Intimal Formation Research Articles

Clinical results of 30 consecutive patients of carotid artery stenosis treated with CASPER stent placement: 1-year follow-up and in-stent findings on intravascular ultrasound examination immediately and 6 months after treatment

BackgroundThe CASPER stent is expected to reduce periprocedural ischemic complications, but there is concern about restenosis in the early period. One-year follow-up results of CASPER stenting and findings on intravascular...

Deficiency of smooth muscle cell ILF3 alleviates intimal hyperplasia via HMGB1 mRNA degradation-mediated regulation of the STAT3/DUSP16 axis

Intimal hyperplasia is a complicated pathophysiological phenomenon attributable to in-stent restenosis, and the underlying mechanism remains unclear. Interleukin enhancer-binding factor 3 (ILF3), a double-stranded RNA-binding protein involved in regulating mRNA stability, has been recently demonstrated to assume a crucial role in cardiovascular disease; nevertheless, its impact on intimal hyperplasia remains unknown. In current study, we used samples of human restenotic arteries and rodent models of intimal hyperplasia, we found that vascular smooth muscle cell (VSMC) ILF3 expression was markedly elevated in human restenotic arteries and murine ligated carotid arteries. SMC-specific ILF3 knockout mice significantly suppressed injury induced neointimal formation. In vitro, platelet-derived growth factor type BB (PDGF-BB) treatment elevated the level of VSMC ILF3 in a dose- and time-dependent manner. ILF3 silencing markedly inhibited PDGF-BB-induced phenotype switching, proliferation, and migration in VSMCs. Transcriptome sequencing and RNA immunoprecipitation sequencing depicted that ILF3 maintained its stability upon binding to the mRNA of the high-mobility group box 1 protein (HMGB1), thereby exerting an inhibitory effect on the transcription of dual specificity phosphatase 16 (DUSP16) through enhanced phosphorylation of signal transducer and activator of transcription 3 (STAT3). Therefore, the results both in vitro and in vivo indicated that the loss of ILF3 in VSMC ameliorated neointimal hyperplasia by regulating the STAT3/DUSP16 axis through the degradation of HMGB1 mRNA. Our findings revealed that vascular injury activates VSMC ILF3, which in turn promotes intima formation. Consequently, targeting specific VSMC ILF3 may present a potential therapeutic strategy for ameliorating cardiovascular restenosis.

Chitosan inhibits vascular intimal hyperplasia via LINC01615/MIR-185-5p/PIK3R2 signaling pathway

The abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) are the main pathological processes which are involved in the formation of new intima. In our previous study, we found that chitosan can inhibit the formation of new intima in the arteriovenous fistulas of uremic patients, and the expression of LINC01615 was significantly increased in patients after treatment with chitosan. Therefore, this study aims to further explore the effect of chitosan on the intimal hyperplasia and elucidate the potential molecular mechanism. In vitro, we found that in chitosan-treated VSMC, the levels of Il-1β, IL-6 and TNF-α decreased, and the intimal hyperplasia was inhibited along with significantly downregulated PIK3R2 and upregualted PI3K, AKT and p-AKT. Meanwhile, we observed the phenotypic transformation of hVSMCs after LINC01615 was upregulated. In addition, inflammatory factors showed the same changes in the process of up-regulating LINC01615. Moreover, only in the LINC01615 overexpression and miR-185-5p mimic experimental group, the inhibition of intimal hyperplasia was the most obvious. The interaction between LINC01615 and miR-185-5p, miR-185-5p and PIK3R2 was further confirmed by the dual luciferase assay. These results suggest that chitosan has a potential preventive effect on neointimal hyperplasia and related vascular remodeling.

