Partial Carotid Artery Ligation Research Articles

Disturbed flow impairs MerTK-mediated efferocytosis in aortic endothelial cells during atherosclerosis.

Background: MER proto-oncogene tyrosine kinase (MerTK) is a key receptor for efferocytosis, a process for the clearance of apoptotic cells. MerTK is mainly expressed in macrophages and immature dendritic cells. There are very limited reports focused on MerTK biology in aortic endothelial cells (ECs). It remains unclear for the role of blood flow patterns in regulating MerTK-mediated efferocytosis in aortic ECs. This study was designed to investigate whether endothelial MerTK and EC efferocytosis respond to blood flow patterns during atherosclerosis. Methods: Big data analytics, RNA-seq and proteomics combined with our in vitro and in vivo studies were applied to reveal the potential molecular mechanisms. Partial carotid artery ligation combined with AAV-PCSK9 and high fat diet were used to set up acute atherosclerosis in 4 weeks. Results: Our data showed that MerTK is sensitive to blood flow patterns and is inhibited by disturbed flow and oscillatory shear stress in primary human aortic ECs (HAECs). The RNA-seq data in HAECs incubated with apoptotic cells showed that d-flow promotes pro-inflammatory pathway and senescence pathway. Our in vivo data of proteomics and immunostaining showed that, compared with WT group, MerTK-/- aggravates atherosclerosis in d-flow areas through upregulation of endothelial dysfunction markers (e.g. IL-1β, NF-κB, TLR4, MAPK signaling, vWF, VCAM-1 and p22phox) and mitochondrial dysfunction. Interestingly, MerTK-/-induces obvious abnormal endothelial thickening accompanied with decreased endothelial efferocytosis, promoting the development of atherosclerosis. Conclusions: Our data suggests that blood flow patterns play an important role in regulating MerTK-mediated efferocytosis in aortic ECs, revealing a new promising therapeutic strategy with EC efferocytosis restoration to against atherosclerosis.

A novel circular RNA, circSQSTM1, protects the endothelial function in atherosclerosis

A novel circRNA named circSQSTM1 (hsa_circRNA_075320) was screened out in atorvastatin (ATV) stimulated endothelial cells (ECs) by our group. Considering the anti-atherosclerotic function of ATV, we hypothesized the circSQSTM1 could protect ECs functions in AS progression. The effects of circSQSTM1 on ECs inflammation, oxidative stress and autophagy were measured by qRT-PCR, Western blotting, monocyte-endothelial adhesion assay, dichloro-dihydro-fluorescein diacetate and mCherry-GFP-LC3 labeling. A luciferase reporter assay, RNA immunoprecipitation, MS2-tagging system and fluorescence in situ hybridization were performed to identify the biological functions of circSQSTM1. The partial left carotid artery ligation model and atherosclerosis model were established to analyze the effects of circSQSTM1 on atherosclerosis progression in vivo. Our results revealed that ATV induced the accumulation of circSQSTM1 in ECs via suppressing m6A modified degradation. In the cytoplasm, circSQSTM1 could relieve Sirt1 by competitively sponging miR-23b-3p. In the nucleus, circSQSTM1 directly interacts with eIF4A3 and promoting the efficient nuclear export of FOXO1 mRNA, which encodes FOXO1 transcription factor to directly activate Sirt1 promoter activity. Hence, circSQSTM1 reduced inflammation, inhibited oxidative stress and promoted autophagy by upregulating Sirt1 in ECs. Moreover, circSQSTM1 overexpression in ECs attenuated the progression of atherosclerosis in ApoE−/− mice. Taken together, the unique noncoding RNA known as circSQSTM1 took a protective role to the ECs in atherosclerosis.

Single-Cell RNA sequencing investigation of female-male differences under PAD conditions.

Peripheral arterial disease (PAD) is an age-related medical condition affecting mostly muscular arteries of the limb. It is the 3rd leading cause of atherosclerotic morbidity. The mechanical environment of endothelial cells (ECs) in PAD is characterized by disturbed blood flow (d-flow) and stiff extracellular matrices. In PAD, the stiffness of arteries is due to decreased elastin function and increased collagen content. These flow and stiffness parameters are largely missing from current models of PAD. It has been previously proven that ECs exposed to d-flow or stiff substrates lead to proatherogenic pathways, but the effect of both, d-flow and stiffness, on EC phenotype has not been fully investigated. In this study, we sought to explore the effect of sex on proatherogenic pathways that could result from exposing endothelial cells to a d-flow and stiff environment. We utilized the scRNA-seq tool to analyze the gene expression of ECs exposed to the different mechanical conditions both in vitro and in vivo. We found that male ECs exposed to different mechanical stimuli presented higher expression of genes related to fibrosis and d-flow in vitro. We validated our findings in vivo by exposing murine carotid arteries to d-flow via partial carotid artery ligation. Since women have delayed onset of arterial stiffening and subsequent PAD, this work may provide a framework for some of the pathways in which biological sex interacts with sex-based differences in PAD.

