Association of Gene Polymorphisms of Some Endothelial Factors with Stent Reendothelization after Elective Coronary Artery Revascularization.

Restenosis remains the main complication after percutaneous coronary interventions in patients with coronary heart disease. The causes of its development include, in particular, genetic factors. We studied polymorphic loci of genes encoding endothelin-1 (EDN1 rs5370), endothelin-1 receptor (EDNRA rs5333), endothelin-converting enzyme (ECE1 rs1076669), and endothelial NO synthase (eNOS rs1549758, eNOS rs1799983, and eNOS rs2070244) in the context of in-stent restenosis development. It was found that the analyzed polymorphisms of the endothelin system genes were more significant for patients aged ≥ 65 years, while the polymorphic loci of the endothelial NO synthase gene (eNOS rs1799983 and eNOS rs1549758) were predominantly associated with time of in-stent restenosis. The obtained results can be useful for comprehensive assessment of the restenosis risk factors and the choice of optimal treatment for patients with coronary heart disease before elective surgical intervention.

Reactive Oxygen Species: Modulators of Phenotypic Switch of Vascular Smooth Muscle Cells.

Reactive oxygen species (ROS) are natural byproducts of oxygen metabolism in the cell. At physiological levels, they play a vital role in cell signaling. However, high ROS levels cause oxidative stress, which is implicated in cardiovascular diseases (CVD) such as atherosclerosis, hypertension, and restenosis after angioplasty. Despite the great amount of research conducted to identify the role of ROS in CVD, the image is still far from being complete. A common event in CVD pathophysiology is the switch of vascular smooth muscle cells (VSMCs) from a contractile to a synthetic phenotype. Interestingly, oxidative stress is a major contributor to this phenotypic switch. In this review, we focus on the effect of ROS on the hallmarks of VSMC phenotypic switch, particularly proliferation and migration. In addition, we speculate on the underlying molecular mechanisms of these cellular events. Along these lines, the impact of ROS on the expression of contractile markers of VSMCs is discussed in depth. We conclude by commenting on the efficiency of antioxidants as CVD therapies.

Dimethylarginine Dimethylaminohydrolase 1 Polymorphisms and Venous Intimal Hyperplasia in Hemodialysis Patients.

BACKGROUND: After angioplasty, veins are more prone to intimal hyperplasia than arteries. Veins tend to produce less nitric oxide (NO), which could lead to endothelial dysfunction. Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of NO synthase and contributes to cardiovascular disease. In humans, dimethylarginine dimethylaminohydrolase 1 (DDAH1) is the major enzyme for ADMA degradation. In this study, we aim to determine whether venous intimal hyperplasia in hemodialysis (HD) vascular access is influenced by common polymorphisms in the DDAH1 genes. METHODS: This is a prospective observational cohort study. A total of 473 HD patients referred for the angioplasty of vascular access were enrolled. There were 190 arteriovenous grafts (AVG) and 283 arteriovenous fistulas (AVF). The follow-up lasted for 2 years after the interventions. Seven single nucleotide polymorphisms (SNPs) in DDAH1 were genotyped and ADMA were measured at baseline. The primary outcome was restenosis after angioplasty. RESULTS: Among the 7 SNPs, plasma ADMA levels were significantly different in DDAH1 rs233112 (GA + GG vs. AA, 0.86 ± 0.23 vs. 0.82 ± 0.19 µM, p = 0.03) and rs1498373 (CT + TT vs. CC, 0.87 ± 0.23 vs. 0.82 ± 0.20 µM, p = 0.02) genotypes. The AVF group with GG + GA genotype of rs233112 and CT + TT genotype of rs1498373 had higher risks of early restenosis at 3 months. In the AVG group, only GG + GA genotype of rs233112 was associated with early restenosis. A combined analysis of AVG and AVF groups showed that patients with rs233112 GA + GG genotype and rs1498373 CT + TT genotype had higher risks of early restenosis (both p < 0.001). The multivariate analysis results showed that the association of these genotypes with early restenosis is independent of clinical, access, or biochemical factors. CONCLUSIONS: Our findings suggest that certain DDAH1 polymorphisms modulate circulating ADMA levels and are associated with venous intimal hyperplasia.

