Wire Injury Research Articles

Loss of platelet BMP4 reduces vascular inflammation and ameliorates vascular remodelling after carotid wire injury

Abstract Background Bone morphogenic proteins (BMPs) are members of the TGFβ superfamily. They have distinct functions during hemostasis and play a central role in various pathologic conditions, including cardiovascular diseases. Recent findings demonstrate that BMPs are also expressed in platelets. However, their function is poorly understood. Here, we investigate the role of platelet BMP4 during vascular inflammation and vascular remodelling. Methods BMP4 floxed mice were crossed with PF4 Cre mice to generate platelet-specific deletion of BMP4 (BMP4Plt−/−). Intravital microscopy of mesenteric veins was performed to evaluate leukocyte adhesion upon stimulation with TNFα. Expression of adhesion molecules and chemokines were analysed by RT-PCR and Western Blot. P-selectin and platelet-leukocyte aggregates were evaluated using flow cytometry. For carotid wire injury, BMP4Plt−/− were further crossed with LDLr−/− mice (BMP4Plt−/−/LDLr−/−). At 8 weeks of age, BMP4Plt−/−/LDLr−/− mice and control littermates received a 2-week diet containing 15.8% wt/wt fat and 1.25% cholesterol. Carotid wire injury was performed at the age of 10 weeks. Re-endothelialisation and neointimal hyperplasia were evaluated. Results Platelet morphology and function did not differ between BMP4Plt−/− and control mice. Stimulation with TNFα resulted in increased rolling and adherence of leukocytes to the vessel wall which was reduced in BMP4Plt−/− mice (175±25 versus 50±7 rolling cells and 16±3 versus 7±2 adherent cells, respectively). Expression of P-selectin, adhesion molecules and the chemokines RANTES and PF-4 were reduced in BMP4Plt−/− mice. Platelet activation by thrombin was reduced in BMP4Plt−/− mice, resulting in diminished P-Selectin and JONA expression. Furthermore, monocyte infiltration and circulating leukocyte-platelet complexes were reduced in BMP4Plt−/− mice. Loss of platelet BMP4 prevented neointima formation after carotid wire injury (4.2x104±0.9x104μm2 versus 14.9x104±2.2x104μm2 in BMP4Plt−/−/LDLr−/− and control mice, respectively). Interestingly, endothelial regeneration after injury was decelerated in BMP4Plt−/− mice. This is further demonstrated in-vitro, where platelet BMP4 promoted endothelial cell proliferation and migration. Conclusion Platelet-BMP4 deficiency reduced vascular inflammation and ameliorated intima hyperplasia after wire injury. This is partly mediated by inhibition of platelet activation, reduced expression of adhesion molecules and inflammatory response. Our finding suggests that BMP4 is a promising target for the treatment of vascular inflammation and restenosis. Funding Acknowledgement Type of funding source: Private grant(s) and/or Sponsorship. Main funding source(s): Else-Kröner Fresenius Foundation; German Cardiac Society

Endothelial-specific Ablation of Non-nuclear Estrogen Receptor alpha Signaling Deteriorates Vascular Remodelling Response

Abstract Background and introduction The difference in cardiovascular disease risk between age-matched women and men narrows as transition through menopause in observational studies. Estrogen exerts complex physiological effects via its non-nuclear and nuclear actions. Experimental studies have shown that endothelial estrogen receptors mediate vasoprotection via endothelial nitric oxide production, reendothelialization, and atherosclerosis. Prior studies in vitro addressed estrogen's effects on endothelial cells and vascular smooth muscle cells, leading to vasoprotection. However, the in vivo evidences are lacking for beneficial effects of endothelium non-nuclear ERα signaling on vascular remodelling in response to injury. Purpose This study aims to clarify the impact of endothelial ERα non-nuclear signaling in the vasoprotection, using a novel mouse model lacking tissue-specific ERα non-nuclear signaling. Methods We identified the amino acids of ERα which were responsible for its binding to p85α subunit of phosphatidylinositol 3-kinase in vitro. We generated a novel mouse model in which non-nuclear signaling of ERα was ablated in endothelial cells by crossing Tie2-Cre transgenic mice with floxed ERα mutants (RR259/260AA) in which p85α and ERα interaction was disrupted. Results In endothelial cells isolated from ERαKI/KITie2 cre/+ animals, E2 failed to induce phosphorylation of Akt, confirming the absence of ERα non-nuclear signaling. Baseline characteristics at 8 to 12 weeks of age were undistinguishable between the genotypes, including body weight, systolic blood pressure, uterine weight and echocardiographic fractional shortening. We then assessed how vascular remodelling process was impacted in a carotid artery wire injury model. Histological analyses with Elastica van Gieson staining two weeks after injury revealed that estrogen dependent suppression of remodelling response (intima to medial ratio) was abolished in ERαki/kiTie2cre/+mice (P=0.0004). Masson's Trichrome staining showed that in the presence of E2 fibrosis was significantly higher in ERαki/kiTie2cre/+ mice than ERαki/kiTie2cre/− mice (P=0.0015). Conclusions We generated a novel mouse model for tissue-specific ablation of ERα non-nuclear signaling by interfering ERα-PI3K interaction. Our results demonstrate that the pivotal role for ERα non-nuclear signaling of endothelial cells in carotid arterial protection following injury with its minimal impact on baseline cardiovascular phenotype. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): Japan Heart Foundation Research Grant, SENSHIN Medical Research Foundation