Abstract 455: Lats1/2 Expression Reduced By The Pathological Flow Triggers Intraplaque Hemorrhage And Distal Pulmonary Thrombosis By Promoting EC Apoptosis And Proliferation In Concert

Atherosclerotic and high-risk plaques are predominantly localized to vessel wall regions with disturbed blood flow (d-flow), leading to the theory that d- and low-flow-driven endothelial cells (EC) dysfunction play a role in this process. It has become clear that high flow (h-flow; high shear stress due to increased flow rate) is also critical to developing high-risk plaques including both rupture and erosion. We aim to determine how the high-risk plaque is formed by pathological flow. The pathological flow (both h- and d-flow) resulted in decreased LATS1/2 expression, but physiological laminar flow did not. Within 14 days after inducing LATS1/2 deletion in tamoxifen-inducible EC-specific LATS1 homo /LATS2 homo knock-out mice (LATS1 homo /LATS2 homo -EKO), all the mice (27/27) had died of severe systemic edema, accompanied by increased EC apoptosis and vascular permeability. We generated EC-specific LATS1 he t /LATS2 homo -EKO mice under hypercholesterolemia (H-chol) with pAAV/D377Y-mPCSK9 injections and a high-fat diet (HFD). After 8weeks of HFD, we detected plaque erosion-like lesions at the h- and d-flow area in the aortic arch and carotids, which revealed strong fibrin/fibrinogen-positive organized thrombus formation without a sizeable necrotic core. Interestingly, we also found a significant increase in distal pulmonary thrombosis and EC proliferation in LATS1 he t /LATS2 homo -EKO mice. H-chol increased the incidents of aortic but not lung lesions. We also performed partial left carotid artery ligation (PLCL) and found that d-flow induced by PLCL developed severe atherosclerosis formation with unique intraplaque hemorrhage in H-chol LATS1 he t /LATS2 homo -EKO mice, which was not observed in wild-type mice. In LATS1 he t /LATS2 homo -EKO mice, we found significant increases in 1) EC proliferation, 2) EC apoptosis with senescent phenotype, 3) tissue factor (TF) expression and 4) inflammation, which are collectively referred to as the “pro-thrombotic phenotype” in vivo . These data suggest that pathological flow-induced LATS1/2 degradation promotes EC apoptosis and proliferation simultaneously. This cycle of EC damage-thrombus formation-re-endothelialization results in rapid thrombus enlargement and distal pulmonary thrombosis.

Vertical Sleeve Gastrectomy Offers Protection against Disturbed Flow-Induced Atherosclerosis in High-Fat Diet-Fed Mice.

Bariatric surgery reduces body weight, enhances metabolic and diabetic control, and improves outcomes on obesity-related comorbidities. However, the mechanisms mediating this protection against cardiovascular diseases remain unclear. We investigated the effect of sleeve gastrectomy (SG) on vascular protection in response to shear stress-induced atherosclerosis using an overweighted and carotid artery ligation mouse model. Eight-week-old male wild-type mice (C57BL/6J) were fed a high-fat diet (HFD) for two weeks to induce weight gain and dysmetabolism. SG was performed in HFD-fed mice. Two weeks after the SG procedure, partial carotid-artery ligation was performed to promote disturbed flow-induced atherosclerosis. Compared with the control mice, HFD-fed wild-type mice exhibited increased body weight, total cholesterol level, hemoglobin A1c, and enhanced insulin resistance; SG significantly reversed these adverse effects. As expected, HFD-fed mice exhibited greater neointimal hyperplasia and atherosclerotic plaques than the control group, and the SG procedure attenuated HFD-promoted ligation-induced neointimal hyperplasia and arterial elastin fragmentation. Besides, HFD promoted ligation-induced macrophage infiltration, matrix metalloproteinase-9 expression, upregulation of inflammatory cytokines, and increased vascular endothelial growth factor secretion. SG significantly reduced the above-mentioned effects. Moreover, HFD restriction partially reversed the intimal hyperplasia caused by carotid artery ligation; however, this protective effect was significantly lower than that observed in SG-operated mice. Our study demonstrated that HFD deteriorates shear stress-induced atherosclerosis and SG mitigates vascular remodeling, and this protective effect was not comparable in HFD restriction group. These findings provide a rationale for using bariatric surgery to counter atherosclerosis in morbid obesity.