Contents of matrix metalloproteinase type 2 (MMP-2) and complexes MMP-9/TIMP-1, MMP-2/TIMP-2, and total cholesterol in practically healthy people and patients with hypertension

Arterial hypertension (AH) is the most common age-associated disease among the working population. Polyetiology of AH requires a more thorough definition and study of its predictors aiming for understanding the role of external influences, as well as detection of genetic polymorphisms at early stages, in order to limit their implementation. The scientific reviews discuss the important role of abnormal vascular remodeling in the pathogenesis of hypertension. The main factor of such alterations is the rearrangement of extracellular matrix due to matrix metalloproteinases (MMP), the zinc-dependent enzymes with a wide substrate specificity. In addition, in more than 70% of cases, AH is combined with lipid metabolism disorders, including an increase in cholesterol levels. These mechanisms should be taken into account when assessing risk factors for development of cardiovascular accidents. The role of MMP-2 and the complexes MMP- 9/ TIMP- 1, MMP-2/TIMP-2 in pathogenesis of vascular restructuring is insufficiently described in the review articles. The article presents our results of studying the system of proteolytic enzymes and the level of total cholesterol in 110 practically healthy people and 90 persons with AH aged 18 to 74 years old. The levels of MMP-2, MMP-9/ TIMP- 1, MMP-2/TIMP-2 complexes in blood serum were studied by the sandwich variant of ELISA technique. Statistical processing of the obtained data was carried out using the analytical software IBM SPSS Statistics, v. 22.0.1. It was found that in the groups of women, regardless of the presence, or absence of pathology, the level of total cholesterol is higher if compared with male subjects. MMP-2 values were low both in the general group of practically healthy people, regardless of gender, and in the group of men with hypertension. Proteolytic activity of MMP-2 shows a broader substrate activity in women with hypertension. In the group of practically healthy men and women, we have registered lower levels of the studied MMP complexes and their tissue inhibitors. An imbalance in the proteolysis/antiproteolysis system is observed both in the group of hypertensive women with more pronounced effects upon migration, proliferation and apoptosis of smooth muscle cells, endothelial and inflammatory cells, thus determining formation of intima and arterial remodeling, as well as in the group of men with hypertension with predominance of remodeling factors that affects the rigidity of vascular wall.

Impact of myocardial bridging on coronary artery plaque formation and long-term mortality after heart transplantation

ObjectivesThis study aimed to explore the impact of myocardial bridging (MB) on early development of cardiac allograft vasculopathy and long-term graft survival after heart transplantation. BackgroundMB has been reported to be associated with acceleration of proximal plaque development and endothelial dysfunction in native coronary atherosclerosis. However, its clinical significance in heart transplantation remains unclear. MethodsIn 103 heart-transplant recipients, serial (baseline and 1-year post-transplant) volumetric intravascular ultrasound (IVUS) analyses were performed in the first 50 mm of the left anterior descending (LAD) artery. Standard IVUS indices were evaluated in 3 equally divided LAD segments (proximal, middle, and distal segments). MB was defined by IVUS as an echolucent muscular band lying on top of the artery. The primary endpoint was death or re-transplantation, assessed for up to 12.2 years (median follow-up: 4.7 years). ResultsIVUS identified MB in 62% of the study population. At baseline, MB patients had smaller intimal volume in the distal LAD than non-MB patients (p = 0.002). During the first year, vessel volume decreased diffusely irrespective of the presence of MB. Intimal growth diffusely distributed in non-MB patients, whereas MB patients demonstrated significantly augmented intimal formation in the proximal LAD. Kaplan-Meier analysis revealed significantly lower event-free survival in patients with versus without MB (log-rank p = 0.02). In multivariate analysis, the presence of MB was independently associated with late adverse events [hazard ratio 5.1 (1.6–22.2)]. ConclusionMB appears to relate to accelerated proximal intimal growth and reduced long-term survival in heart-transplant recipients.

Correlation between stem cell molecular phenotype and atherosclerotic plaque neointima formation and analysis of stem cell signal pathways.

