Neointimal Lesions Research Articles

Correction to: XBP 1-Deficiency Abrogates Neointimal Lesion of Injured Vessels Via Cross Talk With the PDGF Signaling.

Correction to: XBP 1-Deficiency Abrogates Neointimal Lesion of Injured Vessels Via Cross Talk With the PDGF Signaling.

Abstract 3057: Vascular Smooth Muscle Cell Cd47 Contributes To Atherosclerosis Development

Aims: Thrombospondin-1 (TSP1), a secretory protein, has been associated with various cardiovascular diseases. TSP1 mainly functions via its cognate receptors CD47 and CD36. Recently, we observed elevated TSP1 expression in human and murine atherosclerotic arteries. However, the role of vascular smooth muscle cell (VSMC) TSP1-CD47 signaling in regulating VSMC phenotype and atherogenesis is unclear. Methods and Results: Human arterial SMCs were treated with human TSP1 and analyzed for SMC markers and proliferation. The data revealed decreased expression of SMC markers, ACTA, CNN1, and SM22α in TSP1-exposed VSMCs compared with vehicle-treated control cells. Further, TSP1 treatment increased VSMC proliferation in vitro , suggesting TSP1 induces VSMC hyperproliferative phenotype. Additional experiments showed CD47 as the major TSP1’s receptor in VSMCs. To investigate the in vivo role of VSMC Cd47 in atherosclerosis, we generated tamoxifen-inducible SMC-specific Cd47 knockout mice ( Cd47 f/f Myh11 Cre +/– , Cd47 ΔVSMC ) by crossing female Cd47 f/f mice with male Myh11 Cre +/- mice. Male tamoxifen-injected Cd47 ΔVSMC and corn oil-administered Cd47 WT mice were given a single injection of AAV8- hPCSK9 (1х10 11 viral genomes/mouse, IP) and fed a Western diet for 12 weeks. En face oil red O (ORO) staining of whole aortas exhibited reduced atherosclerosis in Cd47 ΔVSMC mice compared with control Cd47 WT mice. Further histochemical staining performed on aortic root sections revealed attenuated neointimal lesion area, lipid accumulation and necrotic area in Cd47 ΔVSMC mice. However, there were no significant differences in weight gain, body composition (fat and lean mass), plasma total cholesterol, and fasting blood glucose between Cd47 ΔVSMC and Cd47 WT mice. Conclusions: Taken together, these findings suggest that TSP1 promotes VSMC phenotype switch and VSMC-specific Cd47 deletion in hypercholesterolemic mice suppresses atherosclerosis progression.

Atlas of Cell Repertoire Within Neointimal Lesions Is Metabolically Altered in Hypertensive Rats.

High blood pressure has been suggested to accelerate vascular injury-induced neointimal formation and progression. However, little is known about the intricate relationships between vascular injury and hypertension in the context of arterial remodeling. Single-cell RNA-sequencing analysis was used to depict the cell atlas of carotid arteries of Wistar Kyoto rats and spontaneously hypertensive rats with or without balloon injury. We found that hypertension significantly aggravated balloon injury-induced arterial stenosis. A total of 36 202 cells from carotid arteries with or without balloon injury were included in single-cell RNA-sequencing analysis. Cell composition analysis showed that vascular injury and hypertension independently induced distinct aortic cell phenotypic alterations including immune cells, endothelial cells (ECs), and smooth muscle cells. Specifically, our data showed that injury and hypertension-induced specific EC phenotypic alterations, and revealed a transition from functional ECs to hypermetabolic, and eventually dysfunctional ECs in hypertensive rats upon balloon injury. Importantly, our data also showed that vascular injury and hypertension-induced different smooth muscle cell phenotypic alterations, characterized by deferential expression of synthetic signatures. Interestingly, pathway analysis showed that dysregulated metabolic pathways were a common feature in monocytes/macrophages, ECs, and smooth muscle cells in response to injury and hypertension. Functionally, we demonstrate that inhibition of mitochondrial respiration significantly ameliorated injury-induced neointimal formation in spontaneously hypertensive rats. This study provides the cell landscape changes of the main aortic cell phenotypic alterations in response to injury and hypertension. Our findings suggest that targeting cellular mitochondrial respiration could be a novel therapeutic for patients with hypertension undergoing vascular angioplasty.