Inhibition of Trimethylamine N-Oxide Attenuates Neointimal Formation Through Reduction of Inflammasome and Oxidative Stress in a Mouse Model of Carotid Artery Ligation.

Aims: Trimethylamine-N-oxide (TMAO) is a metabolite generated from dietary choline, betaine, and l-carnitine, after their oxidization in the liver. TMAO has been identified as a novel independent risk factor for atherosclerosis through the induction of vascular inflammation. However, the effect of TMAO on neointimal formation in response to vascular injury remains unclear. Results: This study was conducted using a murine model of acutely disturbed flow-induced atherosclerosis induced by partial carotid artery ligation. 3,3-Dimethyl-1-butanol (DMB) was used to reduce TMAO concentrations. Wild-type mice were divided into four groups [regular diet, high-TMAO diet, high-choline diet, and high-choline diet+DMB] to investigate the effects of TMAO elevation and its inhibition by DMB. Mice fed high-TMAO and high-choline diets had significantly enhanced neointimal hyperplasia and advanced plaques, elevated arterial elastin fragmentation, increased macrophage infiltration and inflammatory cytokine secretion, and enhanced activation of nuclear factor (NF)-κB, the NLRP3 inflammasome, and endoplasmic reticulum (ER) stress relative to the control group. Mice fed high-choline diets with DMB treatment exhibited attenuated flow-induced atherosclerosis, inflammasome expression, ER stress, and reactive oxygen species expression. Human aortic smooth muscle cells (HASMCs) were used to investigate the mechanism of TMAO-induced injury. The HASMCs were treated with TMAO with or without an ER stress inhibitor to determine whether inhibition of ER stress modulates the TMAO-induced inflammatory response. Innovation: This study demonstrates that TMAO regulates vascular remodeling via ER stress. Conclusion: Our findings demonstrate that TMAO elevation promotes disturbed flow-induced atherosclerosis and that DMB administration mitigates vascular remodeling, suggesting a rationale for a TMAO-targeted strategy for the treatment of atherosclerosis. Antioxid. Redox Signal. 38, 215-233.

Tenascin-X Mediates Flow-Induced Suppression of EndMT and Atherosclerosis.

Endothelial-to-mesenchymal transition (EndMT) has been identified as a critical driver of vascular inflammation and atherosclerosis, and TGF-β (transforming growth factor β) is a key mediator of EndMT. Both EndMT and atherosclerosis are promoted by disturbed flow, whereas unidirectional laminar flow limits EndMT and is atheroprotective. How EndMT and endothelial TGF-β signaling are regulated by different flow patterns is, however, still poorly understood. Flow chamber experiments in vitro and endothelium-specific knockout mice were used to study the role of tenascin-X in the regulation of EndMT and atherosclerosis as well as the underlying mechanisms. In human endothelial cells as well as in human and mouse aortae, unidirectional laminar flow but not disturbed flow strongly increased endothelial expression of the extracellular matrix protein TN-X (tenascin-X) in a KLF4 (Krüppel-like factor 4) dependent manner. Mice with endothelium-specific loss of TN-X (EC-Tnxb-KO) showed increased endothelial TGF-β signaling as well as increased endothelial expression of EndMT and inflammatory marker genes. When EC-Tnxb-KO mice were subjected to partial carotid artery ligation, we observed increased vascular remodeling. EC-Tnxb-KO mice crossed to low-density lipoprotein receptor-deficient mice showed advanced atherosclerotic lesions after being fed a high-fat diet. Treatment of EC-Tnxb-KO mice with an anti-TGF-beta antibody or additional endothelial loss of TGF-beta receptors 1 and 2 normalized endothelial TGF-beta signaling and prevented EndMT. In in vitro studies, we found that TN-X through its fibrinogen-like domain directly interacts with TGF-β and thereby interferes with its binding to the TGF-β receptor. In summary, we show that TN-X is a central mediator of flow-induced inhibition of EndMT, endothelial inflammation and atherogenesis, which functions by binding to and by blocking the activity of TGF-β. Our data identify a novel mechanism of flow-dependent regulation of vascular TGF-β, which holds promise for generating new strategies to prevent vascular inflammation and atherosclerosis.

