Neointimal Lesion Formation Research Articles

MicroRNA-144 silencing attenuates intimal hyperplasia by directly targeting PTEN

ABSTRACT Background Intimal hyperplasia contributed by phenotypic switching of vascular smooth muscle cell (VSMC) plays an important role in the pathogenesis of various cardiovascular diseases. MicroRNA-144 (miR-144) is recently reported to be implicated in the development of atherosclerosis. However, the individual role of miR-144 in VSMCs phenotypic modulation and intimal hyperplasia currently still remains unknown. Methods and Results Here we found that miR-144 expression was upregulated in carotid arteries with intimal hyperplasia that subjected to wire injury and the consistent results were obtained with dedifferentiated VSMCs upon platelet-derived growth factor-BB (PDGF-BB) stimulation. Loss-of-function study showed that miR-144 knockdown decreased the ability of VSMC proliferation tested by Brdu and CCK8, and reduced the migrate capability analyzed by Transwell, whereas increased the differentiated SMC marker gene expression examined by RT-PCR. The above results were reversed by miR-144 overexpression. Mechanistically, we have demonstrated that PTEN was the direct target of miR-144 that was responsible for the alleviated effect of miR-144 inhibition on phenotypic switching of VSMCs. Notably, mice injected with miR-144 inhibitor attenuated the formation of neointimal lesions in response to wire injury and maintained the mature SMC marker expression inhibited the proliferation and migration of VSMCs. Conclusion Our research exhibited that miR-144 knockdown attenuated intimal hyperplasia through inhibiting the VSMC phenotypic switching, which was partially mediated by directly targeting to PTEN. Taken together, these evidences suggested that miR-144 may act as a promising therapeutic target for arterial restenosis.

Cellular mechanisms of oligoclonal vascular smooth muscle cell expansion in cardiovascular disease.

Quiescent, differentiated adult vascular smooth muscle cells (VSMCs) can be induced to proliferate and switch phenotype. Such plasticity underlies blood vessel homeostasis and contributes to vascular disease development. Oligoclonal VSMC contribution is a hallmark of end-stage vascular disease. Here, we aim to understand cellular mechanisms underpinning generation of this VSMC oligoclonality. We investigate the dynamics of VSMC clone formation using confocal microscopy and single-cell transcriptomics in VSMC-lineage-traced animal models. We find that activation of medial VSMC proliferation occurs at low frequency after vascular injury and that only a subset of expanding clones migrate, which together drives formation of oligoclonal neointimal lesions. VSMC contribution in small atherosclerotic lesions is typically from one or two clones, similar to observations in mature lesions. Low frequency (<0.1%) of clonal VSMC proliferation is also observed in vitro. Single-cell RNA-sequencing revealed progressive cell state changes across a contiguous VSMC population at onset of injury-induced proliferation. Proliferating VSMCs mapped selectively to one of two distinct trajectories and were associated with cells showing extensive phenotypic switching. A proliferation-associated transitory state shared pronounced similarities with atypical SCA1+ VSMCs from uninjured mouse arteries and VSMCs in healthy human aorta. We show functionally that clonal expansion of SCA1+ VSMCs from healthy arteries occurs at higher rate and frequency compared with SCA1- cells. Our data suggest that activation of proliferation at low frequency is a general, cell-intrinsic feature of VSMCs. We show that rare VSMCs in healthy arteries display VSMC phenotypic switching akin to that observed in pathological vessel remodelling and that this is a conserved feature of mouse and human healthy arteries. The increased proliferation of modulated VSMCs from healthy arteries suggests that these cells respond more readily to disease-inducing cues and could drive oligoclonal VSMC expansion.

Bves maintains vascular smooth muscle cell contractile phenotype and protects against transplant vasculopathy via Dusp1-dependent p38MAPK and ERK1/2 signaling

Background and aimsVascular smooth muscle cell (VSMC) plasticity is tightly associated with the pathological process of vasculopathy. Blood vessel epicardial substance (Bves) has emerged as an important regulator of intracardiac vasculogenesis and organ homeostasis. However, the involvement and role of Bves in VSMC plasticity and neointimal lesion development remain unclear. MethodsWe used an in vivo rat model of graft arteriosclerosis and in vitro PDGF-treated VSMCs and identified the novel VSMC contractile phenotype-related gene Bves using a transcriptomic analysis and literature search. In vitro knockdown and overexpression approaches were used to investigate the mechanisms underlying VSMC phenotypic plasticity. In vivo, VSMC-specific Bves overexpression in rat aortic grafts was generated to assess the physiological function of Bves in neointimal lesion development. ResultsHere, we found that Bves expression was negatively regulated in aortic allografts in vivo and PDGF-treated VSMCs in vitro. The genetic knockdown of Bves dramatically inhibited, whereas Bves overexpression markedly promoted, the VSMC contractile phenotype. Furthermore, RNA sequencing unraveled a positive correlation between Bves and dual-specificity protein phosphatase 1 (Dusp1) expression in VSMCs. We found that Bves knockdown restrained Dusp1 expression, but enhanced p38MAPK and ERK1/2 activation, resulting in the loss of the VSMC contractile phenotype. In vivo, an analysis of a rat graft model confirmed that VSMC-specific Bves and Dusp1 overexpression in aortic allografts significantly attenuated neointimal lesion formation. ConclusionsBves maintains the VSMC contractile phenotype through Dusp1-dependent p38MAPK and ERK1/2 signaling, and protects against neointimal formation, underscoring the important role of Bves in preventing transplant vasculopathy.