Spatial multiomics of arterial regions from cardiac allograft vasculopathy rejected grafts reveal novel insights into the pathogenesis of chronic antibody-mediated rejection

Cardiac allograft vasculopathy (CAV) causes late graft failure and mortality after heart transplantation. Donor-specific antibodies (DSAs) lead to chronic endothelial cell injury, inflammation, and arterial intimal thickening. In this study, GeoMx digital spatial profiling was used to analyze arterial areas of interest (AOIs) from CAV+DSA+ rejected cardiac allografts (N = 3; 22 AOIs total). AOIs were categorized based on CAV neointimal thickening and underwent whole transcriptome and protein profiling. By comparing our transcriptomic data with that of healthy control vessels of rapid autopsy myocardial tissue, we pinpointed specific pathways and transcripts indicative of heightened inflammatory profiles in CAV lesions. Moreover, we identified protein and transcriptomic signatures distinguishing CAV lesions exhibiting low and high neointimal lesions. AOIs with low neointima showed increased markers for activated inflammatory infiltrates, endothelial cell activation transcripts, and gene modules involved in metalloproteinase activation and TP53 regulation of caspases. Inflammatory and apoptotic proteins correlated with inflammatory modules in low neointima AOIs. High neointima AOIs exhibited elevated TGFβ-regulated transcripts and modules enriched for platelet activation/aggregation. Proteins associated with growth factors/survival correlated with modules enriched for proliferation/repair in high neointima AOIs. Our findings reveal novel insight into immunological mechanisms mediating CAV pathogenesis.

Unilateral Lung Removal in Combination with Monocrotaline or SU5416 in Rodents: A Reliable Model to Mimic the Pathology of the Human Pulmonary Hypertension.

Pulmonary hypertension (PH) is a chronic and progressive disorder characterized by elevated mean pulmonary arterial pressure, pulmonary vascular remodeling, and the development of concentric laminar intimal fibrosis with plexiform lesions. While rodent models have been developed to study PH, they have certain deficiencies and do not entirely replicate the human disease due to the heterogeneity of PH pathology. Therefore, combined models are necessary to study PH. Recent studies have shown that altered pulmonary blood flow is a significant trigger in the development of vascular remodeling and neointimal lesions. One of the most promising rodent models for increased pulmonary flow is the combination of unilateral left pneumonectomy with a "second hit" of monocrotaline (MCT) or SU5416. The removal of one lung in this model forces blood to circulate only in the other lung and induces increased and turbulent pulmonary blood flow. This increased vascular flow leads to progressive remodeling and occlusion of small pulmonary arteries. The second hit by MCT or SU5416 leads to endothelial cell dysfunction, resulting in severe PH and the development of plexiform arteriopathy.

Pharmacological inhibition of MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1) induces ferroptosis in vascular smooth muscle cells

MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1) is a human paracaspase protein with proteolytic activity via its caspase-like domain. The pharmacological inhibition of MALT1 by MI-2, a specific chemical inhibitor, diminishes the response of endothelial cells to inflammatory stimuli. However, it is largely unknown how MALT1 regulates the functions of vascular smooth muscle cells (SMCs). This study aims to investigate the impact of MALT1 inhibition by MI-2 on the functions of vascular SMCs, both in vitro and in vivo. MI-2 treatment led to concentration- and time-dependent cell death of cultured aortic SMCs, which was rescued by the iron chelator deferoxamine (DFO) or ferrostatin-1 (Fer-1), a specific inhibitor of ferroptosis, but not by inhibitors of apoptosis (Z-VAD-fmk), pyroptosis (Z-YVAD-fmk), or necrosis (Necrostatin-1, Nec-1). MI-2 treatment downregulated the expression of glutathione peroxidase 4 (GPX4) and ferritin heavy polypeptide 1 (FTH1), which was prevented by pre-treatment with DFO or Fer-1. MI-2 treatment also activated autophagy, which was inhibited by Atg7 deficiency or bafilomycin A1 preventing MI-2-induced ferroptosis. MI-2 treatment reduced the cleavage of cylindromatosis (CYLD), a specific substrate of MALT1. Notably, MI-2 treatment led to a rapid loss of contractility in mouse aortas, which was prevented by co-incubation with Fer-1. Moreover, local application of MI-2 significantly reduced carotid neointima lesions and atherosclerosis in C57BL/6J mice and apolipoprotein-E knockout (ApoE−/−) mice, respectively, which were both ameliorated by co-treatment with Fer-1. In conclusion, the present study demonstrated that MALT1 inhibition induces ferroptosis of vascular SMCs, likely contributing to its amelioration of proliferative vascular diseases.

3,4-dimethoxychalcone induces autophagy and reduces neointimal hyperplasia and aortic lesions in mouse models of atherosclerosis

Autophagy inducers can prevent cardiovascular aging and age-associated diseases including atherosclerosis. Therefore, we hypothesized that autophagy-inducing compounds that act on atherosclerosis-relevant cells might have a protective role in the development of atherosclerosis. Here we identified 3,4-dimethoxychalcone (3,4-DC) as an inducer of autophagy in several cell lines from endothelial, myocardial and myeloid/macrophagic origin, as demonstrated by the aggregation of the autophagosome marker GFP-LC3 in the cytoplasm of cells, as well as the downregulation of its nuclear pool indicative of autophagic flux. In this respect, 3,4-DC showed a broader autophagy-inducing activity than another chalcone (4,4- dimethoxychalcone), spermidine and triethylene tetramine. Thus, we characterized the potential antiatherogenic activity of 3,4-DC in two different mouse models, namely, (i) neointima formation with smooth muscle expansion of vein segments grafted to the carotid artery and (ii) genetically predisposed ApoE−/− mice fed an atherogenic diet. In the vein graft model, local application of 3,4-DC was able to maintain the lumen of vessels and to reduce neointima lesions. In the diet-induced model, intraperitoneal injections of 3,4-DC significantly reduced the number of atherosclerotic lesions in the aorta. In conclusion, 3,4-DC stands out as an autophagy inducer with potent antiatherogenic activity.

Pharmacology and Rationale for Seralutinib in the Treatment of Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a complex disorder characterized by vascular remodeling and a consequent increase in pulmonary vascular resistance. The histologic hallmarks of PAH include plexiform and neointimal lesions of the pulmonary arterioles, which are composed of dysregulated, apoptosis-resistant endothelial cells and myofibroblasts. Platelet-derived growth factor receptors (PDGFR) α and β, colony stimulating factor 1 receptor (CSF1R), and mast/stem cell growth factor receptor kit (c-KIT) are closely related kinases that have been implicated in PAH progression. In addition, emerging data indicate significant crosstalk between PDGF signaling and the bone morphogenetic protein receptor type 2 (BMPR2)/transforming growth factor β (TGFβ) receptor axis. This review will discuss the importance of the PDGFR-CSF1R-c-KIT signaling network in PAH pathogenesis, present evidence that the inhibition of all three nodes in this kinase network is a potential therapeutic approach for PAH, and highlight the therapeutic potential of seralutinib, currently in development for PAH, which targets these pathways.