Nur77 Deficiency Exacerbates Macrophage NLRP3 Inflammasome-Mediated Inflammation and Accelerates Atherosclerosis.

Purpose Activation of NLR (nucleotide-binding and leucine-rich repeat immune receptor) family pyrin domain containing 3 (NLRP3) inflammasome mediating interleukin- (IL-) 1β secretion has emerged as an important component of inflammatory processes in atherogenesis. The nuclear receptor Nur77 is highly expressed in human atherosclerotic lesions; however, its functional role in macrophage NLRP3 inflammasome activation has not yet been clarified. Methods, Materials, and Results. Eight-week-old apolipoprotein E (ApoE)−/− and ApoE−/− Nur77−/− mice that were fed a Western diet underwent partial ligation of the left common carotid artery (LCCA) and left renal artery (LRA) to induce atherogenesis. Four weeks later, severe plaque burden associated with increased lipid deposition, reduced smooth muscle cells, macrophage infiltration, and decreased collagen expression was identified in ApoE−/− Nur77−/− mice compared with those in ApoE−/− mice. ApoE−/− Nur77−/− mice showed increased macrophage inflammatory responses in carotid atherosclerotic lesions. In vitro studies demonstrated that oxidized low-density lipoprotein cholesterol (ox-LDL) increased the release of lactate dehydrogenase (LDH) and upregulated the expressions of cleaved caspase-1, cleaved IL-1β and gasdermin D (GSMD) in WT peritoneal macrophages (PMs) in a NLRP3-dependent manner. Nur77−/− PMs exhibited a further increased level of NLRP3 inflammasome-mediated inflammation under ox-LDL treatment compared with WT PMs. Mechanistically, Nur77 could bind to the promoter of NLRP3 and inhibit its transcriptional activity. Conclusions This study demonstrated that Nur77 deletion promotes atherogenesis by exacerbating NLRP3 inflammasome-mediated inflammation.

3-Dimensional electrical impedance spectroscopy for in situ endoluminal mapping of metabolically active plaques

Electrical impedance spectroscopy (EIS) has been recognized to characterize oxidized low-density lipoprotein (oxLDL) in the metabolically active plaque. However, intravascular deployment of 3-D EIS-derived electrical impedance tomography (EIT) for endoluminal mapping of oxLDL-laden arterial walls remains an unmet clinical challenge. To this end, we designed the 6-point microelectrode arrays that were circumferentially configurated onto the balloon catheter for 15 intravascular EIS permutations. In parallel, we created the metabolically active plaques by performing partial ligation of right carotid artery in Yorkshire mini-pigs (n = 6 males), followed by demonstrating the plaque progression at baseline, 8 weeks, and 16 weeks of high-fat diet via computed tomography (CT) angiogram. Next, we deployed the 3-D EIS sensors to the right and left carotid arteries, and we demonstrated 3-D EIS mapping of metabolically active endolumen in the right but not left carotid arteries as evidenced by the positive E06 immunostaining for oxLDL-laden regions. By considering electrical conductivity (σ) and permittivity (ε) properties of collagen, lipid, and smooth muscle presence in the arterial wall, we further validated the 3-D EIS-derived EIT by reconstructing the histology of right and left carotid arteries for the finite element modeling of the oxLDL-laden endolumen, and we accurately predicted 3-D EIS mapping. Thus, we establish the capability of 3-D EIS-derived EIT to detect oxLDL-laden arterial walls with translational implication to predict metabolically active plaques prone to acute coronary syndromes or stroke.

Abstract 13231: Semaphorin3F Reduces Atherosclerosis by Decreasing Endothelial Activation and Barrier Permeability and Smooth Muscle Cell Migration