Targeting platelet-derived CXCL12 impedes arterial thrombosis

AbstractThe prevention and treatment of arterial thrombosis continue to be clinically challenging, and understanding the relevant molecular mechanisms in detail may facilitate the quest to identify novel targets and therapeutic approaches that improve protection from ischemic and bleeding events. The chemokine CXCL12 augments collagen-induced platelet aggregation by activating its receptor CXCR4. Here we show that inhibition of CXCR4 attenuates platelet aggregation induced by collagen or human plaque homogenate under static and arterial flow conditions by antagonizing the action of platelet-secreted CXCL12. We further show that platelet-specific CXCL12 deficiency in mice limits arterial thrombosis by affecting thrombus growth and stability without increasing tail bleeding time. Accordingly, neointimal lesion formation after carotid artery injury was attenuated in these mice. Mechanistically, CXCL12 activated via CXCR4 a signaling cascade involving Bruton's tyrosine kinase (Btk) that led to integrin αIIbβ3 activation, platelet aggregation, and granule release. The heterodimeric interaction between CXCL12 and CCL5 can inhibit CXCL12-mediated effects as mimicked by CCL5-derived peptides such as [VREY]4. An improved variant of this peptide, i[VREY]4, binds to CXCL12 in a complex with CXCR4 on the surface of activated platelets, thereby inhibiting Btk activation and preventing platelet CXCL12-dependent arterial thrombosis. In contrast to standard antiplatelet therapies such as aspirin or P2Y12 inhibition, i[VREY]4 reduced CXCL12-induced platelet aggregation and yet did not prolong in vitro bleeding time. We provide evidence that platelet-derived CXCL12 is involved in arterial thrombosis and can be specifically targeted by peptides that harbor potential therapeutic value against atherothrombosis.

Elevated expression of CDKN1A-interacting zinc finger protein 1 in intimal hyperplasia after endovascular arterial injury

Restenosis formation is initiated from intimal hyperplasia when arterial stretch and injury exist. In this study, we examined the role of CDKN1A-interacting zinc finger protein 1 (CIZ1) in neointimal hyperplasia in injured arteries. CIZ1 protein was up-regulated, and p21Cip1/Waf1 (p21) was down-regulated in the neointimal hyperplasia from a mouse femoral artery wire-injury model. In vitro, proliferation and migration were reduced in vascular smooth muscle cells transformed with Ciz1-shRNA lentiviral particles. In addition, p21 expression is increased, and MMP9 expression is decreased in vascular smooth muscle cells with CIZ1 knockdown. These data imply that CIZ1 might be a novel repressor of neointimal lesion formation caused by interventional therapy.

Chronic Daily House Dust Mite Exposure in Mice is an Effective Model to Quantify the Effect of Pharmacologic Agents on Discrete Stages of Artery Remodeling in Pulmonary Hypertension.

Pulmonary hypertension (PH) is a heterogenous and incurable disease marked by varying degrees of pulmonary vascular remodeling. This vascular remodeling, which includes thickening of the smooth muscle layer (an early finding) and formation of occlusive neointimal lesions (a late finding) in the pulmonary arteries, is a major driver of morbidity and mortality in PH. Available PH therapies consist of vasodilators that do not specifically target lesion formation or expansion and neither prevent progression nor reverse disease. This paucity of curative treatments highlights the need for new drug discovery targeting crucial steps of artery remodeling in PH. The cell dynamics and molecular signals driving neointimal lesion formation have been difficult to elucidate as classic mouse models of PH do not develop neointima. Here, we detail the methods to generate a robust and non-genetic mouse model of PH with medial thickening and neointimal lesion formation in the pulmonary arteries, through chronic exposure to an inflammatory stimulus-house dust mite (HDM). This model rapidly generates human-like pulmonary arterial lesions following a reproducible time course, allowing scrutiny of the cellular and molecular mechanisms controlling each stage of artery remodeling. Further, we outline optimal tissue handling, sectioning, and staining methodologies for detailed quantitative analysis of artery medial thickening and neointimal lesion formation and expansion. Finally, we present a method for staged pharmacologic intervention to identify molecules and pathways required at each step of the pulmonary arterial remodeling process. The advantages of this mouse model of PH over currently available animal models are five-fold. (i) It allows the use of the full range of genetic and single cell tools available in mice to manipulate and study the process of vascular remodeling seen in human disease, including the formation of neointimal lesions in a controlled and cell specific manner. (ii) The vascular lesions develop in a stereotyped manner with predictable timing, allowing for pharmacologic manipulation at discrete stages of vessel remodeling. (iii) It is rapid, with development of PH and vascular remodeling in a timeframe of two to eight weeks. (iv) It uses simple techniques and requires neither surgery, unusual equipment, or extensive personnel training. (v) The staining and quantitation methodologies we present are a significant improvement over those currently in use in the field. We hope that dissemination of this model and the associated detailed methods will speed up the development of novel and more effective PH therapeutics. Graphic abstract: Chronic perivascular inflammation induces medial thickening and neointima formation in pulmonary arteries, following a stereotyped time course, and allowing staged pharmacologic intervention during specific remodeling events, as well as quantitative assessment of vascular changes.