Hsa_circ_0001402 alleviates vascular neointimal hyperplasia through a miR-183-5p-dependent regulation of vascular smooth muscle cell proliferation, migration, and autophagy

IntroductionVascular neointimal hyperplasia, a pathological process observed in cardiovascular diseases such as atherosclerosis and pulmonary hypertension, involves the abundant presence of vascular smooth muscle cells (VSMCs). The proliferation, migration, and autophagy of VSMCs are associated with the development of neointimal lesions. Circular RNAs (circRNAs) play critical roles in regulating VSMC proliferation and migration, thereby participating in neointimal hyperplasia. However, the regulatory roles of circRNAs in VSMC autophagy remain unclear. ObjectivesWe aimed to identify circRNAs that are involved in VSMC autophagy-mediated neointimal hyperplasia, as well as elucidate the underlying mechanisms. MethodsDual-luciferase reporter gene assay was performed to validate two competing endogenous RNA axes, hsa_circ_0001402/miR-183-5p/FKBP prolyl isomerase like (FKBPL) and hsa_circ_0001402/miR-183-5p/beclin 1 (BECN1). Cell proliferation and migration analyses were employed to investigate the effects of hsa_circ_0001402, miR-183-5p, or FKBPL on VSMC proliferation and migration. Cell autophagy analysis was conducted to reveal the role of hsa_circ_0001402 or miR-183-5p on VSMC autophagy. The role of hsa_circ_0001402 or miR-183-5p on neointimal hyperplasia was evaluated using a mouse model of common carotid artery ligation. ResultsHsa_circ_0001402 acted as a sponge for miR-183-5p, leading to the suppression of miR-183-5p expression. Through direct interaction with the coding sequence (CDS) of FKBPL, miR-183-5p promoted VSMC proliferation and migration by decreasing FKBPL levels. Besides, miR-183-5p reduced BECN1 levels by targeting the 3′-untranslated region (UTR) of BECN1, thus inhibiting VSMC autophagy. By acting as a miR-183-5p sponge, overexpression of hsa_circ_0001402 increased FKBPL levels to inhibit VSMC proliferation and migration, while simultaneously elevating BECN1 levels to activate VSMC autophagy, thereby alleviating neointimal hyperplasia. ConclusionHsa_circ_0001402, acting as a miR-183-5p sponge, increases FKBPL levels to inhibit VSMC proliferation and migration, while enhancing BECN1 levels to activate VSMC autophagy, thus alleviating neointimal hyperplasia. The hsa_circ_0001402/miR-183-5p/FKBPL axis and hsa_circ_0001402/miR-183-5p/BECN1 axis may offer potential therapeutic targets for neointimal hyperplasia.

Inflammatory Cell Dynamics after Murine Femoral Artery Wire Injury: A Multi-Parameter Flow Cytometry-Based Analysis.

An acute inflammatory response following arterial surgery for atherosclerosis, such as balloon angioplasty, stenting, and surgical bypass, is an important driver of neointimal hyperplasia after arterial injury, which leads to recurrent ischemia. However, a comprehensive understanding of the dynamics of the inflammatory infiltrate in the remodeling artery is difficult to attain due to the shortcomings of conventional methods such as immunofluorescence. We developed a 15-parameter flow cytometry method to quantitate leukocytes and 13 leukocyte subtypes in murine arteries at 4 time points after femoral artery wire injury. Live leukocyte numbers peaked at 7 days, which preceded the peak neointimal hyperplasia lesion at 28 days. Neutrophils were the most abundant early infiltrate, followed by monocytes and macrophages. Eosinophils were elevated after 1 day, while natural killer and dendritic cells gradually infiltrated over the first 7 days; all decreased between 7 and 14 days. Lymphocytes began accumulating at 3 days and peaked at 7 days. Immunofluorescence of arterial sections demonstrated similar temporal trends of CD45+ and F4/80+ cells. This method allows for the simultaneous quantitation of multiple leukocyte subtypes from small tissue samples of injured murine arteries and identifies the CD64+Tim4+ macrophage phenotype as being potentially important in the first 7 days post-injury.