Vascular stem cells exist in the three-layer structure of blood vessel walls and play an indispensable role in angiogenesis under physiological conditions and vascular remodeling under pathological conditions. Vascular stem cells are mostly quiescent, but can be activated in response to injury and participate in endothelial repair and neointima formation. Extensive studies have demonstrated the differentiation potential of stem/progenitor cells to repair endothelium and participate in neointima formation during vascular remodeling. The stem cell population has markers on the surface of the cells that can be used to identify this cell population. The main positive markers include Stem cell antigen-1 (Sca1), Sry-box transcription factor 10 (SOX10). Stromal cell antigen 1 (Stro-1) and Stem cell growth factor receptor kit (c-kit) are still controversial. Different parts of the vessel have different stem cell populations and multiple markers. In this review, we trace the role of vascular stem/progenitor cells in the progression of atherosclerosis and neointima formation, focusing on the expression of stem cell molecular markers that occur during neointima formation and vascular repair, as well as the molecular phenotypic changes that occur during differentiation of different stem cell types. To explore the correlation between stem cell molecular markers and atherosclerotic diseases and neointima formation, summarize the differential changes of molecular phenotype during the differentiation of stem cells into smooth muscle cells and endothelial cells, and further analyze the signaling pathways and molecular mechanisms of stem cells expressing different positive markers participating in intima formation and vascular repair. Summarizing the limitations of stem cells in the prevention and treatment of atherosclerotic diseases and the pressing issues that need to be addressed, we provide a feasible scheme for studying the signaling pathways of vascular stem cells involved in vascular diseases.

Abstract 321: Endothelial Cell Translational Control Is A Key Mediator Of Intima Growth In Response To Disturbed Flow

Background: Carotid intima-media thickening (IMT) is a good surrogate for atherosclerosis and CVD. Previously we used the IMT response of inbred mouse strains to disturbed flow (d-flow) in the partial carotid ligation (PCL) model to identify an intima modifier locus Im3. Microarray and QTL analysis revealed ribosomal protein large subunit (RpL17) as the primary candidate gene for IMT within Im3; further, low RpL17 levels correlate with enhanced intima formation. RpL17 is located in the nascent protein exit tunnel, and serves as a docking site for the NAC chaperone in protein folding. We generated endothelial cell (EC)-specific conditional heterozygous RpL17 mice (EC-RpL17+/-, termed EC-RpL17) to study the IMT response to d-flow using PCL. Results: EC-RpL17 mice had increased IMT relative to controls (RpL17+/+) after PCL. Data showed induction of oxidative stress and endoplasmic reticulum (ER) stress in EC-RpL17 with activation of the integrated stress response (ISR), a stress-support pathway promoting hyperproliferation in cancer, including: 1) increased levels of reactive oxygen species (ROS), BiP, Chop, and Atf4 expression, activation of PERK (p-PERK), and inactivation of eIF2 alpha (p-eIF2 alpha) in cultured RpL17 microvascular EC. The in vitro data correlated well with in vivo data from post-PCL carotid immunostaining, including: increased p-PERK, BiP, Chop, 4-hydroxynonenal and 8-hydroxyguanosine. EM analysis revealed dilation of the rough ER (indicating ER stress), and decreased cytosolic polyribosomes in EC-RpL17 EC. Conclusion: Decreased EC RpL17 increases d-flow mediated IMT, likely due to induction of growth-promoting ISR by ER and oxidative stress. These data define a novel pathophysiological role for RpL17.

USP10 exacerbates neointima formation by stabilizing Skp2 protein in vascular smooth muscle cells

The underlying mechanism of neointima formation remains unclear. Ubiquitin-specific peptidase 10 (USP10) is a deubiquitinase that plays a major role in cancer development and progression. However, the function of USP10 in arterial restenosis is unknown. Herein, USP10 expression was detected in mouse arteries and increased after carotid ligation. The inhibition of USP10 exhibited thinner neointima in the model of mouse carotid ligation. In vitro data showed that USP10 deficiency reduced proliferation and migration of rat thoracic aorta smooth muscle cells (A7r5) and human aortic smooth muscle cells (HASMCs). Mechanically, USP10 can bind to Skp2 and stabilize its protein level by removing polyubiquitin on Skp2 in the cytoplasm. The overexpression of Skp2 abrogated cell cycle arrest induced by USP10 inhibition. Overall, the current study demonstrated that USP10 is involved in vascular remodeling by directly promoting VSMC proliferation and migration via stabilization of Skp2 protein expression.

Dichloroacetate inhibits the degeneration of decellularized cardiovascular implants.