MicroRNA-17-5p, a novel endothelial cell modulator, controls vascular re-endothelialization and neointimal lesion formation.

The functions of miR-17-5p in tumorigenesis have been explored. However, their functionalities in arterial endothelial cells (ECs) have not been investigated. Besides, the issue of vascular remodelling is barely addressed. The study aimed to determine the effect of overexpression or inhibition of miR-17-5p on arterial endothelial cells' (ECs) function and vascular remodelling in vitro and the rat carotid arteries model. Quantitative RT-PCR analysis was performed to examine the expression of miR-17-5p. Then, gain-of-function and loss-of-function approaches were employed to investigate the functional roles of miR-17-5p in cultured human coronary artery endothelial cells (HCAECs); further, TargetScan software analysis and luciferase reporter activity assay were performed to investigate the potential mechanism. Lastly, the results of the cell segment were verified in a rat carotid artery balloon injury model by Western blot analysis, measurement of the vascular cGMP level and plasma 8-iso-prostaglandin F2 (8-iso-PGF2) testing. Moreover, morphometric analysis was implemented to detect the re-endothelialization and neointimal formation in rat carotid artery after balloon injury. This study firstly found that miR-17-5p expression was upregulated in the injured vascular walls and highly expressive in ECs; overexpression of miR-17-5p inhibited HCAECs' proliferation and migration, whereas miR-17-5p knockdown strengthened its proliferative and migratory roles, influenced inflammatory response, through regulating VEGRA and VEGFR2. It was found that miR-17-5p bind to VEGFA and VEGFR2 at the 3'UTR. Next, downregulation of miR-17-5p promotes re-endothelialization, and attenuates neointimal formation as measured by the I/M ratio (0.63±0.05 vs 1.45±0.06, antagomiR-17-5p vs. Lenti-NC, p < 0.05). In addition, the functional recovery of the endothelium was also accelerated by miR-17-5p knockdown. Our study suggests that miR-17-5p is a feasible strategy for the selective modulation of endothelialization and vascular remodelling through regulating VEGFA and VEGFR2.

Ruxolitinib attenuates intimal hyperplasia via inhibiting JAK2/STAT3 signaling pathway activation induced by PDGF-BB in vascular smooth muscle cells

BackgroundCardiovascular diseases are associated with proliferation and phenotypic switch. Platelet-derived growth factor-BB (PDGF-BB) is a major initiating factor for proliferative vascular diseases, such as neointimal lesion formation, restenosis after angioplasty, and atherosclerosis. Ruxolitinib, a potent Janus kinase (JAK) 1 and 2 inhibitor, has been reported to significantly block the proliferation-related signaling pathway of JAK2/signal transducers and activators of transcription 3 (STAT3) and harbor a broad spectrum of anti-cancer activities, including proliferation inhibition, apoptosis induction, and anti-inflammation. However, the role of ruxolitinib in regulating PDGF-BB-induced VSMC proliferation remains to be elucidated. Thus, this study investigates the role of ruxolitinib in regulating PDGF-BB-induced VSMC proliferation and its underlying mechanisms. MethodsIn vivo, the medial thickness of the carotid artery was evaluated using a mouse carotid ligation model, ruxolitinib was administered orally to the mice every other day, and the mice were euthanized on day 28 to evaluate the therapeutic effects of ruxolitinib. Cell proliferation markers were measured using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blotting. In vitro, VSMCs were treated with ruxolitinib with or without PDGF-BB at an indicated time and concentration. Cell proliferation and apoptosis were measured using Cell Counting Kit-8 assay, MTS assays and flow cytometry. The JAK2/STAT3 signaling pathway involved in the effects of ruxolitinib on VSMCs was detected by western blotting with the specific pathway inhibitor AG490. ResultsIn vivo, ruxolitinib significantly decreased the ratio-of-intima ratio (I/M ratio) by inhibiting the expression of PCNA and cyclinD1 (p <0.05). In vitro, ruxolitinib inhibited PDGF-BB-induced VSMC proliferation compared with the PDGF-BB treatment group (p <0.05). In addition, ruxolitinib inhibited the PDGF-BB-induced activation of the JAK2/STAT3 signaling pathway and decreased the expression of proliferation related-proteins cyclinD1 and PCNA in VSMCs (p <0.05). ConclusionOur findings suggest that ruxolitinib inhibits VSMC proliferation in vivo and in vitro by suppressing the activation of the JAK2/STAT3 signaling pathway. Therefore, ruxolitinib has a therapeutic potential for proliferative vascular diseases.