Endovascular Biopsy and Endothelial Cell Gene Expression Analysis of Dialysis Arteriovenous Fistulas: A Feasibility Study.

PURPOSE: To demonstrate feasibility of endothelial cell (EC) biopsy from dialysis arteriovenous fistulas (AVFs) with the use of guidewires and to characterize gene expression differences between ECs from stenotic and nonstenotic outflow vein segments. MATERIALS AND METHODS: Nine consecutive patients undergoing fistulography for AVF dysfunction from June to August 2016 were enrolled. ECs were biopsied with the use of guidewires from venous outflow stenoses and control outflow veins central to the stenoses. ECs were sorted with the use of flow cytometry, and the Fluidigm Biomark HD system was used for single-cell quantitative polymerase chain reaction (qPCR) analysis of gene expression. Forty-eight genes were assessed and were selected based on different cellular functions and previous literature. Linear mixed models (LMMs) were used to identify differential gene expression between the groups, and self-organizing maps (SOMs) were used to identify cell clusters based on gene coexpression profiles. RESULTS: A total of 219 and 213 ECs were sampled from venous outflow stenoses and control vein segments, respectively. There were no immediate biopsy-related complications. Forty-eight cells per patient were sorted for qPCR analysis. LMM identified 7 genes with different levels of expression at stenotic segments (P < .05), including AGTR-2, HMOX-2, MTHFR, SERPINC-1, SERPINE-1, SMAD-4, and VWF. SOM analysis identified 4 cell clusters with unique gene expression profiles, each containing stenotic and control ECs. CONCLUSIONS: EC biopsy from dialysis AVFs with the use of guidewires is feasible. Gene expression data suggest that genes involved in multiple cellular functions are dysregulated in stenotic areas. SOMs identified 4 unique clusters of cells, indicating EC phenotypic heterogeneity in outflow veins.

Vascular restenosis in coronary artery bypass grafting might be associated with VEGF-C/VEGFR-3 signaling pathway.

The vascular endothelial growth factor (VEGF) family of peptides and caveolins (CAVs) are reported to contribute, in early graft failure in patients, a coronary artery bypass grafting (CABG). To investigate the possible association of ultimate luminal occlusion to VEGFs and CAVs expression, a functional analysis (based on the molecular biology, bioinformatics, histology, and clinical studies) was performed. Twenty-four hundred and sixty-eight CABG patients diagnosed with multivessel stable coronary artery disease (CAD) were enrolled into prospective study and assigned to two subgroups: double- and triple-vessel CAD subjects. Distal parts of all the harvested saphenous vein (SV) and internal thoracic artery (ITA) segments were used for further tests. ITA graft failure did not differ between double-vessel and triple-vessel CAD patients. The number of SV occlusions was significantly higher in triple-vessel CAD subjects. The microarray analysis performed on SV and ITA samples obtained exclusively from triple-vessel CAD patients who developed early graft occlusion revealed 383 genes with increased and 301 genes with decreased expression in ITA samples as compared to SV grafts. This was followed by functional analysis of 'blood vessel development' group of genes. Average VEGF-C expression in ITA grafts was higher than in corresponding SV grafts; FLT4 expression was significantly higher in SV than in ITA transplants. VEGFR-3 and CAV3 expression demonstrated immunohistochemically in SMCs of the tunica media of SV grafts predicted their early restenosis in triple-vessel CAD patients. CAV2 protein expression in SMCs of ITA grafts indicated the risk of early graft failure both in double-vessel and triple-vessel CAD subjects.

Role of Gene Polymorphisms/Haplotypes and Plasma Level of TGF-ß1 in Susceptibility to In-Stent Restenosis Following Coronary Implantation of Bare Metal Stent in Chinese Han Patients.

