellular mechanisms

Context: Geniposidic acid, one of the main active ingredients in Gardenia jasminoides J. Ellis (Rubiaceae), may also possess important pharmacological activities for cardiovascular disorders similar to other derivatives, such as geniposide.Objective: To evaluate its anti-atherosclerosis (anti-AS) effect, the related pharmacological activities and possible cellular mechanisms were studied.Materials and methods: Thirty rabbits were randomly divided into normal control group, model control group, and geniposidic acid subgroups. In the AS model, its effects on the intima/media thickness ratio and aortic morphology were observed. In the study of primary cultured endothelial cells (ECs) and human umbilical artery smooth muscle cells (HUASMCs), its activities on both ECs and HUASMCs proliferation, HUASMCs' migration were also studied.Results: Compared with the model control group, the plaque area, intima/media thickness ratio, and intimal foam cells number in geniposidic acid (80, 160, and 240 mg/kg) subgroups were significantly improved (p < 0.05). By HE staining, the activities of geniposidic acid on relieving ECs shedding and improving aortic morphology disorders were also demonstrated. From the results of CCK-8 testing, only 100 μg/ml geniposidic acid performed significant inhibition on SMC proliferation. The relative IC50 of geniposidic acid on SMC inhibition was 87.73 μg/ml. Geniposide acid also showed promotion effect on ECs proliferation, and the related ED50 of geniposidic acid was 86.05 μg/ml. Besides, only 50 and 100 μg/ml geniposidic acid showed obvious inhibition on SMC migration from the upper chamber (p < 0.05).Discussion and conclusion: The effects of geniposidic acid on protecting vascular endothelium and reversing plaque formation in an atherosclerotic model were demonstrated.

Injury-induced vascular intimal hyperplasia is mediated through a HMGB1- and TLR4- driven mechanism (HUM1P.304)

Abstract Aim: To determine whether high-mobility group box 1 (HMGB1) and Toll like receptor 4 (TLR4) drive the inflammatory cascade that promotes intimal hyperplasia (IH) following acute vascular injury. Methods and Results: Carotid artery wire injury in C57BL/6 mice induced a significant increase in intima to media (I/M) ratio at four weeks. Global deletion of HMGB1 using an inducible knockout mouse strain caused prevention of IH. IH was decreased by over 50% in WT mice treated with HMGB1 neutralizing antibody. Of knockout mouse strains deficient in putative receptors for HMGB1, TLR4-/- mice showed the greatest inhibition of IH. Both anti-HMGB1 treated mice and TLR4-/- mice exhibited a marked decrease in monocytic recruitment. Mice with selective depletion of TLR4 from macrophage exhibited a similar level of inhibition of IH to that seen in the global TLR4-/-. In vitro, dithiol HMGB1 dose-dependently promoted SMC migration and MCP-1/CCR2 expression, which was abolished by TLR4 inhibitory peptide. Conditioned media from HMGB1 treated macrophage induced SMC proliferation and SMC mitogen production, which was also diminished by blocking TLR4 on macrophages. Conclusion: Innate immune mechanism known to be involved in the sterile inflammatory response are involved in the acute vascular injury responses leading to IH. HMGB1 promotes IH by inducing vascular SMC migration and chemokine production via TLR4 pathway while simultaneously triggering SMC mitogen release by infiltrating monocytes.

Thromboxane A(2) receptor stimulation promotes closure of the rat ductus arteriosus through enhancing neointima formation.