A Notch3-Marked Subpopulation of Vascular Smooth Muscle Cells Is the Cell of Origin for Occlusive Pulmonary Vascular Lesions.

Pulmonary arterial hypertension (PAH) is a fatal disease characterized by profound vascular remodeling in which pulmonary arteries narrow because of medial thickening and occlusion by neointimal lesions, resulting in elevated pulmonary vascular resistance and right heart failure. Therapies targeting the neointima would represent a significant advance in PAH treatment; however, our understanding of the cellular events driving neointima formation, and the molecular pathways that control them, remains limited. We comprehensively map the stepwise remodeling of pulmonary arteries in a robust, chronic inflammatory mouse model of pulmonary hypertension. This model demonstrates pathological features of the human disease, including increased right ventricular pressures, medial thickening, neointimal lesion formation, elastin breakdown, increased anastomosis within the bronchial circulation, and perivascular inflammation. Using genetic lineage tracing, clonal analysis, multiplexed in situ hybridization, immunostaining, deep confocal imaging, and staged pharmacological inhibition, we define the cell behaviors underlying each stage of vascular remodeling and identify a pathway required for neointima formation. Neointima arises from smooth muscle cells (SMCs) and not endothelium. Medial SMCs proliferate broadly to thicken the media, after which a small number of SMCs are selected to establish the neointima. These neointimal founder cells subsequently undergoing massive clonal expansion to form occlusive neointimal lesions. The normal pulmonary artery SMC population is heterogeneous, and we identify a Notch3-marked minority subset of SMCs as the major neointimal cell of origin. Notch signaling is specifically required for the selection of neointimal founder cells, and Notch inhibition significantly improves pulmonary artery pressure in animals with pulmonary hypertension. This work describes the first nongenetically driven murine model of pulmonary hypertension (PH) that generates robust and diffuse occlusive neointimal lesions across the pulmonary vascular bed and does so in a stereotyped timeframe. We uncover distinct cellular and molecular mechanisms underlying medial thickening and neointima formation and highlight novel transcriptional, behavioral, and pathogenic heterogeneity within pulmonary artery SMCs. In this model, inflammation is sufficient to generate characteristic vascular pathologies and physiological measures of human PAH. We hope that identifying the molecular cues regulating each stage of vascular remodeling will open new avenues for therapeutic advancements in the treatment of PAH.

BET bromodomain-containing epigenetic reader proteins regulate vascular smooth muscle cell proliferation and neointima formation.

Recent studies revealed that the bromodomain and extra-terminal (BET) epigenetic reader proteins resemble key regulators in the underlying pathophysiology of cancer, diabetes, or cardiovascular disease. However, whether they also regulate vascular remodelling processes by direct effects on vascular cells is unknown. In this study, we investigated the effects of the BET proteins on human smooth muscle cell (SMC) function in vitro and neointima formation in response to vascular injury in vivo. Selective inhibition of BETs by the small molecule (+)-JQ1 dose-dependently reduced proliferation and migration of SMCs without apoptotic or toxic effects. Flow cytometric analysis revealed a cell cycle arrest in the G0/G1 phase in the presence of (+)-JQ1. Microarray- and pathway analyses revealed a substantial transcriptional regulation of gene sets controlled by the Forkhead box O (FOXO1)1-transcription factor. Silencing of the most significantly regulated FOXO1-dependent gene, CDKN1A, abolished the antiproliferative effects. Immunohistochemical colocalization, co-immunoprecipitation, and promoter-binding ELISA assay data confirmed that the BET protein BRD4 directly binds to FOXO1 and regulates FOXO1 transactivational capacity. In vivo, local application of (+)-JQ1 significantly attenuated SMC proliferation and neointimal lesion formation following wire-induced injury of the femoral artery in C57BL/6 mice. Inhibition of the BET-containing protein BRD4 after vascular injury by (+)-JQ1 restores FOXO1 transactivational activity, subsequent CDKN1A expression, cell cycle arrest and thus prevents SMC proliferation in vitro and neointima formation in vivo. Inhibition of BET epigenetic reader proteins might thus represent a promising therapeutic strategy to prevent adverse vascular remodelling.