OBJECTIVESIntima hyperplasia is a major issue of biological cardiovascular grafts resulting in progressive in vivo degeneration that particularly decreases the durability of coronary and peripheral vascular bypasses. Previously, dichloroacetate (DCA) has been reported to prevent the formation of hyperplastic intima in injured arteries. In this study, the effect of DCA on the neointima formation and degeneration of decellularized small-caliber implants was investigated in a rat model.METHODSDonor rat aortic grafts (n = 22) were decellularized by a detergent-based technique, surface-coated with fibronectin (50 µl ml−1, 24 h incubation) and implanted via anastomoses to the infrarenal aorta of the recipients. Rats in the DCA group (n = 12) received DCA via drinking water during the whole follow-up period (0.75 g l−1), while rats without DCA treatment served as controls (n = 10). At 2 (n = 6 + 5) and 8 (n = 6 + 5) weeks, the grafts were explanted and examined by histology and immunofluorescence.RESULTSSystemic DCA treatment inhibited neointima hyperplasia, resulting in a significantly reduced intima-to-media ratio (median 0.78 [interquartile range, 0.51–1.27] vs 1.49 [0.67–2.39] without DCA, P < 0.001). At 8 weeks, neointima calcification, as assessed by an established von Kossa staining-based score, was significantly decreased in the DCA group (0 [0–0.25] vs 0.63 [0.06–1.44] without DCA, P < 0.001). At 8 weeks, explanted grafts in both groups were luminally completely covered by an endothelial cell layer. In both groups, inflammatory cell markers (CD3, CD68) proved negative.CONCLUSIONSSystemic DCA treatment reduces adverse neointima hyperplasia in decellularized small-caliber arterial grafts, while allowing for rapid re-endothelialization. Furthermore, DCA inhibits calcification of the implants.

Disease-Relevant Single Cell Photonic Signatures Identify S100\u03b2 Stem Cells and their Myogenic Progeny in Vascular Lesions

A hallmark of subclinical atherosclerosis is the accumulation of vascular smooth muscle cell (SMC)-like cells leading to intimal thickening and lesion formation. While medial SMCs contribute to vascular lesions, the involvement of resident vascular stem cells (vSCs) remains unclear. We evaluated single cell photonics as a discriminator of cell phenotype in vitro before the presence of vSC within vascular lesions was assessed ex vivo using supervised machine learning and further validated using lineage tracing analysis. Using a novel lab-on-a-Disk(Load) platform, label-free single cell photonic emissions from normal and injured vessels ex vivo were interrogated and compared to freshly isolated aortic SMCs, cultured Movas SMCs, macrophages, B-cells, S100β+ mVSc, bone marrow derived mesenchymal stem cells (MSC) and their respective myogenic progeny across five broadband light wavelengths (λ465 - λ670 ± 20 nm). We found that profiles were of sufficient coverage, specificity, and quality to clearly distinguish medial SMCs from different vascular beds (carotid vs aorta), discriminate normal carotid medial SMCs from lesional SMC-like cells ex vivo following flow restriction, and identify SMC differentiation of a series of multipotent stem cells following treatment with transforming growth factor beta 1 (TGF- β1), the Notch ligand Jagged1, and Sonic Hedgehog using multivariate analysis, in part, due to photonic emissions from enhanced collagen III and elastin expression. Supervised machine learning supported genetic lineage tracing analysis of S100β+ vSCs and identified the presence of S100β+vSC-derived myogenic progeny within vascular lesions. We conclude disease-relevant photonic signatures may have predictive value for vascular disease.Graphical abstract

Roles of LmCDA1 and LmCDA2 in cuticle formation in the foregut and hindgut of Locusta migratoria.

Chitin deacetylases (CDAs, including CDA1 and CDA2) are considered key enzymes for body cuticle formation and tracheal morphogenesis in various insect species. However, their functions in the formation of the cuticular intima of the foregut and hindgut are unclear. Here, we investigated the roles of their respective genes LmCDA1 and LmCDA2 in this process, in the hemimetabolous insect Locusta migratoria. Transcripts of LmCDA1 and LmCDA2 were highly expressed both before and after molting in the foregut. In the hindgut, their expression was high only before molting. In both the foregut and hindgut, LmCDA1 protein was localized in the basal half of the chitin matrix (procuticle), whereas LmCDA2 was detected in the upper half of the procuticle. Knockdown of LmCDA1 by RNA interference (RNAi) in 5th-instar nymphs caused no visible defects of the hindgut cuticle. By contrast, the chitinous lamellae of the cuticular intima in the foregut of knockdown animals were less compact than in control animals. RNAi against LmCDA2 led to thickening of both the foregut and hindgut cuticles, with a greater number of thinner laminae than in the respective control cuticles. Taken together, our results show that LmCDA1 and LmCDA2 have distinct, but overlapping, functions in chitin organization in the foregut cuticle. However, in the hindgut, this process seems independent of LmCDA1 activity but requires LmCDA2 function. Thus, the CDAs reflect tissue-specific differences in cuticular organization and function, which need further detailed molecular and histological analyses for full comprehension.