Inhibitory effect of CP-25 on intimal formation and vascular hyperplasia via suppression of GRK2/ERK1/2/EVI1 signaling

Excessive proliferation, migration and dedifferentiation of vascular smooth muscle cells (VSMCs) are the center of intimal formation during in-stent restenosis and vein graft disease. Paeoniflorin-6′-O-benzene sulfonate (CP-25) is known to suppress inflammation and atherogenesis. However, the potential effect of CP-25 on intimal formation remains elusive. In the present study, we found that CP-25 significantly attenuated wire injury-induced intimal formation in C57BL/6 mice (intimal area: 2.64 ± 0.25 × 104 μm2 vs. 1.53 ± 0.21 × 104 μm2, P < 0.05) and vascular hyperplasia indicated by PCNA staining. In vitro experiments showed that CP-25 significantly alleviated human aortic smooth muscle cell (HASMC) proliferation, migration and dedifferentiation induced by PDGF-BB. Mechanistically, CP-25 inhibited GRK2 phosphorylation through PDGF receptor in the presence of PDGF-BB. In accordance with these results, CP-25 disrupted the interaction of GRK2 with ERK1/2 and suppressed the activation of ERK1/2 signaling in HASMCs. EVI1, which is considered as a downstream of ERK1/2 signaling and a novel transcription factor for VSMC differentiation, was also downregulated by CP-25 treatment. Moreover, overexpression of EVI1 partly restored the decreased proliferation and dedifferentiation of HASMCs treated by CP-25. Collectively, these findings suggested that CP-25 could alleviate intimal formation in response to wire injury via suppression of the interaction of GRK2 and ERK1/2 and EVI1 activation, indicating CP-25 might serve as a potent pharmaceutical for intimal formation.

The cyclic adenosine monophosphate elevating medicine, forskolin, reduces neointimal formation and atherogenesis in mice.

Neointimal formation and atherogenesis are major vascular complications following percutaneous coronary intervention, and there is lack of pharmacological therapy. This study was aimed to examine the effect of forskolin (FSK), a cyclic adenosine monophosphate (cAMP)‐elevating agent, on vascular response to angioplasty wire injury and on atherogenesis in mice. Forskolin treatment reduced neointima formation at 7 and 28 days after wire injury. Early morphometrics of the injured vessels revealed that FSK treatment enhanced endothelial repair and reduced inflammatory cell infiltration. In vitro treatment of primary aortic cells with FSK, at 3‐100 μmol/L, increased endothelial cell proliferation, whereas FSK, at 30‐100 μmol/L, inhibited smooth muscle cell proliferation. FSK inhibited lipopolysaccharide‐induced leucocyte‐endothelial interaction in vitro and in vivo. In a mouse model of atherosclerosis driven by dyslipidaemia and hypertension, FSK administration increased endothelial repair and reduced atherosclerotic plaque formation, without affecting blood pressure, plasma lipids or aortic aneurysms formation. In summary, FSK, at doses relevant to human therapeutic use, protects against neointimal hyperplasia and atherogenesis, and this is attributable to its activities on pro‐endothelial repair and anti‐inflammation. This study raises a potential of clinical use of FSK as an adjunct therapy to prevent restenosis and atherosclerosis after percutaneous coronary intervention.