Transforming growth factor (TGF)-ß1 has been implicated in the pathogenesis of restenosis. However, the role of TGF-ß1 polymorphisms in development of in-stent restenosis (ISR) after coronary bare metal stent (BMS) implantation in Chinese Han population has not been reported to date. The aim of this study was to explore the association between TGF-ß1 gene polymorphisms (-509C/T and 869T/C) and its plasma level in Chinese Han patients with BMS-ISR.We investigated 419 patients after successful coronary stent placement. All patients were reexamined by angiography. Genotyping for the two TGF-ß1 gene polymorphisms was performed using polymerase chain reaction-restriction fragment length polymorphism analysis. Plasma TGF-ß1 levels were measured by enzyme-linked immunosorbent assay.Ninety-two patients (21.96%) developed ISR during the follow-up period. The multivariable analysis adjusted for potential confounders and it revealed that the C allele of TGF-ß1 869T/C polymorphism was linked to an increased risk of ISR in both additive (Per each C allele) and dominant (TC+CC versus TT) models with odds ratios (ORs) of 1.88 (95% confidence interval [CI]: 1.21-2.84, P = 0.008) and 2.52 (95% CI: 1.40-4.80, P = 0.005), respectively. In accord with this, C-dominant CC/CT genotype was linked to higher plasma TGF-ß1 level compared to TT genotype. One haplotype (TC) (-509T, +869C) was associated with an increased risk for ISR (OR = 1.48, 95% CI: 1.06-2.06, P = 0.010).The C allele of TGF-ß1 869T/C polymorphism, correlated with high plasma TGF-ß1 level, represented an independent risk factor for BMS-ISR in Chinese Han patients with coronary artery disease.

A protective role of IRF3 and IRF7 signalling downstream TLRs in the development of vein graft disease via type I interferons.

BACKGROUND: Toll like receptors (TLR) play an important role in vein graft disease (VGD). Interferon regulatory factors (IRF) 3 and 7 are the transcriptional regulators of type I interferons (IFN) and type I IFN responsive genes and are downstream factors of TLRs. Relatively little is known with regard to the interplay of IRFs and TLRs in VGD development. The aim of this study was to investigate the role of IRF3 and IRF7 signaling downstream TLRs and the effect of IRF3 and IRF7 in VGD. METHODS AND RESULTS: In vitro activation of TLR3 induced IRF3 and IRF7 dependent IFNß expression in bone marrow macrophages and vascular smooth muscle cells. Activation of TLR4 showed to regulate pro-inflammatory cytokines via IRF3. Vein graft surgery was performed in Irf3-/- , Irf7-/- and control mice. After 14 days Irf3-/- vein grafts had an increased vessel wall thickness compared to both control (P = 0.01) and Irf7-/- (P = 0.02) vein grafts. After 28 days, vessel wall thickness increased in Irf3-/- (P = 0.0003) and Irf7-/- (P = 0.04) compared to control vein grafts and also increased in Irf7-/- compared to Irf3-/- vein grafts (P = 0.02). Immunohistochemical analysis showed a significant higher influx of macrophages after 14 days in Irf3-/- vein grafts and after 28 days in Irf7-/- vein grafts compared to control vein grafts. CONCLUSIONS: The present study is the first to describe a protective role of both IRF3 and IRF7 in VGD. IRFs regulate VGD downstream TLRs since Irf3-/- and Irf7-/- vein grafts show increased vessel wall thickening after respectively 14 and 28 days after surgery.

Tumor necrosis factor alpha-stimulated gene-6 (TSG-6) inhibits the inflammatory response by inhibiting the activation of P38 and JNK signaling pathway and decreases the restenosis of vein grafts in rats.

This study aims to explore the effects of tumor necrosis factor alpha-stimulated gene-6 (TSG-6) on vascular inflammatory response and vascular injury in grafted vein wall of rats and its possible mechanism. Vascular grafting model was established by modified cuff. The effect of TSG-6 on the inflammatory response and vascular injury of vein graft was investigated. The activation of mast cells and macrophages after LPS stimulation was observed by lentivirus-mediated upregulation or downregulation of TSG-6 expression. The results showed that rhTSG-6 treatment could significantly inhibit the proliferation of venous bridge, decrease macrophage infiltration and smooth muscle cell proliferation. The expression levels of TNF-α and IL-1 in treated group were significantly lower than that of untreated group (P < 0.05), while the expression of IL-10 in treated group were significantly higher than that of untreated group (P < 0.05). The expression levels of P38, p-P38, JNK and p-JNK in venous bridge of rats were significantly lower than those of untreated rats (P < 0.05), while there was no significant difference in the expression level of ERK and p-ERK (P > 0.05). TSG-6 could inhibit the proliferation of mast cells and macrophages and the release of inflammatory cytokines by down regulating the expression levels of P38, p-P38, JNK and p-JNK. TSG-6 can inhibit the inflammatory response of transplanted vein grafts in rats and reduce vascular injury by downregulation of P38 and JNK signaling pathway.