Coronary artery disease (CAD) remains a leading cause of death worldwide. To identify genes causally linked to CAD, we previously conducted large-scale genetic analyses, and identified an association between genes in the semaphorin pathway and CAD. Expression profiling in human atherosclerosis-relevant cell types and atherosclerotic plaques identified semaphorin3F (SEMA3F) as a possible candidate causally linked to atherosclerosis. Administration of SEMA3F reduced aortic lesion area in western-type diet-fed Ldlr -/- mice (Control: 12.1±1.4; SEMA3F: 8.1±0.8, % lesion area/total aortic sinus area, p=0.02). In contrast, neointimal formation was increased in Sema3f -/- mice with partial carotid artery ligation ( WT : 22.8±2.8; Sema3f -/- : 39.2±4.0; % intimal area/total vessel area, p=0.004). Additionally, SEMA3F administration increased re-endothelialization and reduced restenosis in Ldlr -/- mice that underwent carotid endothelial denudation (Control: 53.3±3.5; SEMA3F: 21.4±3.1; % intimal plaque area/vessel area, p=0.0006). Monocyte adhesion to aortic vascular endothelial cells (VECs) from Sema3f -/- mice was elevated, driven by increased NF-κB activation and increased expression of its target adhesion molecules, VCAM1 and ICAM1, in VECs. Monocyte transmigration through VECs from Sema3f -/- mice was also increased, likely due to increased membrane permeability, assessed by in-vivo intravital two-photon microscopy. The increased membrane permeability was modulated in part by decreased mTOR phosphorylation that in turn suppressed vascular endothelial cadherin protein expression. SEMA3F increased thick actomyosin filaments and elicited larger focal adhesions in VECs, suggesting decreased migration. In line with this, SEMA3F inhibited vascular smooth muscle cell (VSMC) migration and maintained the contractile phenotype of VSMCs, which was mediated by the Rho pathway. SEMA3F is causally atheroprotective in models of atherosclerosis and restenosis, mediated, in part, through the modulation of vascular endothelial cell permeability and adhesion molecule expression, and vascular smooth muscle cell migration and phenotype switching.

Single-cell RNA-seq reveals cellular heterogeneity of mouse carotid artery under disturbed flow

Disturbed blood flow (d-flow) has been known to induce changes of the cells in the arterial wall, increasing the risk of atherosclerosis. However, the heterogeneity of the vascular cell populations under d-flow remains less understood. To generate d-flow in vivo, partial carotid artery ligation (PCL) was performed. Seven days after ligation, single-cell RNA sequencing of nine left carotid arteries (LCA) from the PCL group (10,262 cells) or control group (14,580 cells) was applied and a single-cell atlas of gene expression was constructed. The integrated analysis identified 15 distinct carotid cell clusters, including 10 d-flow-relevant subpopulations. Among endothelial cells, at least four subpopulations were identified, including Klk8hi ECs, Lrp1hi ECs, Dkk2hi ECs, and Cd36hi ECs. Analysis of GSVA and single-cell trajectories indicated that the previously undescribed Dkk2hi ECs subpopulation was mechanosensitive and potentially transformed from Klk8hi ECs under d-flow. D-flow-induced Spp1hi VSMCs subpopulation that appeared to be endowed with osteoblast differentiation, suggesting a role in arterial stiffness. Among the infiltrating cell subpopulations, Trem2hi Mφ, Birc5hi Mφ, DCs, CD4+ T cells, CXCR6+ T cells, NK cells, and granulocytes were identified under d-flow. Of note, the novel Birc5hi Mφ was identified as a potential contributor to the accumulation of macrophages in atherosclerosis. Finally, Dkk2hi ECs, and Cd36hi ECs were also found in the proatherosclerotic area of the aorta where the d-flow occurs. In conclusion, we presented a comprehensive single-cell atlas of all cells in the carotid artery under d-flow, identified previously unrecognized cell subpopulations and their gene expression signatures, and suggested their specialized functions.

F-18 FDG PET/CT CAN BE USED AS AN IMAGING BIOMARKER FOR REFLECTING SYNTHETIC VASCULAR SMOOTH MUSCLE CELL ACTIVITY IN THE ANIMAL MODEL OF VASCULAR REMODELING DISORDER

Objective: Synthetic vascular smooth muscle cells (VSMCs) play important roles in vascular remodeling disorders including atherosclerosis, restenosis, and transplant vasculopathy. 18F-fluorodeoxyglucose positron emission tomography (F-18 FDG PET/CT) is a non-invasive molecular imaging modality widely used in clinical practice for reflecting glucose metabolism. The aim of this study was to demonstrate the feasibility of measuring the activity of synthetic VSMCs using F-18 FDG PET/CT in an atherosclerotic rat model of partial carotid artery ligation. Design and method: Using Sprague-Dawley rats, neointimal hyperplasia was made by right partial carotid ligation method. After 1 month, all animal models undertook F-18 FDG PET/CT images. Then the vessels were harvested and analyzed with autoradiography and histomolecular approaches. Results: The affected carotid artery exhibited prominent neointinal hyperplasia region with a narrowed lumen. Known surrogate marker of synthetic VSMCs including matrix metallopeptidase-9, cyclophilin A, and collagen type III were increased in neointimal hyperplasia region. There were no immune cells such as macrophages or neutrophils. F-18 FDG PET/CT and autoradiography showed increased tracer uptake along the same affected arteries. Glucose transporter-1 was also increased in neointimal hyperplasia region. Conclusions: It is feasible to use F-18 FDG PET/CT as an imaging surrogate-marker for reflecting synthetic VSMC activities in vascular remodeling disorders.