Management of colorectal anastomotic stricture with multidiameter balloon dilation: long-term results

BackgroundPostoperative colorectal anastomotic strictures are quite common. As such, many techniques have been available to address such a problem, one of which is endoscopic dilation. The aim of the present study was to evaluate the long-term outcomes following endoscopic dilation using a multidiameter balloon.MethodsA retrospective study was conducted on patients with postoperative anastomotic stenosis treated with endoscopic dilation using a multidiameter balloon at our institution, in January 2005–December 2019 were retrospectively reviewed, excluding those with tumor recurrence. Perioperative factors, complications, and recurrence rates were analyzed.ResultsThere were 40 patients, (22 males and 18 females, mean age 64.6 ± 10.7 years, range 33–84 years). The median follow-up period was 56 months (interquartile range 22.5–99 months). Only 1 complication occurred, micro-perforation due to guided wire injury, which was managed conservatively. Five (12.5%) patients developed restenosis and underwent repeat balloon dilation. None of the five recurrences required more aggressive management, such as redo anastomosis.ConclusionsEndoscopic multidiameter balloon dilation is a safe and effective method for treating benign colorectal anastomotic strictures.

Macrophage NCOR1 Deficiency Ameliorates Myocardial Infarction and Neointimal Hyperplasia in Mice.

BackgroundNCOR1 (nuclear receptor corepressor 1) is an essential coregulator of gene transcription. It has been shown that NCOR1 in macrophages plays important roles in metabolic regulation. However, the function of macrophage NCOR1 in response to myocardial infarction (MI) or vascular wire injury has not been elucidated.Methods and ResultsHere, using macrophage Ncor1 knockout mouse in combination with a mouse model of MI, we demonstrated that macrophage NCOR1 deficiency significantly reduced infarct size and improved cardiac function after MI. In addition, macrophage NCOR1 deficiency markedly inhibited neointimal hyperplasia and vascular remodeling in a mouse model of arterial wire injury. Inflammation and macrophage proliferation were substantially attenuated in hearts and arteries of macrophage Ncor1 knockout mice after MI and arterial wire injury, respectively. Cultured primary macrophages from macrophage Ncor1 knockout mice manifested lower expression of inflammatory genes upon stimulation by interleukin‐1β, interleukin‐6, or lipopolysaccharide, together with much less activation of inflammatory signaling cascades including signal transducer and activator of transcription 1 and nuclear factor‐κB. Furthermore, macrophage Ncor1 knockout macrophages were much less proliferative in culture, with inhibited cell cycle progression compared with control cells.ConclusionsCollectively, our data have demonstrated that NCOR1 is a critical regulator of macrophage inflammation and proliferation and that deficiency of NCOR1 in macrophages attenuates MI and neointimal hyperplasia. Therefore, macrophage NCOR1 may serve as a potential therapeutic target for MI and restenosis.

A Rat Carotid Artery Pressure-Controlled Segmental Balloon Injury with Periadventitial Therapeutic Application.

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the luminal surface area in arteries thereby reducing adequate blood flow to organs and distal tissues. Clinically, revascularization procedures such as balloon angioplasty with or without stent placement aim to restore blood flow. Although these procedures reestablish blood flow by reducing plaque burden, they damage the vessel wall, which initiates the arterial healing response. The prolonged healing response causes arterial restenosis, or re-narrowing, ultimately limiting the long-term success of these revascularization procedures. Therefore, preclinical animal models are integral for analyzing the pathophysiological mechanisms driving restenosis, and provide the opportunity to test novel therapeutic strategies. Murine models are cheaper and easier to operate on than large animal models. Balloon or wire injury are the two commonly accepted injury modalities used in murine models. Balloon injury models in particular mimic the clinical angioplasty procedure and cause adequate damage to the artery for the development of restenosis. Herein we describe the surgical details for performing and histologically analyzing the modified, pressure-controlled rat carotid artery balloon injury model. Additionally, this protocol highlights how local periadventitial application of therapeutics can be used to inhibit neointimal hyperplasia. Lastly, we present light sheet fluorescence microscopy as a novel approach for imaging and visualizing the arterial injury in three-dimensions.

Restoring the Platelet miR-223 by Calpain Inhibition Alleviates the Neointimal Hyperplasia in Diabetes.

Platelet hyperactivity is the hallmark of diabetes, and platelet activation plays a crucial role in diabetic vascular complications. Recent studies have shown that upon activation, platelet-derived miRNAs are incorporated into vascular smooth muscle cells (VSMCs), regulating the phenotypic switch of VSMC. Under diabetes, miRNA deficiency in platelets fails to regulate the VSMC phenotypic switch. Therefore, manipulation of platelet-derived miRNAs expression may provide therapeutic option for diabetic vascular complications. We seek to investigate the effect of calpeptin (calpain inhibitor) on the expression of miRNAs in diabetic platelets, and elucidate the downstream signaling pathway involved in protecting from neointimal formation in diabetic mice with femoral wire injury model. Using human cell and platelet coculture, we demonstrate that diabetic platelet deficient of miR-223 fails to suppress VSMC proliferation, while overexpression of miR-223 in diabetic platelets suppressed the proliferation of VSMC to protect intimal hyperplasia. Mechanistically, miR-223 directly targets the insulin-like growth factor-1 receptor (IGF-1R), which inhibits the phosphorylation of GSK3β and activates the phosphorylation of AMPK, resulting in reduced VSMC dedifferentiation and proliferation. Using a murine model of vascular injury, we show that calpeptin restores the platelet expression of miR-223 in diabetes, and the horizontal transfer of platelet miR-223 into VSMCs inhibits VSMC proliferation in the injured artery by targeting the expression of IGF-1R. Our data present that the platelet-derived miR-223 suppressed VSMC proliferation via the regulation miR-223/IGF-1R/AMPK signaling pathways, and inhibition of calpain alleviates neointimal formation by restoring the expression of miR-223 in diabetic platelet.