BFGF blockade reduces intraplaque angiogenesis and macrophage infiltration in atherosclerotic vein graft lesions in ApoE3*Leiden mice

Intraplaque angiogenesis increases the chance of unstable atherosclerotic plaque rupture and thrombus formation leading to myocardial infarction. Basic Fibroblast Growth Factor (bFGF) plays a key role in angiogenesis and inflammation and is involved in the pathogenesis of atherosclerosis. Therefore, we aim to test K5, a small molecule bFGF-inhibitor, on remodelling of accelerated atherosclerotic vein grafts lesions in ApoE3*Leiden mice. K5-mediated bFGF-signalling blockade strongly decreased intraplaque angiogenesis and intraplaque hemorrhage. Moreover, it reduced macrophage infiltration in the lesions by modulating CCL2 and VCAM1 expression. Therefore, K5 increases plaque stability. To study the isolated effect of K5 on angiogenesis and SMCs-mediated intimal hyperplasia formation, we used an in vivo Matrigel-plug mouse model that reveals the effects on in vivo angiogenesis and femoral artery cuff model to exclusively looks at SMCs. K5 drastically reduced in vivo angiogenesis in the matrigel plug model while no effect on SMCs migration nor proliferation could be seen in the femoral artery cuff model. Moreover, in vitro K5 impaired endothelial cells functions, decreasing migration, proliferation and tube formation. Our data show that K5-mediated bFGF signalling blockade in hypercholesterolemic ApoE3*Leiden mice reduces intraplaque angiogenesis, haemorrhage and inflammation. Therefore, K5 is a promising candidate to stabilize advanced atherosclerotic plaques.

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

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

Possible Roles of Periostin in the Formation of Hemodialysis Vascular Access Stenosis after Polytetrafluoroethylene Graft Implantation in Dogs

Periostin, a recently found matricellular protein, has been implicated in neointima formation after balloon injury. However, the relationship between periostin and hyperplastic intima formation after PTFE graft implantation is unclear. Under mixed anesthesia, PTFE grafts were implanted between the canine carotid artery and jugular vein, and PTFE graft samples were harvested 1, 2, and 4 months after implantation. Intima formation started on the luminal surface of PTFE grafts at the venous anastomotic region 1 month after implantation. Thereafter, the increase in intimal volume was not only observed in the venous and arterial anastomotic regions, but also in the middle region of the PTFE grafts. In accordance with the increased intimal formation, time-dependent increases in mRNA expressions of periostin and transforming growth factor beta 1 (TGF-β1), as well as a strong positive correlation between periostin and TGF-β1, were observed. These findings suggest that periostin may play a very important role in the pathogenesis of hemodialysis vascular access stenosis through the acceleration of intimal formation. Thus, periostin may be a very important therapeutic target for the treatment of vascular access graft dysfunction in hemodialysis patients.

The expression of survivin in irreversible pulmonary arterial hypertension rats and its value in evaluating the reversibility of pulmonary arterial hypertension secondary to congenital heart disease.