Altered microRNA expression in stenoses of native arteriovenous fistulas in hemodialysis patients.

OBJECTIVE: Arteriovenous fistula (AVF) disfunction is largely due to venous stenosis characterized by a marked amount of intima-media hyperplasia. However, the molecular mechanisms are currently poorly understood. MicroRNAs (miRNAs), small noncoding RNAs that are post-transcriptional regulators of gene expression, could provide insights into a mechanism for the differential expression of genes in stenotic AVFs. METHODS: A microarray study was done to detect differences in miRNA levels between stenotic AVF (n = 8) and controls (n = 4). Real-time quantitative reverse-transcription polymerase chain reaction assays with 12 stenotic AVF veins and eight control veins from predialytic patients were used for verification. Putative gene targets were retrieved from miRNA target prediction databases. Networks from the target gene set were created and examined. Western blotting and immunohistochemical staining were performed to confirm the bioinformatic findings. RESULTS: A microarray study identified 33 miRNAs with markedly different expression levels between stenotic AVFs and control veins. Among them, nine miRNAs were upregulated and 24 miRNAs were downregulated in the stenotic AVFs. Real-time reverse-transcription polymerase chain reaction confirmed statistically consistent expression of six selected miRNAs with microarray analysis. The predicted miRNA target genes differentially expressed in stenotic AVF based on databases were identified. The mitogen-activated protein kinase signaling pathway might be regulated by miRNAs according to bioinformatic analyses and further confirmed by Western blotting and immunohistochemical staining. CONCLUSIONS: Our genome-wide approach identified several differentially expressed miRNAs in stenotic AVFs. This study also suggested that the mitogen-activated protein kinase signaling pathway might play a role in the pathogenesis of stenotic AVF.

Scavenger receptor class A member 5 (SCARA5) and suprabasin (SBSN) are hub genes of coexpression network modules associated with peripheral vein graft patency.

OBJECTIVE: Approximately 30% of autogenous vein grafts develop luminal narrowing and fail because of intimal hyperplasia or negative remodeling. We previously found that vein graft cells from patients who later develop stenosis proliferate more in vitro in response to growth factors than cells from patients who maintain patent grafts. To discover novel determinants of vein graft outcome, we have analyzed gene expression profiles of these cells using a systems biology approach to cluster the genes into modules by their coexpression patterns and to correlate the results with growth data from our prior study and with new studies of migration and matrix remodeling. METHODS: RNA from 4-hour serum- or platelet-derived growth factor (PDGF)-BB-stimulated human saphenous vein cells obtained from the outer vein wall (20 cell lines) was used for microarray analysis of gene expression, followed by weighted gene coexpression network analysis. Cell migration in microchemotaxis chambers in response to PDGF-BB and cell-mediated collagen gel contraction in response to serum were also determined. Gene function was determined using short-interfering RNA to inhibit gene expression before subjecting cells to growth or collagen gel contraction assays. These cells were derived from samples of the vein grafts obtained at surgery, and the long-term fate of these bypass grafts was known. RESULTS: Neither migration nor cell-mediated collagen gel contraction showed a correlation with graft outcome. Although 1188 and 1340 genes were differentially expressed in response to treatment with serum and PDGF, respectively, no single gene was differentially expressed in cells isolated from patients whose grafts stenosed compared with those that remained patent. Network analysis revealed four unique groups of genes, which we term modules, associated with PDGF responses, and 20 unique modules associated with serum responses. The "yellow" and "skyblue" modules, from PDGF and serum analyses, respectively, correlated with later graft stenosis (P = .005 and P = .02, respectively). In response to PDGF, yellow was also associated with increased cell growth. For serum, skyblue was also associated with inhibition of collagen gel contraction. The hub genes for yellow and skyblue (ie, the gene most connected to other genes in the module), scavenger receptor class A member 5 (SCARA5) and suprabasin (SBSN), respectively, were tested for effects on proliferation and collagen contraction. Knockdown of SCARA5 increased proliferation by 29.9% ± 7.8% (P < .01), whereas knockdown of SBSN had no effect. Knockdown of SBSN increased collagen gel contraction by 24.2% ± 8.6% (P < .05), whereas knockdown of SCARA5 had no effect. CONCLUSIONS: Using weighted gene coexpression network analysis of cultured vein graft cell gene expression, we have discovered two small gene modules, which comprise 42 genes, that are associated with vein graft failure. Further experiments are needed to delineate the venous cells that express these genes in vivo and the roles these genes play in vein graft healing, starting with the module hub genes SCARA5 and SBSN, which have been shown to have modest effects on cell proliferation or collagen gel contraction.