Microbe-Derived Butyrate and Its Receptor, Free Fatty Acid Receptor 3, But Not Free Fatty Acid Receptor 2, Mitigate Neointimal Hyperplasia Susceptibility After Arterial Injury.

BackgroundArterial restenosis after vascular surgery is a common cause of midterm restenosis and treatment failure. Herein, we aim to investigate the role of microbe‐derived butyrate, FFAR2 (free fatty acid receptor 2), and FFAR3 (free fatty acid receptor 3) in mitigating neointimal hyperplasia development in remodeling murine arteries after injury.Methods and ResultsC57BL/6 mice treated with oral vancomycin before unilateral femoral wire injury to deplete gut microbiota had significantly diminished serum and stool butyrate and more neointimal hyperplasia development after arterial injury, which was reversed by concomitant butyrate supplementation. Deficiency of FFAR3 but not FFAR2, both receptors for butyrate, exacerbated neointimal hyperplasia development after injury. FFAR3 deficiency was also associated with delayed recovery of the endothelial layer in vivo. FFAR3 gene expression was observed in multiple peripheral arteries, and expression was increased after arterial injury. Treatment of endothelial but not vascular smooth muscle cells with the pharmacologic FFAR3 agonist 1‐methylcyclopropane carboxylate stimulated cellular migration and proliferation in scratch assays.ConclusionsOur results support a protective role for butyrate and FFAR3 in the development of neointimal hyperplasia after arterial injury and delineate activation of the butyrate‐FFAR3 pathway as a valuable strategy for the prevention and treatment of neointimal hyperplasia.

Omega-3 Polyunsaturated Fatty Acids Decrease Aortic Valve Disease Through the Resolvin E1 and ChemR23 Axis.

Aortic valve stenosis (AVS), which is the most common valvular heart disease, causes a progressive narrowing of the aortic valve as a consequence of thickening and calcification of the aortic valve leaflets. The beneficial effects of omega-3 polyunsaturated fatty acids (n-3 PUFAs) in cardiovascular prevention have recently been demonstrated in a large randomized, controlled trial. In addition, n-3 PUFAs serve as the substrate for the synthesis of specialized proresolving mediators, which are known by their potent beneficial anti-inflammatory, proresolving, and tissue-modifying properties in cardiovascular disease. However, the effects of n-3 PUFA and specialized proresolving mediators on AVS have not yet been determined. The aim of this study was to identify the role of n-3 PUFA-derived specialized proresolving mediators in relation to the development of AVS. Lipidomic and transcriptomic analyses were performed in human tricuspid aortic valves. Apoe-/- mice and wire injury in C57BL/6J mice were used as models for mechanistic studies. We found that n-3 PUFA incorporation into human stenotic aortic valves was higher in noncalcified regions compared with calcified regions. Liquid chromatography tandem mass spectrometry-based lipid mediator lipidomics identified that the n-3 PUFA-derived specialized proresolving mediator resolvin E1 was dysregulated in calcified regions and acted as a calcification inhibitor. Apoe-/- mice expressing the Caenorhabditis elegans Fat-1 transgene (Fat-1tg×Apoe-/-), which enables the endogenous synthesis of n-3 PUFA and increased valvular n-3 PUFA content, exhibited reduced valve calcification, lower aortic valve leaflet area, increased M2 macrophage polarization, and improved echocardiographic parameters. Finally, abrogation of the resolvin E1 receptor ChemR23 enhanced disease progression, and the beneficial effects of Fat-1tg were abolished in the absence of ChemR23. n-3 PUFA-derived resolvin E1 and its receptor ChemR23 emerge as a key axis in the inhibition of AVS progression and may represent a novel potential therapeutic opportunity to be evaluated in patients with AVS.

Macrophage (Drp1) Dynamin-Related Protein 1 Accelerates Intimal Thickening After Vascular Injury.