The reversibility of pulmonary arterial hypertension (PAH) determines the operability of congenital heart disease (CHD) complicating with PAH, but it lacks a method for evaluating the reversibility. The current study aims to investigate the serum survivin level in irreversible PAH rats and to explore its potential as a biomarker for evaluating the reversibility of PAH in CHD patients. Irreversible PAH rats were characterized by prominent obstructive lesions resulting from the intimal formation, which was associated with decreased apoptosis and increased survivin expression, while reversible PAH rats were featured by medial hypertrophy resulting in mild occlusion, with increased apoptosis and unchanged survivin expression. In addition, the serum survivin was significantly increased in irreversible PAH rats when compared to both reversible PAH and control rats, and a positive correlation of serum survivin with survivin expression in the lung was confirmed. Third, the preoperative serum survivin was significantly higher in patients with irreversible CHD-PAH than in these with reversible CHD-PAH, and significant correlations between the serum survivin and BNP, preoperative pulmonary vascular resistance index, and postoperative mean pulmonary arterial pressure were also identified. In conclusion, the increased survivin level is a feature of irreversible PAH and the serum survivin represents a candidate biomarker reflecting the operability of CHD-PAH patients.

LncRNA expression profile and ceRNA analysis in tomato during flowering.

Long non-coding RNAs (lncRNAs) are a class of non-coding RNAs that play essential regulatory roles in various developmental processes and stress responses. However, the functions of lncRNAs during the flowering period of tomato are largely unknown. To explore the lncRNA profiles and functions during flowering in tomato, we performed strand-specific paired-end RNA sequencing of tomato leaves, flowers and roots, with three biological replicates. We identified 10919 lncRNAs including 248 novel lncRNAs, of which 65 novel lncRNAs were significantly differentially expressed (DE) in the flowers, leaves, and roots. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were carried out to identify the cis target gene of DE lncRNAs. The results showed that the lncRNAs might play an important role in the growth, development, and apoptosis of flowering tomato plant by regulating the formation of intima in flower tissues, binding to various molecules, influencing metabolic pathways, and inducing apoptosis. Moreover, we identified the interaction between 32, 78, and 397 kinds of miRNAs, lncRNAs, and mRNAs. The results suggest that the lncRNAs can regulate the expression of mRNA during flowering period in tomato by forming competitive endogenous RNA, and further regulate various biological metabolism pathways in tomato.

Abstract 16886: Caspase-4/11 Mediates Intima Formation by Increasing Inflammation and Smooth Muscle Cell Migration and Proliferation

Introduction: Intima formation after vessel injury occurs via vascular smooth muscle cell(VSMC) proliferation, migration, dedifferentiation and inflammatory response. Recently, it was demonstrated that caspase-4 (Casp4, human)/caspase11(Casp11, mouse) increases the synthesis and release of inflammatory factors in macrophages, indicating a potential role of Casp4/Casp11 in intima formation. However, to date, the biological role of Casp4/Casp11 in vivo has not been investigated. Therefore, we hypothesize that Casp4/Casp11 deficiency will inhibit intima formation. Method and Results: We measured Casp4 expression and location by immunohistochemistry (IHC) staining in human coronary artery intima lesions from coronary artery disease patients. Surprisingly, we found Casp4 expression was remarkably increased in smooth muscle cells (SMC) and macrophages. We observed similar phenomena of Casp11 expression and location in intima of complete carotid ligation mouse model. These findings imply an important role of Casp4/11 in SMC function. Thus, we performed complete carotid ligation on Casp11 wild type (WT) and knockout (KO) mice.Morphometric analysis demonstrated that Casp11 depletion robustly attenuated intima area and intima/media ratio (I/M) compared with WT mice . Inflammation was quantified by CD45 IHC staining . SMC proliferation was detected by PCNA staining and there was a marked decrease of PCNA positive SMCs. In vitro, Casp4 expression was time- and dose-dependently increased by platelet-derived growth factor(PDGF), angiotensin II(AngII) and TNF-α in human coronary artery SMCs(HCASMCs). Depletion of Casp4 by siRNA decreased expression of cytokines, e.g. IL-1α, IL-1β, IL-6, as well as protein expression of adhesion factors (VCAM-1, ICAM-1, MCP-1) induced by PDGF, AngII or TNF-α. Moreover, HCASMC proliferation stimulated by PDGF was measured by EdU cell proliferation assay and was inhibited by 65% after knockdown of Casp4. Wound scratch assay demonstrated impaired HCASMC migration in Casp4 depleted cells. However, Casp4 has no effects on HCASMCs dedifferentiation. Mechanistically, Casp4 knockdown significantly inhibited NF-κB signaling. Conclusions: Casp4/11 promotes intima formation by increasing SMC proliferation, migration and inflammation through NF κB signaling. Specific inhibition of the Casp4/11 will provide novel therapeutic strategy for vascular remodeling related diseases.