PRAS40 suppresses atherogenesis through inhibition of mTORC1-dependent pro-inflammatory signaling in endothelial cells

Endothelial pro-inflammatory activation plays a pivotal role in atherosclerosis, and many pro-inflammatory and atherogenic signals converge upon mechanistic target of rapamycin (mTOR). Inhibitors of mTOR complex 1 (mTORC1) reduced atherosclerosis in preclinical studies, but side effects including insulin resistance and dyslipidemia limit their clinical use in this context. Therefore, we investigated PRAS40, a cell type-specific endogenous modulator of mTORC1, as alternative target. Indeed, we previously found PRAS40 gene therapy to improve metabolic profile; however, its function in endothelial cells and its role in atherosclerosis remain unknown. Here we show that PRAS40 negatively regulates endothelial mTORC1 and pro-inflammatory signaling. Knockdown of PRAS40 in endothelial cells promoted TNFα-induced mTORC1 signaling, proliferation, upregulation of inflammatory markers and monocyte recruitment. In contrast, PRAS40-overexpression blocked mTORC1 and all measures of pro-inflammatory signaling. These effects were mimicked by pharmacological mTORC1-inhibition with torin1. In an in vivo model of atherogenic remodeling, mice with induced endothelium-specific PRAS40 deficiency showed enhanced endothelial pro-inflammatory activation as well as increased neointimal hyperplasia and atherosclerotic lesion formation. These data indicate that PRAS40 suppresses atherosclerosis via inhibition of endothelial mTORC1-mediated pro-inflammatory signaling. In conjunction with its favourable effects on metabolic homeostasis, this renders PRAS40 a potential target for the treatment of atherosclerosis.

P1940Reciprocal regulation of Endothelial-Mesenchymal Transition by MAPK7 and EZH2 activity in Intimal Hyperplasia and Coronary Artery Disease

Abstract Introduction Endothelial cells play a pivotal role in the formation of neointimal lesions by the acquisition of a fibro-proliferative phenotype through endothelial-to-mesenchymal transition (EndMT). Uniform laminar shear stress activates the mitogen-activated protein kinase 7 (MAPK7) which suppresses EndMT. It is elusive how MAPK7 activity is regulated in fibroproliferative disease. We recently found in intimal hyperplasia the signaling activity of MAPK7 is rapidly lost through the activation microRNA-374b. The histone methyltransferase Enhancer of Zeste Homolog 2 (EZH2), which is the catalytic subunit of the Polycomb Repressive Complex 2, plays a pivotal role in endothelial dysfunction. EZH2 trimethylates lysine 27 on histone 3, which silences gene expression and is elevated in endothelial cells in atherosclerotic lesions. Here, we found the reciprocity that exists between MAPK7 and EZH2 in the regulation of EndMT and in human coronary artery stenosis. Materials and results In endothelial cells, activation of MAPK7 increases the expression of microRNA-101, which represses the expression of EZH2. Reciprocally, the loss of EZH2 coincides with a decreased expression of the Dual Specificity Phosphatase (DUSP)-1 and DUSP-6 – the phosphatases responsible for the dephosphorylation of MAPK7 - which facilitates the activation of MAPK7. H3K27Me3, the repressive histone mark placed by EZH2, is abundantly present in the promoter regions of the miR-200b/a/429 and miR-200c/141 gene clusters, which are responsible for the loss of DUSP-1 and DUSP-6 expression. Endothelial cells deficient in EZH2 have reduced levels of H3K27Me3 at these gene promoters, which associates with the increased expression of miR-200b and miR-200c and concurrent increased MAPK7 activation. In endothelial cells with constitutively active MAPK7 signaling (MEK5D), the enrichment of H3K27Me3 at the promoter regions of miR-200b/a/429 (1.6-fold, p=0.034) and miR-200c/141 (1.9-fold, p=0.035) is decreased, suggesting that MAPK7 activation results in a decreased EZH2 activity. Disbalances in this reciprocal signaling circuit culminate in the induction of EndMT and associate to the severity of human coronary artery stenosis. Conclusion In summary, we show that in endothelial cells there is reciprocity between MAPK7 signaling and EZH2 expression and that disturbances in this reciprocal signaling circuit associate with the induction of EndMT and severity of human coronary artery stenosis. The reciprocity between MAPK7 and EZH2 is governed by a complex mechanism involving microRNAs and the phosphatases DUSP-1 and DUSP-6. Our study contributes to a better understanding of the molecular and epigenetic cascades that underlie EndMT during coronary artery stenosis and might identify novel targets for therapy. Acknowledgement/Funding Mongolian Government Scholarship #621

261Decisive role of microRNA-494 in smooth muscle cell proliferation and vascular remodeling