Mitochondria consistently change their morphology in a process regulated by proteins, including Drp1 (dynamin-related protein 1), a protein promoting mitochondrial fission. Drp1 is involved in the mechanisms underlying various cardiovascular diseases, such as myocardial ischemia/reperfusion injury, heart failure, and pulmonary arterial hypertension. However, its role in macrophages, which promote various vascular diseases, is poorly understood. We therefore tested our hypothesis that macrophage Drp1 promotes vascular remodeling after injury. To explore the selective role of macrophage Drp1, we created macrophage-selective Drp1-deficient mice and performed femoral arterial wire injury. In these mice, intimal thickening and negative remodeling were attenuated at 4 weeks after injury when compared with control mice. Deletion of macrophage Drp1 also attenuated the macrophage accumulation and cell proliferation in the injured arteries. Gain- and loss-of-function experiments using cultured macrophages indicated that Drp1 induces the expression of molecules associated with inflammatory macrophages. Morphologically, mitochondrial fission was induced in inflammatory macrophages, whereas mitochondrial fusion was induced in less inflammatory/reparative macrophages. Pharmacological inhibition or knockdown of Drp1 decreased the mitochondrial reactive oxygen species and chemotactic activity in cultured macrophages. Co-culture experiments of macrophages with vascular smooth muscle cells indicated that deletion of macrophage Drp1 suppresses growth and migration of vascular smooth muscle cells induced by macrophage-derived soluble factors. Macrophage Drp1 accelerates intimal thickening after vascular injury by promoting macrophage-mediated inflammation. Macrophage Drp1 may be a potential therapeutic target of vascular diseases.

MiR-17 Knockdown Promotes Vascular Smooth Muscle Cell Phenotypic Modulation Through Upregulated Interferon Regulator Factor 9 Expression

MiR-17 is a small noncoding RNA that plays an important role in the development of tumorgenesis, which recently has emerged to be involved in regulation of inflammatory responses and angiogenesis. However, the effect and underlying mechanism of miR-17 on vascular smooth muscle cell (VSMC) phenotypic modulation have not been investigated. In the current study, we observed that miR-17 expression tested by real-time polymerase chain reaction (RT-PCR) was downregulated in VSMCs administrated with platelet-derived growth factor-BB stimulation and carotid arteries subjected to wire injury, which were accompanied with decreased VSMC differentiation markers. Loss-of-function strategy demonstrated that miR-17 knockdown promoted VSMC phenotypic modulation characterized as decreased VSMC differentiation marker genes, increased proliferated and migrated capability of VSMC examined by RT-PCR and western blot analysis. Mechanistically, the bioinformatics analysis and luciferase assay demonstrated that miR-17 directly targeted Interferon Regulator Factor 9 (IRF9) and the upregulated IRF9 expression was responsible for the promoted effect miR-17 knockdown on VSMC phenotypic modulation. Taken together, our results demonstrated that miR-17 knockdown accelerated VSMC phenotypic modulation partially through directly targeting to IRF9, which suggested that miR-17 may act as a novel therapeutic target for intimal hyperplasia management.

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.

456-P: Targeted Deletion of Insulin Receptor in Vascular Smooth Muscle Cells Accelerated Thinning of Plaque Cap and Apoptosis and Atherosclerosis

Vascular smooth muscle cells (VSMCs) play an important role in the development of the stability of atherosclerotic plaque. We reported that knockout of insulin receptor (IR), but not IGF1 receptor, in VSMCs reduced intimal hyperplasia of femoral artery after wire injury. To investigate the role of IR in VSMCs on atherosclerotic plaque, ApoE and smooth muscle cells IR double knockout (SMIRKO/ApoE-/-) mice were generated. Extent of lipid deposition and atherosclerosis were increased in the aorta of SMIRKO/ApoE-/- mice compared to control mice (p&amp;lt;0.05). However, total cell numbers and proliferation of VSMCs, cap thickness and collagen of the plaque were decreased, whereas VSMCs apoptosis, macrophage content and necrotic area were increased in the aortic plaques of SMIRKO/ApoE-/- mice compared to ApoE-/- mice. VSMCs cultured from SMIRKO/ApoE-/- mice exhibited more apoptosis by withdrawing growth factors which were inhibited by insulin compared to VSMCs from ApoE-/- mice. Analysis of genes in VSMCs subsets which were reported to be increased in aortic plaques showed MMP2, decorin, thrombospondin 1 and 5, sFRP3 and IGFBP4 were increased in the aortic media of SMIRKO/ApoE-/- mice vs. ApoE-/- mice, with MMP2, thrombospondin 1 and 5 and sFRP3 were also increased in the aortic media of mice fed with high fat diet or diabetic mice due to streptozotocin injection. Insulin downregulated MMP2 and thrombospondin 1 in cultured VSMCs, whereas overexpression of Foxo1 in VSMCs increased the expression of thrombospondin 1. Reanalysis of existed microarray data base and found MMP2 and thrombospondin 1 were also elevated in internal mammary artery of diabetic compared to nondiabetic people. Thus, insulin receptor action on VSMCs can change expression of extracellular genes and its turnover to increase the stability of atherosclerotic plaque. Disclosure Q. Li: None. J. Fu: None. K. Park: None. Y. Zaitsu: Employee; Self; Sunstar Group. R. St-Louis: None. C. Rask-Madsen: None. G.L. King: Research Support; Self; Janssen Pharmaceuticals, Inc. Funding National Institutes of Health (R01DK053105)