Abstract 317: Nlrc5 Positively Regulates Intimal Formation via Interaction With PPARã in Vascular Smooth Muscle Cells

Background: Innate immunity and vascular smooth muscle cells play crucial role in the development of vascular remodeling and intimal hyperplasia. NLR family CARD domain containing 5 (Nlrc5), known as a pattern recognition receptor, has been noted for its implication in immune disease in recent studies. However, whether Nlrc5 regulates intimal hyperplasia is still unclear. Aim: We aimed to determine the role of Nlrc5 in intimal hyperplasia and its underlying molecular mechanism. Methods and Results: We showed that an elevated expression of Nlrc5 in neointima induced by carotid ligation compared normal carotid artery. In human aortic smooth muscle cells (HASMCs), PDGF induced Nlrc5 expression and nuclear translocation. By utilizing Nlrc5 knockout mice, we found Nlrc5 deficiency prominently aggravated neointimal formation compared to wild type (WT) mice. On the contrary, gain of function of Nlrc5 based on local adenovirus injection attenuated intimal hyperplasia. The neointimal area was comparable in WT mice receiving either WT or Nlrc5-/- bone marrow transplantation, demonstrating that hematopoietic cell-derived Nlrc5 did not affect vascular hyperplasia. In vitro experiments unveiled that knockdown of Nlrc5 resulted in excessive proliferation, migration and dedifferentiation of HASMCs. Nevertheless, depletion of Nlrc5 in HASMCs did not influence proinflammatory cytokine excretion and immune activation reflected by major histocompatibility complex-I expression. Mechanistic study revealed that Nlrc5 bound to nuclear transcription factor PPARγ and positively regulated PPARγ luciferase activity, ensuing contributing to KLF4 ubiquitination and degradation. Furthermore, we generated five deletion constructs of Nlrc5 and identified that the functional domain containing DD and NACHT constructs was responsible for the interaction with PPARγ. Conclusions: Our study reveals that Nlrc5 alleviates intimal formation through interaction with PPARγ and inhibition of smooth muscle cell dedifferentiation, suggesting that Nlrc5 constitutes a novel molecular target for vascular remodeling.

P6570Ambivalent role of eNOS in murine intima formation depends on caveolin-1 expression

P6570Ambivalent role of eNOS in murine intima formation depends on caveolin-1 expression

Graft Protective Effect of HMG-CoA Reductase Inhibitor Pravastatin in Murine Cardiac Allograft Transplantation

The HMG-CoA reductase inhibitor (statin), which reduces serum cholesterol, has been demonstrated in the control of immune responses and may potentially play an important role in the regulation of acute and chronic rejection in organ transplantations. We investigated the graft-protective effect of a kind of statin, pravastatin, in the survival of fully major histocompatibility complex--mismatched murine cardiac allograft transplantation. Fully vascularized heterotopic hearts from C57BL/6 donors were transplanted into CBA recipients through microsurgical techniques. CBA recipients transplanted with a C57BL/6 heart received oral administration of 40, 120, or 400 μg/kg/day of pravastatin from the day of transplantation to 7 days afterward. Immunohistochemical staining studies were performed to determine whether intimal formation of coronary arteries in the transplanted cardiac allografts was preserved and also to conduct morphometric analysis. Untreated CBA recipients rejected C57BL/6 cardiac grafts acutely (median survival time [MST] 7 days). CBA recipients exposed with 40 and 120 μg/kg/day of pravastatin had a small prolonged allograft survival (MSTs of 10 and 9 days, respectively). However, the MST of CBA recipients exposed to 400 μg/kg/day of pravastatin was significantly effective for allograft survival (MST 50 days). Immunohistochemical staining assessments on 4 weeks after grafting showed suppression of intimal hyperplasia in allograft coronary arteries. Pravastatin could induce the prolongation of fully major histocompatibility complex--mismatched cardiac allograft through the protection of the coronary artery.