Protective role of serpina3c as a novel thrombin inhibitor against atherosclerosis in mice.

Abnormal vascular smooth muscle cell (VSMC) proliferation is a critical step in the development of atherosclerosis. Serpina3c is a serine protease inhibitor (serpin) that plays a key role in metabolic diseases. The present study aimed to investigate the role of serpina3c in atherosclerosis and regulation of VSMC proliferation and possible mechanisms. Serpina3c is down-regulated during high-fat diet (HFD)-induced atherosclerosis. An Apoe-/-/serpina3c-/--double-knockout mouse model was used to determine the role of serpina3c in atherosclerosis after HFD for 12 weeks. Compared with Apoe-/- mice, the Apoe-/-/serpina3c-/- mice developed more severe atherosclerosis, and the number of VSMCs and macrophages in aortic plaques was significantly increased. The present study revealed serpina3c as a novel thrombin inhibitor that suppressed thrombin activity. In circulating plasma, thrombin activity was high in the Apoe-/-/serpina3c-/- mice, compared with Apoe-/- mice. Immunofluorescence staining showed thrombin and serpina3c colocalization in the liver and aortic cusp. In addition, inhibition of thrombin by dabigatran in serpina3c-/- mice reduced neointima lesion formation due to partial carotid artery ligation. Moreover, an in vitro study confirmed that thrombin activity was also decreased by serpina3c protein, supernatant and cell lysate that overexpressed serpina3c. The results of experiments showed that serpina3c negatively regulated VSMC proliferation in culture. The possible mechanism may involve serpina3c inhibition of ERK1/2 and JNK signaling in thrombin/PAR-1 system-mediated VSMC proliferation. Our results highlight a protective role for serpina3c as a novel thrombin inhibitor in the development of atherosclerosis, with serpina3c conferring protection through the thrombin/PAR-1 system to negatively regulate VSMC proliferation through ERK1/2 and JNK signaling.

Abstract 16504: Low Disturbed Flow Induces Dynamic Immune Compartment Alterations in Atherogenesis as Revealed by Single Cell Rna-seq

Low disturbed blood flow (LDF) is a critical contributing factor to atherogenesis but its direct impact on the immune compartment was not well-depict. To fill this knowledge gap, we adopted scRNA-seq to capture sheer-stress induced immune responses during atherogenesis. A partial carotid artery ligation (PCAL) model was selected for its paired comparison of carotid arteries with normal flow (NF) or LDF. Indeed, we observed drastic changes in both endothelial and immune compartment. Macrophages were the most significantly increased population induced by LDF (from 4% to 12% of CD45+ cells) with two well-separated subsets (Mac-c8, Mac-c9). MacSpectrum analyses revealed that Mac-c8 displayed higher inflammatory states than the lipid-laden Mac-c9. Interestingly, three T subtypes displayed unique flow-induced enrichment patterns that were selectively enriched in LDF but not in the NF condition. Furthermore, we created an original algorithms to evaluate the impact of sheer-stress on membrane protein-mediated cell-cell interaction among all cell types in the atheroma. Several pairs of molecular interactions were identified, including multiple APP-ligands interaction pairs and those in BAG2 Signaling. Moreover, signature genes identified in these LDF-induced T cells displayed high correlation to the plaque severity in human artery-aorta samples. Collectively, our study provided a high-resolution and focused analyses of sheer-stress induced immune cell action during atherogenesis. This is also the first identification of unique T subsets, to our knowledge, that are enriched in arterial wall exposed to low and disturbed flow. Further characterization of these cells will provide valuable information to understand and therapeutically treat atherogenesis.