Endothelial Scaffolding Protein ENH (Enigma Homolog Protein) Promotes PHLPP2 (Pleckstrin Homology Domain and Leucine-Rich Repeat Protein Phosphatase 2)-Mediated Dephosphorylation of AKT1 and eNOS (Endothelial NO Synthase) Promoting Vascular Remodeling.

A decrease in nitric oxide, leading to vascular smooth muscle cell proliferation, is a common pathological feature of vascular proliferative diseases. Nitric oxide synthesis by eNOS (endothelial nitric oxide synthase) is precisely regulated by protein kinases including AKT1. ENH (enigma homolog protein) is a scaffolding protein for multiple protein kinases, but whether it regulates eNOS activation and vascular remodeling remains unknown. Approach and Results: ENH was upregulated in injured mouse arteries and human atherosclerotic plaques and was associated with coronary artery disease. Neointima formation in carotid arteries, induced by ligation or wire injury, was greatly decreased in endothelium-specific ENH-knockout mice. Vascular ligation reduced AKT and eNOS phosphorylation and nitric oxide production in the endothelium of control but not ENH-knockout mice. ENH was found to interact with AKT1 and its phosphatase PHLPP2 (pleckstrin homology domain and leucine-rich repeat protein phosphatase 2). AKT and eNOS activation were prolonged in VEGF (vascular endothelial growth factor)-induced ENH- or PHLPP2-deficient endothelial cells. Inhibitors of either AKT or eNOS effectively restored ligation-induced neointima formation in ENH-knockout mice. Moreover, endothelium-specific PHLPP2-knockout mice displayed reduced ligation-induced neointima formation. Finally, PHLPP2 was increased in the endothelia of human atherosclerotic plaques and blood cells from patients with coronary artery disease. ENH forms a complex with AKT1 and its phosphatase PHLPP2 to negatively regulate AKT1 activation in the artery endothelium. AKT1 deactivation, a decrease in nitric oxide generation, and subsequent neointima formation induced by vascular injury are mediated by ENH and PHLPP2. ENH and PHLPP2 are thus new proatherosclerotic factors that could be therapeutically targeted.

Stimulator of Interferon Genes (STING) Regulates Endothelial Healing and Reendothelialization Following Angioplasty‐Mediated Vascular Injury

Angioplasty and stent implantation are common clinical interventions to improve blood flow in Peripheral Artery Disease, which is rising in prevalence in America. However, these interventions result in vascular endothelial damage, leading to the activation of signaling pathways that can have a major effect on short‐ and long‐term vessel health. We hypothesized that endothelial cell‐intrinsic Stimulator of Interferon Genes (STING), a crucial regulator of immune responses triggered by pathogen or self‐derived DNA, regulates reendothelialization and healing in response to vascular injury. To study whether STING‐mediated reendothelialization was an endothelial cell specific effect, in vitro wound healing assay was performed on WT and STING deficient primary mouse heart endothelial cells (MHEC) using live imaging and quantification with Cytation and IncuCyte. We found that, while WT MHEC completely covered the scratch area within 24 hours of initial scratch, only 75.59% ± 5.8 of the initial wound was healed in STING deficient MHEC monolayers. Furthermore, our in vivo experiments using the wire injury‐mediated experimental model of angioplasty in mice followed by Evans Blue Dye staining to distinguish the areas with denuded endothelium, demonstrate a decrease in reendothelialization in injured carotid arteries in STING deficient mice as compared to WT mice five days post injury (43.57% ± 9.79 area reendothelialized vs 95.16% ± 2.77, respectively). Seven days post wire injury, WT carotid arteries were completely healed, in contrast to those of STING deficient mice that remained unhealed (67.74% ± 5.80 of reendothelialized vessel area). Additionally, multi‐parameter flow cytometry analysis of the enzymatically digested arteries from the seven days post wire injury group demonstrated an increase in myeloid cell infiltration in WT mice, which was not observed in either the injured STING deficient arteries or in the uninjured contralateral WT carotid arteries, indicating an early localized and differential immune response likely affecting long term vascular remodeling. Our results support that endothelial STING contributes to endothelial healing in vitro and in vivo, and that myeloid cell infiltration in the carotid arteries is required for reendothelialization and may be responsible for lack of healing in STING deficient mice. Ongoing experiments aim to definitively quantify temporal recruitment of immune cells over the course of reendothelialization and chronic vascular remodeling following wire injury and the mechanisms involved in short‐ and long‐term vascular health.Support or Funding InformationThis work was supported by the NIH‐RO1‐HL123658