Gene-repressing epigenetic reader EED unexpectedly enhances cyclinD1 gene activation

Epigenetically switched, proliferative vascular smooth muscle cells (SMCs) form neointima, engendering stenotic diseases. Histone-3 lysine-27 trimethylation (H3K27me3) and acetylation (H3K27ac) marks are associated with gene repression and activation, respectively. The polycomb protein embryonic ectoderm development (EED) reads H3K27me3 and also enhances its deposition, hence is a canonical gene repressor. However, herein we found an unexpected role for EED in activating the bona fide pro-proliferative gene Ccnd1 (cyclinD1). EED overexpression in SMCs increased Ccnd1 mRNA, seemingly contradicting its gene-repressing function. However, consistently, EED co-immunoprecipitated with gene-activating H3K27ac reader BRD4, and they co-occupied at both mitogen-activated Ccnd1 and mitogen-repressed P57 (bona fide anti-proliferative gene), as indicated by chromatin immunoprecipitation qPCR. These results were abolished by an inhibitor of either the EED/H3K27me3 or BRD4/H3K27ac reader function. In accordance, elevating BRD4 increased H3K27me3. Invivo, while EED was upregulated in rat and human neointimal lesions, selective EED inhibition abated angioplasty-induced neointima and reduced cyclinD1 in rat carotid arteries. Thus, results uncover a previously unknown role for EED in Ccnd1 activation, likely via its cooperativity with BRD4 that enhances each other's reader function; i.e., activating pro-proliferative Ccnd1 while repressing anti-proliferative P57. As such, this study confers mechanistic implications for the epigenetic intervention of neointimal pathology.

Polo-like kinase 4 inhibitor CFI-400945 inhibits carotid arterial neointima formation but increases atherosclerosis

Neointima lesion and atherosclerosis are proliferative vascular diseases associated with deregulated proliferation of vascular smooth muscle cells (SMCs). CFI-400945 is a novel, highly effective anticancer drug that inhibits polo-like kinase 4 (PLK4) and targets mitosis. In this study, we aim to investigate how CFI-400945 affects the development of proliferative vascular diseases. In C57BL/6 mice, neointima formation was generated by complete carotid ligation. In apolipoprotein E knockout (ApoE−/−) mice fed a high-fat diet, atherosclerosis was induced by partial carotid ligation. CFI-400945 was directly applied to carotid arteries via a perivascular collar. Our results showed that CFI-400945 drastically inhibited neointima formation but significantly accelerated atherosclerosis. In vitro studies showed that CFI-400945 treatment induced SMC polyploidization and arrested cells in the G2/M phase. CFI-400945 treatment upregulated p53 and p27 expression but decreased p21 and cyclin B1 expression. CFI-400945 also induced SMC apoptosis, which was inhibited by hydroxyurea, a DNA synthesis inhibitor that inhibits polyploidization. Furthermore, CFI-400945 caused supernumerary centrosomes, leading to mitotic failure, resulting in polyploidization. In conclusion, CFI-400945 prevents carotid arterial neointima formation in C57BL/6 mice but accelerates atherosclerosis in ApoE−/− mice, likely through mitotic arrest and subsequent induction of polyploidization and apoptosis.

Abstract 15731: Rpl13a Small Nucleolar RNAs Promote Atherosclerosis and Oxidative Stress