Ductus arteriosus (DA) closure follows constriction and remodeling of the entire vessel wall. Patent ductus arteriosus occurs when the DA does not close after birth, and this condition is currently treated using cyclooxygenase inhibitors. However, the efficacy of cyclooxygenase inhibitors is often limited. Our previous study demonstrated that low-dose thromboxane A2 receptor (TP) stimulation constricted the DA with minimal adverse effects in rat neonates. However, its effect on DA remodeling remains unknown. In this study, we focused on the impact of the exogenous TP stimulation on the DA remodeling, especially intimal thickening. Using DA explants from rat fetuses at embryonic day 19 as a ex vivo model and primary cultured rat DA smooth muscle cells from embryonic day 21 as a in vitro model, we evaluated the effect of TP stimulation on the DA remodeling. The selective TP agonists U46619 and I-BOP promoted neointima formation in the ex vivo DA explants, and TP stimulation increased DA SMC migration in a dose-dependent manner. Both effects were inhibited by the selective TP antagonist SQ29548 or the siRNA against TP. TP stimulation also increased DA SMC proliferation in the presence of 10% fetal bovine serum. LC/MS/MS analysis revealed that TP stimulation increased secretion of several extracellular matrix proteins that may contribute to an increase in neointima formation. In conclusion, we uncovered that exogenous administration of TP agonist promotes neointima formation through the induction of migration and proliferation of DA SMC, which could contribute to DA closure and also to its vasoconstrictive action.

Integration of proteomic and transcriptomic profiles identifies a novel PDGF-MYC network in human smooth muscle cells

Platelet-derived growth factor-BB (PDGF-BB) has been implicated in the proliferation, migration and synthetic activities of smooth muscle cells that characterize physiologic and pathologic tissue remodeling in hollow organs. However, neither the molecular basis of PDGFR-regulated signaling webs, nor the extent to which specific components within these networks could be exploited for therapeutic benefit has been fully elucidated. Expression profiling and quantitative proteomics analysis of PDGF-treated primary human bladder smooth muscle cells identified 1,695 genes and 241 proteins as differentially expressed versus non-treated cells. Analysis of gene expression data revealed MYC, JUN, EGR1, MYB, RUNX1, as the transcription factors most significantly networked with up-regulated genes. Forty targets were significantly altered at both the mRNA and protein levels. Proliferation, migration and angiogenesis were the biological processes most significantly associated with this signature, and MYC was the most highly networked master regulator. Alterations in master regulators and gene targets were validated in PDGF-stimulated smooth muscle cells in vitro and in a model of bladder injury in vivo. Pharmacologic inhibition of MYC and JUN confirmed their role in SMC proliferation and migration. Network analysis identified the diaphanous-related formin 3 as a novel PDGF target regulated by MYC and JUN, which was necessary for PDGF-stimulated lamellipodium formation. These findings provide the first systems-level analysis of the PDGF-regulated transcriptome and proteome in normal smooth muscle cells. The analyses revealed an extensive cohort of PDGF-dependent biological processes and connected key transcriptional effectors to their regulation, significantly expanding current knowledge of PDGF-stimulated signaling cascades. These observations also implicate MYC as a novel target for pharmacological intervention in fibroproliferative expansion of smooth muscle, and potentially in cancers in which PDGFR-dependent signaling or MYC activation promote tumor progression.

Decorin mimic inhibits vascular smooth muscle proliferation and migration.

Over the past 10 years, the number of percutaneous coronary intervention procedures performed in the United States increased by 33%; however, restenosis, which inhibits complete functional recovery of the vessel wall, complicates this procedure. A wide range of anti-restenotic therapeutics have been developed, although many elicit non-specific effects that compromise vessel healing. Drawing inspiration from biologically-relevant molecules, our lab developed a mimic of the natural proteoglycan decorin, termed DS-SILY, which can mask exposed collagen and thereby effectively decrease platelet activation, thus contributing to suppression of vascular intimal hyperplasia. Here, we characterize the effects of DS-SILY on both proliferative and quiescent human SMCs to evaluate the potential impact of DS-SILY-SMC interaction on restenosis, and further characterize in vivo platelet interactions. DS-SILY decreased proliferative SMC proliferation and pro-inflammatory cytokine secretion in vitro in a concentration dependent manner as compared to untreated controls. The addition of DS-SILY to in vitro SMC cultures decreased SMC migration and protein synthesis by 95% and 37%, respectively. Furthermore, DS-SILY decreased platelet activation, as well as reduced neointimal hyperplasia by 60%, in vivo using Ossabaw swine. These results indicate that DS-SILY demonstrates multiple biological activities that may all synergistically contribute to an improved treatment paradigm for balloon angioplasty.