IRF-1 mediates the suppressive effects of mTOR inhibition on arterial endothelium

AimsMammalian target of rapamycin (mTOR) inhibitors used in drug-eluting stents (DES) to control restenosis have been found to delay endothelialization and increase incidence of late-stent thrombosis through mechanisms not completely understood. We revealed that mTOR inhibition (mTORi) upregulated the expression of cell growth suppressor IRF-1 in primary human arterial endothelial cells (HAEC). This study aimed to examine how mTOR-regulated IRF-1 expression contributes to the suppressive effect of mTORi on arterial endothelial proliferation. Methods and resultsWestern blotting, quantitative PCR, and a dual-luciferase reporter assay indicated that mTOR inhibitors rapamycin and torin 1 upregulated IRF-1 expression and increased its transcriptional activity. IRF-1 in turn contributed to the suppressive effect of mTORi by mediating HAEC apoptosis and cell cycle arrest in part through upregulation of caspase 1 and downregulation of cyclin D3, as revealed by CCK-8 assay, Annexin V binding assay, measurement of activated caspase 3, BrdU incorporation assay, and matrigel tube formation assay. In a mouse model of femoral artery wire injury, administration of rapamycin inhibited EC recovery, an effect alleviated by EC deficiency of IRF-1. Chromatin immunoprecipitation assay with HAEC and rescue expression of wild type or dominant-negative IRF-1 in EC isolated from Irf1−/− mice confirmed transcriptional regulation of IRF-1 on the expression of CASP1 and CCND3. Furthermore, mTORi activated multiple PKC members, among which PKCζ was responsible for the growth-inhibitory effect on HAEC. Activated PKCζ increased IRF1 transcription through JAK/STAT-1 and NF-κB signaling. Finally, overexpression of wild type or mutant raptor incapable of binding mTOR indicated that mTOR-free raptor contributed to PKCζ activation in mTOR-inhibited HAEC. ConclusionsThe study reveals an IRF-1-mediated mechanism that contributes to the suppressive effects of mTORi on HAEC proliferation. Further study may facilitate the development of effective strategies to reduce the side effects of DES used in coronary interventions.

Osteoprotegerin promotes intimal hyperplasia and contributes to in-stent restenosis: Role of an αVβ3/FAK dependent YAP pathway

ObjectiveAbnormal proliferation and migration of vascular smooth muscle cells (VSMCs) are related to in-stent-restenosis (ISR) following percutaneous coronary intervention (PCI). Osteoprotegerin (OPG) has been implicated in various vascular diseases. However, the effects of OPG on ISR and the underlying mechanism remained elusive. We here investigated the association between OPG and ISR, and to demonstrate the role and potential mechanisms of OPG in neointimal hyperplasia. Approach and resultsFrom 2962 patients who received coronary angiography and follow-up coronary angiography at approximately one year, 291 patients were diagnosed with ISR, and another 291 gender- and age- matched patients without ISR were selected as controls. Serum OPG levels were significantly increased in patients with ISR. Multivariable logistic regression analysis indicated that OPG level was independently associated with the increased risk of ISR. In a mouse femoral artery wire injury model, upregulated OPG was evidenced in vascular tissue after injury. OPG deletion attenuated the vascular injury-induced neointimal hyperplasia and related gene expression in mice. OPG promoted neointimal hyperplasia and human aortic smooth muscle cell (hASMC) proliferation and migration through activation of yes-associated protein (YAP), a major downstream effector of the Hippo signaling pathway, whereas knockdown or inhibition of YAP in hASMCs blunted OPG-induced above effects. Moreover, we found that OPG, as a ligand for integrin αVβ3, mediated phosphorylation of focal adhesion kinase (FAK) and actin cytoskeleton reorganization, resulting in YAP dephosphorylation in hASMCs. OPG-dependent YAP and VSMC activation was prevented by treatment with αVβ3-blocking antibodies and inhibitors of FAK and actin stress fibers. ConclusionsIncreased serum OPG levels are associated with increased risk of ISR following PCI and OPG could promote neointimal hyperplasia in response to injury through integrin αVβ3 mediated FAK and YAP activation, indicating OPG/YAP inhibition might serve as an attractive novel target for the prevention of ISR after PCI.