Reactive oxygen species (ROS) exacerbate atherosclerosis (athero). ROS levels are elevated by specific non-coding, small nucleolar (sno) RNAs encoded within introns of the Rpl13a gene. We therefore tested the hypothesis that these snoRNAs promote athero, using “snoKO” mice deficient in Rpl13a snoRNAs, but not in Rpl13a itself. ROS levels assessed by CellROX Orange were 35% lower in snoKO than snoRNA +/+ aorta frozen sections ( p <0.01). After 14 wk on Western diet, female snoKO/ Apoe -/- unexpectedly showed total cholesterol levels 20% higher than Apoe -/- mice in (1,046 vs 869 mg/dl, p <0.05). Despite this, neointimal lesions in brachiocephalic artery (BCA) cross-sections were 50% smaller in snoKO/ Apoe -/- than in Apoe -/- mice, and lumen size was 45% larger (both p <0.01, n=8/group). Similar data were obtained in males: snoKO/ Apoe -/- mice had 40% smaller BCA lesion areas ( p <0.02, n=8/group). After being stained for cholesteryl ester with BODIPY, for ACTA2 by immunofluorescence and for DNA (Hoechst), BCAs from female snoKO/ Apoe -/- mice (n=9) demonstrated 50% less foam cell-positive and 95% more ACTA2 + area, 40% less necrotic core area, and a 60% lower prevalence of ACTA2 + foam cells ( p <0.05 for each). Thus, Rpl13a snoRNAs promote vascular ROS and athero. Assessed by MitoSOX Red, ROS levels were 25% lower in snoKO than WT M1-polarized bone marrow-derived Mϕs in vitro (n=3, p<0.05). To identify mechanisms linking the Rpl13a snoRNAs to ROS and athero, we performed LC-MS/MS on WT and snoKO aortic SMCs. COX4I2 was expressed 5.7-fold higher in snoKO than WT SMCs by MS/MS, and 2.5-fold higher in snoKO SMCs by immunoblot (n=3/group, p <0.05). As part of mitochondrial complex IV, COX4I2 lowers cellular ROS levels. We used CRISPR/Cas9 to create 293T cells lacking either the RPL13a -snoRNA U34A or the irrelevant snoRNA U25 . With mRNA from these cells we performed reverse transcription at low [dNTP] followed by qPCR (RTL-P) for COX4I2 . Inversely proportional to the degree of mRNA 2’- O -methylation (mediated by snoRNAs), the RTL-P efficiency was 4-fold higher in U34A -knockout than control cells (3 clones/genotype, p <0.01). Thus, COX4I2 mRNA appears to be regulated by RPL13A -snoRNA-guided 2’- O -methylation in a manner that could link Rpl13a snoRNAs, vascular ROS, and athero.

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.

TAK1 accelerates transplant arteriosclerosis in rat aortic allografts by inducing autophagy in vascular smooth muscle cells

Background and aimsThe proliferation and migration of vascular smooth muscle cells (VSMCs) are fundamental hallmarks of vasculopathy. Transforming growth factor β-activated kinase-1 (TAK1) plays a crucial role in mediating cellular functions, including autophagy, which has been recently linked to the regulation of VSMC functions and the development of vasculopathy. This study aims to better dissect how TAK1 controls VSMC proliferation and migration. MethodsA rat model of graft arteriosclerosis was employed to explore the influence of TAK1 signaling activation on VSMC proliferation, migration, autophagy, and neointima formation in vivo. Knockdown and pharmacological inhibition of TAK1 were utilized in cultured VSMCs to investigate the mechanisms underlying the progression of VSMC proliferation and migration. ResultsIncreased phosphorylation of TAK1 (Thr-184/Thr-187) was examined in SMα-actin positive cells in the medial and neointimal lesions of aortic allografts. Lentivirus-mediated Tak1 shRNA transfection of aortic allografts robustly suppressed neointimal formation and lumen stenosis, as well as autophagy and cell proliferative responses. In cultured PDGF-BB-incubated VSMCs, genetic and pharmacological inhibition of TAK1 markedly attenuated autophagy activation, and blocked the progression of cell cycle, proliferation, and migration responses. ConclusionsActivation of TAK1 in VSMCs in the setting of aortic transplantation is an early and critical event in VSMC proliferation and migration, as well as neointima formation, because it controls autophagy activation, constituting a potential molecular mechanism and target for preventing transplant vasculopathy.

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.