Semaphorin 7A promotes endothelial to mesenchymal transition through ATF3 mediated TGF-\u03b22/Smad signaling

Endothelial to mesenchymal transition (EndMT) is an important pathological change in many diseases. Semaphorin7A (Sema7A) has been reported to regulate nerve and vessel homeostasis, but its role in EndMT remains unclear. Here we investigate the effect of Sema7A on EndMT and the underlying mechanism. Sema7A-overexpressed human umbilical vein endothelial cells (Sema7A-HUVECs) were generated and showed lower levels of endothelial cell markers and higher levels of mesenchymal cell markers indicating the occurrence of EndMT. RNA-sequencing analysis showed a total of 1168 upregulated genes and 886 downregulated genes. Among them, most of the molecules associated with EndMT were upregulated in Sema7A-HUVECs. Mechanistically, Sema7A-HUVECs showed a higher TGF-β2 expression and activated TGF-β/Smad Signaling. Importantly, Sema7A overexpression upregulated activating transcription factor 3 (ATF3) that was found to selectively bind the promotor region of TGF-β2, but not TGF-β1, promoting TGF-β2 transcription, which was further confirmed by ATF3-siRNA knockdown approach. Blocking β1 integrin, a known Sema7A receptor, alleviated the expression of ATF3, TGF-β2, and EndMT in Sema7A-overexpressed HUVECs, implying a role of β1 integrin/ATF3/TGF-β2 axis in mediating Sema7A-induced EndMT. Using Sema7A-deficient mice and the partial carotid artery ligation (PCL) model, we showed that Sema7A deletion attenuated EndMT induced by blood flow disturbance in vivo. In conclusion, Sema7A promotes TGF-β2 secretion by upregulating transcription factor ATF3 in a β1 integrin-dependent manner, and thus facilitates EndMT through TGF/Smad signaling, implying Sema7A as a potential therapeutic target for EndMT-related vascular diseases.

SRGN, a new identified shear-stress-responsive gene in endothelial cells.

Endothelial cells (ECs) play an important role in the pathogenesis of cardiovascular disease, especially atherosclerosis (AS). The abnormal wall shear stress (WSS) which directly contacts with ECs is the key stimulating factor leading to AS. However, the underlying mechanism of ECs responding to WSS is still incompletely understood. This study aims to explore the novel mechano-sensitive genes and its potential mechanism in response to WSS in ECs by employing bioinformatics methods based on previously available high-throughput data from zebrafish embryos, both before and after blood flow formation. Six common differentially expressed genes (DEGs) (SRGN, SLC12A3, SLC25A4, PVALB1, ITGAE.2, zgc:198419) were selected out from two high-throughput datasets (GSE126617 and GSE20707) in the GEO database. Among them, SRGN was chosen for further verification through the in vitro shear stress loading experiments with human umbilical vein endothelial cells (HUVECs) and the in vivo partial ligation of carotid artery in mice. Our data indicated that low shear stress (LSS) could enhance the expression of SRGN via the PKA/CREB-dependent signaling pathway. The proportion of Ki67+ cells and the concentration of nitric oxide (NO) were high in SRGN high expression cells, suggesting that SRGN may be involved in the proliferation of HUVECs. Furthermore, in the partial ligation of the carotid artery mice model, we observed that the expression of SRGN was significantly increased in atherosclerotic plaques induced by abnormal shear stress. Taken together, this study demonstrated that SRGN is a key gene in the response of ECs to WSS and could be involved in AS.

Anti-Atherogenic Effect of Stem Cell Nanovesicles Targeting Disturbed Flow Sites.

Atherosclerosis development leads to irreversible cascades, highlighting the unmet need for improved methods of early diagnosis and prevention. Disturbed flow formation is one of the earliest atherogenic events, resulting in increased endothelial permeability and subsequent monocyte recruitment. Here, a mesenchymal stem cell (MSC)-derived nanovesicle (NV) that can target disturbed flow sites with the peptide GSPREYTSYMPH (PREY) (PMSC-NVs) is presented which is selected through phage display screening of a hundred million peptides. The PMSC-NVs are effectively produced from human MSCs (hMSCs) using plasmid DNA designed to functionalize the cell membrane with PREY. The potent anti-inflammatory and pro-endothelial recovery effects are confirmed, similar to those of hMSCs, employing mouse and porcine partial carotid artery ligation models as well as a microfluidic disturbed flow model with human carotid artery-derived endothelial cells. This nanoscale platform is expected to contribute to the development of new theragnostic strategies for preventing the progression of atherosclerosis.