Abstract Background Proliferation of vascular smooth muscle cells triggered by cytokines and growth factors is a main driver in the development of vascular proliferative diseases such as atherosclerosis and in-stent-restenosis after angioplasty. MicroRNAs (miR) are small noncoding RNAs that can inhibit the expression of multiple genes simultaneously. However, the contribution of microRNAs to the differential gene regulation that triggers vascular remodeling processes is not well understood. Methods and results Neointima formation was induced by a wire-mediated injury of the femoral artery in C57BL/6 mice. Microarray analysis of the developing neointimal lesion showed a strong reduction of miR-494 (0.411±0.04; p&lt;0.05) at 7 days after injury. In order to investigate the expression levels of miRs in vascular cells, human coronary artery smooth muscle cells (SMC), human coronary artery endothelial cells and human monocytes were analyzed via microarray analysis. Intriguingly, miR-494 was found to be predominantly expressed in SMC via microarray and qPCR analysis. The regulation of miR-494 expression was further analyzed after stimulation of SMC with 10%FCS. Following this mitogenic stimulation, mir-494 expression dropped robustly and significantly in a time-dependent manner at 6, 9, and 24 hours. To investigate the functional impact of miR-494 on SMC proliferation, miR-494 was overexpressed using miR-494-mimics (20μM). Overexpression of miR-494 significantly reduced the FCS-induced proliferation of SMC as assessed by BrdU-incorporation. In silico analyses of potential target genes for miR-494 identified ROCK1 and Survivin, both important molecules in the mitogenic response of SMC to cytokines and growth factors, as potential targets of miR-494. Indeed, ROCK1 and Survivin were found down-regulated on the mRNA and protein level after transfection of SMC with miR-494 mimics and both mulecules could be identified as direct targets using luciferase reporter assays. Following the specific inhibition of miR-494 by local application (in a perivascular thermos-responsive, self-degrading pluronic gel) of an in vivo stabilized Pre-miR-494 after wire-induced injury of the mouse femoral artery, SMC proliferation was significantly reduced, as assessed by Ki67 immunofluorescence (26.3% vs 11.2%; p&lt;0.05). Consistently, local application of Pre-miR-494 significantly reduced neointima formation (neointima/media ratio 2.31 in control vs 1.01 in treated animals; p&lt;0.01). Conclusion Our results show that mir-494 is strongly down-regulated in proliferating SMC in vitro as well as during neointimal lesion formation in vivo. Moreover, overexpression/ reconstitution of miR-494 levels effectively prevented SMC proliferation and neointima formation, indicating an important functional role of miR-494 in these processes. Hence, miR-494 may represent an attractive SMC-specific target for future therapeutic interventions for the treatment of vascular proliferative diseases.

1439PRAS40 attenuates mTOR-dependent endothelial inflammation and atherosclerosis

Abstract Introduction Endothelial inflammation plays a pivotal role in atherosclerosis. Many inflammatory and metabolic signals converge upon mechanistic target of rapamycin (mTOR), and inhibition of mTOR has been shown to reduce atherosclerosis. However, clinical use of mTOR-inhibitors is limited by serious adverse effects, of which insulin resistance and dyslipidemia are particularly troubling in the context of atherosclerosis. In that respect, targeting PRAS40, an endogenous modulator of mTOR complex 1 (mTORC1) with highly cell type-specific effects on mTOR signaling, may be a more promising approach. In fact, we have previously demonstrated that, in contrast to conventional mTOR inhibitors, PRAS40 gene therapy substantially improves metabolic profile in obese mice. However, the function of PRAS40 in endothelial cells and its role in atherosclerosis have never been investigated. Methods and results To define the impact of PRAS40 on endothelial mTORC1-signaling in this context, cultured human umbilical vein endothelial cells (HUVECs) were exposed to the atherogenic cytokine TNFα. TNFα induced mTOR signaling as evidenced by increased phosphorylation of S6 kinase and ribosomal S6 protein. Interestingly, this effect was strongly augmented upon siRNA-mediated knock-down of PRAS40, indicating a negative regulation of mTORC1 by PRAS40 in endothelial cells. Moreover, PRAS40-knockdown promoted TNFα-induced inflammatory signaling as reflected by increased proliferative activity, upregulation of atherogenic markers like CCL2 and VCAM-1, as well as enhanced monocyte recruitment in the THP-1 adhesion assay. In contrast, PRAS40-overexpression blocked TNFα-induced activation of mTORC1 and consistently suppressed all of these measures of inflammatory activation. All effects of PRAS40-overexpression could be reproduced by the mTORC1 inhibitors rapamycin and torin1. Thus, our in vitro studies suggest that in endothelial cells PRAS40 exerts anti-atherogenic effects by negative regulation of mTORC1. To validate these findings in vivo in the context of atherosclerosis we created transgenic mice with tamoxifen-inducible endothelium-specific PRAS40-deficiency (EC-PRAS40-KO). These mice were exposed to a model of accelerated atherosclerosis based on western diet and partial carotid ligation: Four weeks after partial carotid ligation, neointimal and atherosclerotic lesion formation was strongly enhanced in EC-PRAS40-KO mice. Moreover, mTORC1 activity as well as CCL2 and VCAM-1 expression were markedly increased compared to control mice. Conclusion Our data indicate that PRAS40 suppresses atherosclerosis via inhibition of mTORC1-mediated inflammatory signaling in endothelial cells. In conjunction with its favourable effects on metabolic homeostasis, the overall therapeutic profile of PRAS40-treatment appears to be beneficial compared to conventional mTOR-inhibitors. Taken together PRAS40 may qualify as a promising therapeutic target for the treatment of atherosclerosis. Acknowledgement/Funding German Federal Ministry of Education and Research, DZHK (German Centre for Cardiovascular Research)

Notch2 and Proteomic Signatures in Mouse Neointimal Lesion Formation.