Canonical transient receptor potential 6 channel deficiency promotes smooth muscle cells dedifferentiation and increased proliferation after arterial injury

ObjectivePrevious studies showed the benefit of canonical transient receptor potential 6 (TRPC6) channel deficiency in promoting endothelial healing of arterial injuries in hypercholesterolemic animals. Long-term studies utilizing a carotid wire-injury model were undertaken in wild-type (WT) and TRPC6-/- mice to determine the effects of TRPC6 on phenotypic modulation of vascular smooth muscle cells (SMC) and neointimal hyperplasia. We hypothesized that TRPC6 was essential in the maintenance or reexpression of a differentiated SMC phenotype and minimized luminal stenosis following arterial injury. MethodsThe common carotid arteries (CCA) of WT and TRPC6-/- mice were evaluated at baseline and 4 weeks after wire injury. At baseline, CCA of TRPC6-/- mice had reduced staining of MYH11 and SM22, fewer elastin lamina, luminal dilation, and wall thinning. After carotid wire injury, TRPC6-/- mice developed significantly more pronounced luminal stenosis compared with WT mice. Injured TRPC6-/- CCA demonstrated increased medial/intimal cell number and active cell proliferation when compared with WT CCA. Immunohistochemistry suggested that expression of contractile biomarkers in medial SMC were essentially at baseline levels in WT CCA at 28 days after wire injury. By contrast, at 28 days after injury medial SMC from TRPC6-/- CCA showed a significant decrease in the expression of contractile biomarkers relative to baseline levels. To assess the role of TRPC6 in systemic arterial SMC phenotype modulation, SMC were harvested from thoracic aortae of WT and TRPC6-/- mice and were characterized. TRPC6-/- SMC showed enhanced proliferation and migration in response to serum stimulation. Expression of contractile phenotype biomarkers, MYH11 and SM22, was attenuated in TRPC6-/- SMC. siRNA-mediated TRPC6 deficiency inhibited contractile biomarker expression in a mouse SMC line. ConclusionsThese results suggest that TRPC6 contributes to the restoration or maintenance of arterial SMC contractile phenotype following injury. Understanding the role of TRPC6 in phenotypic modulation may lead to mechanism-based therapies for attenuation of IH.

Activation of heme oxygenase-1 by Ginkgo biloba extract differentially modulates endothelial and smooth muscle-like progenitor cells for vascular repair

Vascular progenitors such as endothelial progenitor cells (EPCs) and smooth muscle-like progenitor cells (SMPCs) may play different roles in vascular repair. Ginkgo biloba extract (GBE) is an exogenous activator of heme oxygenase (HO)-1, which has been suggested to improve vascular repair; however, the detailed mechanisms have yet to be elucidated. This study aimed to investigate whether GBE can modulate different vascular progenitor cells by activating HO-1 for vascular repair. A bone marrow transplantation mouse model was used to evaluate the in vivo effects of GBE treatment on wire-injury induced neointimal hyperplasia, which is representative of impaired vascular repair. On day 14 of GBE treatment, the mice were subjected to wire injury of the femoral artery to identify vascular reendothelialization. Compared to the mice without treatment, neointimal hyperplasia was reduced in the mice that received GBE treatment for 28 days in a dose-dependent manner. Furthermore, GBE treatment increased bone marrow-derived EPCs, accelerated endothelial recovery, and reduced the number of SMPCs attached to vascular injury sites. The effects of GBE treatment on neointimal hyperplasia could be abolished by co-treatment with zinc protoporphyrin IX, an HO-1 inhibitor, suggesting the in vivo role of HO-1. In this in vitro study, treatment with GBE activated human early and late EPCs and suppressed SMPC migration. These effects were abolished by HO-1 siRNA and an HO-1 inhibitor. Furthermore, GBE induced the expression of HO-1 by activating PI3K/Akt/eNOS signaling in human late EPCs and via p38 pathways in SMPCs, suggesting that GBE can induce HO-1 in vitro through different molecular mechanisms in different vascular progenitor cells. Accordingly, GBE could activate early and late EPCs, suppress the migration of SMPCs, and improve in vivo vascular repair after mechanical injury by activating HO-1, suggesting the potential role of pharmacological HO-1 activators, such as GBE, for vascular protection in atherosclerotic diseases.