A Novel Small Molecule Compound SP-8356 Targeting CD147 Suppresses Plaque Progression and Promotes Plaque Stability in ApoE-Deficient Mice

Background: Plaque vulnerability is the important therapeutic target in atherosclerosis and its related cardiovascular disease. CD147 has been suggested to play key roles in plaque vulnerability through interacting with cyclophilin A (CypA) and resultant activation of matrix metalloproteinase-9 (MMP-9). Here we report that the novel synthetic CD147 inhibitor SP-8356 ((1S,5R)-4-(3,4-dihydroxy-5-methoxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one) inhibits CD147/MMP-9 pathways and reduces plaque progression and stabilizes plaque vulnerability. Methods: Advanced atherosclerotic plaque was induced in apolipoprotein E-deficient (ApoE KO) mice by partial ligation of the right carotid artery coupled with an atherogenic diet. SP-8356 (50 mg/kg) was orally given daily for 3 weeks. Histomolecular analysis was carried out on harvested carotid arteries. Results: Surface plasmon resonance assay showed the specific binding of SP-8356 with CD147. SP-8356 inhibited CypA-CD147 interaction and MMP-9 activation. In ApoE KO mice model, SP-8356 inhibited plaque formation, reduced the number of macrophages, increased the number of vascular smooth muscle cells, increased the fibrous cap thickness, and increased the collagen type I contents in fibrous cap. SP-8356 also reduced the apoptotic cells in the plaque lesion. Conclusions: Owing to its improvement of plaque stability and inhibitory effect on plaque development, SP-8356 could be a potential therapeutic drug candidate for atherosclerosis and related cardiovascular diseases.

SP-8356, a Novel Inhibitor of CD147-Cyclophilin A Interactions, Reduces Plaque Progression and Stabilizes Vulnerable Plaques in apoE-Deficient Mice.

Interactions between CD147 and cyclophilin A (CypA) promote plaque rupture that causes atherosclerosis-related cardiovascular events, such as myocardial infarction and stroke. Here, we investigated whether SP-8356 ((1S,5R)-4-(3,4-dihydroxy-5-methoxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one), a novel drug, can exert therapeutic effects against plaque progression and instability through disruption of CD147-CypA interactions in apolipoprotein E-deficient (ApoE KO) mice. Immunocytochemistry and immunoprecipitation analyses were performed to assess the effects of SP-8356 on CD147-CypA interactions. Advanced plaques were induced in ApoE KO mice via partial ligation of the right carotid artery coupled with an atherogenic diet, and SP-8356 (50 mg/kg) orally administrated daily one day after carotid artery ligation for three weeks. The anti-atherosclerotic effect of SP-8356 was assessed using histological and molecular approaches. SP-8356 interfered with CD147-CypA interactions and attenuated matrix metalloproteinase-9 activation. Moreover, SP-8356 induced a decreased in atherosclerotic plaque size in ApoE KO mice and stabilized plaque vulnerability by reducing the necrotic lipid core, suppressing macrophage infiltration, and enhancing fibrous cap thickness through increasing the content of vascular smooth muscle cells. SP-8356 exerts remarkable anti-atherosclerotic effects by suppressing plaque development and improving plaque stability through inhibiting CD147-CypA interactions. Our novel findings support the potential utility of SP-8356 as a therapeutic agent for atherosclerotic plaque.

A Novel Epigenetic Drug-Eluting Balloon Angioplasty Device: Evaluation in a Large Animal Model of Neointimal Hyperplasia

Drug-eluting balloon catheters (DEBc) coated with paclitaxel (PTX) have been associated with potential safety concerns. An efficacious but less toxic balloon coating may reduce these outcomes. We evaluated a novel DEBc, Epi-Solve, coated with metacept-3 (MCT-3), a member of the histone deacetylase inhibitor (HDACi) class of epigenetic agents, in a large animal model of neointimal hyperplasia (NIH). Plain balloon angioplasty (PABA) catheters were ultrasonically coated with MCT-3 to generate Epi-Solve DEBc. An ovine model of NIH formation was established utilising partial left common carotid artery (LCA) ligation. Twenty-eight days post neointima (NI) induction, PABA, Epi-Solve or PTX-coated DEBc were deployed at the site of induced NI formation. Twenty-eight days post-intervention, ligated vessels were evaluated for attenuation of NI formation, gene expression profiles and immunohistochemical analysis. Epi-Solve DEBc demonstrated attenuation of NIH over no intervention and a trend to inhibition of NIH over PABA. Gene expression analysis and immunohistochemical studies identified significant anti-proliferative and anti-inflammatory signatures and reduced vascular endothelial cell activation compared to PABA. Epi-Solve is a novel HDACi-coated DEBc which demonstrates significant anti-proliferative and anti-inflammatory signatures and reduced vascular endothelial cell activation compared to PABA in an ovine model and may afford endothelial protection.