Vascular remodeling is associated with complex molecular changes, including increased Notch2, which promotes quiescence in human smooth muscle cells. We used unbiased protein profiling to understand molecular signatures related to neointimal lesion formation in the presence or absence of Notch2 and to test the hypothesis that loss of Notch2 would increase neointimal lesion formation because of a hyperproliferative injury response. Murine carotid arteries isolated at 6 or 14 days after ligation injury were analyzed by mass spectrometry using a data-independent acquisition strategy in comparison to uninjured or sham injured arteries. We used a tamoxifen-inducible, cell-specific Cre recombinase strain to delete the Notch2 gene in smooth muscle cells. Vessel morphometric analysis and immunohistochemical staining were used to characterize lesion formation, assess vascular smooth muscle cell proliferation, and validate proteomic findings. Loss of Notch2 in smooth muscle cells leads to protein profile changes in the vessel wall during remodeling but does not alter overall lesion morphology or cell proliferation. Loss of smooth muscle Notch2 also decreases the expression of enhancer of rudimentary homolog, plectin, and annexin A2 in vascular remodeling. We identified unique protein signatures that represent temporal changes in the vessel wall during neointimal lesion formation in the presence and absence of Notch2. Overall lesion formation was not affected with loss of smooth muscle Notch2, suggesting compensatory pathways. We also validated the regulation of known injury- or Notch-related targets identified in other vascular contexts, providing additional insight into conserved pathways involved in vascular remodeling.

Roles of MicroRNAs in Atherosclerosis and Neointimal Lesion Formation under Flow

Atherosclerosis, a vascular pathology responsible for most cardiovascular‐related morbidity and mortality, develops predictably in regions of the arterial tree in which wall shear stresses are generated by complex patterns of blood flow. It has been recognized that hemodynamic characteristics determine the location of lesions and contribute to the pathogenesis of atherosclerosis. The key cells involved in atherogenesis include vascular endothelial cells (ECs) and smooth muscle cells (SMCs). Recent evidence suggests that laminar blood flow in the straight part of the arterial tree and high shear stress modulate cellular signaling and EC function, and protect against atherogenesis. In contrast, disturbed flow in bifurcations of the arterial tree and the associated oscillatory low shear stress enhance leukocyte infiltration of the arterial wall and thus are atherogenic. However, little is known about the effect of disturbed flow on ECs, especially on their interactions with SMCs, whose phenotypic switching is significantly implicated in atherosclerosis and neointimal lesion formation. Our recent studies using microRNA (miR) assay, in vitro EC‐SMC co‐culture flow system, experimental animal models, and human specimens from patients with coronary artery disease (CAD) have identified several miRs to be involved in the formation and progression of atherosclerosis. Among these miRs, miR‐146a and 10a play atheroprotective roles in ECs against atherogenesis induced by disturbed flow. MiR‐451, by targeting Rab5a, inhibits vascular SMC proliferation and inflammation and suppresses injury‐induced neointimal lesion formation. Our findings help the discovery of new target biomarkers and elucidation of functional mechanisms underlying atherosclerosis, thereby facilitating the development of new approaches for therapeutic interventions.Support or Funding InformationThis work was supported by grants from the Ministry of Science and Technology in Taiwan (grant numbers: MOST‐106‐2633‐B‐009‐001, 105‐2321‐B‐400‐007, 106‐3114‐Y‐043‐021)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Rubiarbonone C inhibits platelet-derived growth factor-induced proliferation and migration of vascular smooth muscle cells through the focal adhesion kinase, MAPK and STAT3 Tyr705 signalling pathways.

The proliferation and migration of vascular smooth muscle cells (VSMCs) induced by platelet-derived growth factor (PDGF) are important steps in cardiovascular diseases, including neointimal lesion formation, myocardial infarction and atherosclerosis. Here, we evaluated the rubiarbonone C-mediated signalling pathways that regulate PDGF-induced VSMC proliferation and migration. Cell proliferation and migration were measured in cells treated with rubiarbonone C followed by PDGF BB using the MTT assay, [3 H]-thymidine incorporation, flow cytometry and wound-healing migration assay, MMP gelatin zymography, a fluorescence assay for F-actin. Western blotting of molecules including MAPK, focal adhesion kinase (FAK) and STAT3 and an immunofluorescence assay using anti-PCNA and -STAT3 antibodies were performed to evaluate rubiarbonone C signalling pathway(s). The medial thickness of the carotid artery was evaluated using a mouse carotid ligation model. Rubiarbonone C inhibited PDGF-induced VSMC proliferation and migration and diminished the ligation-induced increase in medial thickness of the carotid artery. In PDGF-stimulated VSMCs rubiarbonone C decreased the following: (i) levels of cyclin-dependent kinases, cyclins, PCNA and hyperphosphorylated retinoblastoma protein; (ii) levels and activity of MMP2 and MMP9; (iii) activation of MAPK; (iv) F-actin reorganization, by reducing FAK activation; (v) activation of STAT3. These findings suggest that rubiarbonone C inhibits the proliferation and migration of VSMCs by inhibiting the FAK, MAPK and STAT3 signalling pathways. Therefore, rubiarbonone C could be a good candidate for the treatment of cardiovascular disease.

The novel mineralocorticoid receptor antagonist finerenone attenuates neointima formation after vascular injury.

BackgroundThe novel nonsteroidal mineralocorticoid receptor (MR) antagonist finerenone holds promise to be safe and efficient in the treatment of patients with heart failure and/or chronic kidney disease. However, its effects on vascular function remain elusive.PurposeThe aim of this study was to determine the functional effect of selective MR antagonism by finerenone in vascular cells in vitro and the effect on vascular remodeling following acute vascular injury in vivo.Methods and resultsIn vitro, finerenone dose-dependently reduced aldosterone-induced smooth muscle cell (SMC) proliferation, as quantified by BrdU incorporation, and prevented aldosterone-induced endothelial cell (EC) apoptosis, as measured with a flow cytometry based caspase 3/7 activity assay.In vivo, oral application of finerenone resulted in an accelerated re-endothelialization 3 days following electric injury of the murine carotid artery. Furthermore, finerenone treatment inhibited intimal and medial cell proliferation following wire-induced injury of the murine femoral artery 10 days following injury and attenuated neointimal lesion formation 21 days following injury.ConclusionFinerenone significantly reduces apoptosis of ECs and simultaneously attenuates SMC proliferation, resulting in accelerated endothelial healing and reduced neointima formation of the injured vessels. Thus, finerenone appears to provide favorable vascular effects through restoring vascular integrity and preventing adverse vascular remodeling.

Novel Pathological Role of hnRNPA1 (Heterogeneous Nuclear Ribonucleoprotein A1) in Vascular Smooth Muscle Cell Function and Neointima Hyperplasia.

hnRNPA1 (heterogeneous nuclear ribonucleoprotein A1) plays a variety of roles in gene expression. However, little is known about the functional involvement of hnRNPA1 in vascular smooth muscle cell (VSMC) function and neointima hyperplasia. In this study, we have attempted to investigate the functional roles of hnRNPA1 in the contexts of VSMC function, injury-induced vessel remodeling, and human atherosclerotic lesions, as well as discern the molecular mechanisms involved. APPROACH AND RESULTS: hnRNPA1 expression levels were consistently modulated during VSMC phenotype switching and neointimal lesion formation induced by wire injury. Functional studies showed that VSMC-specific gene expression, proliferation, and migration were regulated by hnRNPA1. Our data show that hnRNPA1 exerts its effects on VSMC functions through modulation of IQGAP1 (IQ motif containing GTPase activating protein 1). Mechanistically, hnRNPA1 regulates IQGAP1 mRNA degradation through 2 mechanisms: upregulating microRNA-124 (miR-124) and binding to AU-rich element of IQGAP1 gene. Further evidence suggests that hnRNPA1 upregulates miR-124 by modulating miR-124 biogenesis and that IQGAP1 is the authentic target gene of miR-124. Importantly, ectopic overexpression of hnRNPA1 greatly reduced VSMC proliferation and inhibited neointima formation in wire-injured carotid arteries. Finally, lower expression levels of hnRNPA1 and miR-124, while higher expression levels of IQGAP1, were observed in human atherosclerotic lesions. Our data show that hnRNPA1 is a critical regulator of VSMC function and behavior in the context of neointima hyperplasia, and the hnRNPA1/miR-124/IQGAP1 regulatory axis represents a novel therapeutic target for the prevention of cardiovascular diseases.

Perivascular mast cells promote neointimal elastin deposition and suppress chronic vein graft restenosis in hyperlipidaemic mice

Aims: Mast cells are versatile innate immune cells and are reported to promote vascular inflammation and neointimal lesion formation, thereby contributing to the development of vascular stenosis and atherosclerosis. However, it is not clear whether mast cells also regulate vascular matrix remodelling in established neointima. This study addressed the hypothesis that perivascular mast cells regulate neointimal matrix remodelling using a mouse vein graft model. Methods: The impact of mast cells on neointimal remodelling was investigated using mast cell-deficient animals in both normolipidaemic (KitW-sh/W-sh) and hyperlipidaemic (apoE-/-KitW-sh/W-sh) conditions. The effect of perivascular mast cells on vascular matrix remodelling, including collagen and elastin deposition, was investigated using a local mast cell reconstitution method that selectively repopulated mast cells around the carotid artery (where the vein graft was inserted) in KitW-sh/W-sh mice. Results: In normolipidaemic vein grafts (KitW-sh/W-sh vs. the wild type control C57BL/6J), collagen synthesis was not affected by mast cell deficiency at 4 weeks. In contrast, neointimal elastin was reduced by 6.5-fold in mast cell-deficient KitW-sh/W-sh mice, which was prevented by perivascular mast cell reconstitution. Mast cell deficiency induced a similar reduction in neointimal elastin in hyperlipidaemic mice (apoE-/-KitW-sh/W-sh vs. apoE-/-), with a significant increase in cell proliferation and neointimal area at 4 week. Conclusion: Mast cells appear to promote elastin deposition in vein grafts and this may lead to further suppression of cell proliferation and neointimal thickening under hyperlipidaemic conditions.