The Role of Repeat Administration of Adventitial Delivery of Lentivirus-shRNA-Vegf-A in Arteriovenous Fistula to Prevent Venous Stenosis Formation.

PURPOSE: To determine if a second dose of a lentivirus mediated small hairpin RNA that inhibits Vegf-A gene expression (LV-shRNA-Vegf-A) can improve lumen vessel area (LVA) of the outflow vein of an arteriovenous fistula (AVF) and decrease venous neointimal hyperplasia. MATERIALS AND METHODS: Chronic kidney disease was created in C57BL/6 mice; 28 days later, an AVF was created by connecting the right carotid artery to the ipsilateral jugular vein. Immediately after AVF creation, 5 × 10(6) plaque-forming units of LV-shRNA-Vegf-A or control shRNA was administered to the adventitia of the outflow vein, and a second dose of the same treatment was administered 14 days later. Animals were sacrificed at 21 days, 28 days, and 42 days after AVF creation for reverse transcription polymerase chain reaction and histomorphometric analyses. RESULTS: By day 21, there was a 125% increase in the average LVA (day 21, P = .11), with a decrease in cell proliferation (day 21, P = .0079; day 28, P = .28; day 42, P = .5), decrease in α-smooth muscle cell actin staining (day 21, P < .0001; day 28, P < .05; day 42, P = .59), and decrease in hypoxic stress (day 21, P < .001; day 28, P = .28; day 42, P = .46) in LV versus control shRNA vessels. CONCLUSIONS: A second dose of LV-shRNA-Vegf-A administration results in a moderate improvement in LVA at day 21.

Local MicroRNA Modulation Using a Novel Anti-miR-21-Eluting Stent Effectively Prevents Experimental In-Stent Restenosis.

OBJECTIVE: Despite advances in stent technology for vascular interventions, in-stent restenosis (ISR) because of myointimal hyperplasia remains a major complication. APPROACH AND RESULTS: We investigated the regulatory role of microRNAs in myointimal hyperplasia/ISR, using a humanized animal model in which balloon-injured human internal mammary arteries with or without stenting were transplanted into Rowett nude rats, followed by microRNA profiling. miR-21 was the only significantly upregulated candidate. In addition, miR-21 expression was increased in human tissue samples from patients with ISR compared with coronary artery disease specimen. We systemically repressed miR-21 via intravenous fluorescein-tagged-locked nucleic acid-anti-miR-21 (anti-21) in our humanized myointimal hyperplasia model. As expected, suppression of vascular miR-21 correlated dose dependently with reduced luminal obliteration. Furthermore, anti-21 did not impede reendothelialization. However, systemic anti-miR-21 had substantial off-target effects, lowering miR-21 expression in liver, heart, lung, and kidney with concomitant increase in serum creatinine levels. We therefore assessed the feasibility of local miR-21 suppression using anti-21-coated stents. Compared with bare-metal stents, anti-21-coated stents effectively reduced ISR, whereas no significant off-target effects could be observed. CONCLUSION: This study demonstrates the efficacy of an anti-miR-coated stent for the reduction of ISR.

Hyperglycemia-Induced Modulation of the Physiognomy and Angiogenic Potential of Fibroblasts Mediated by Matrix Metalloproteinase-2: Implications for Venous Stenosis Formation Associated with Hemodialysis Vascular Access in Diabetic Milieu.

PURPOSE: It is hypothesized that venous stenosis formation associated with hemodialysis vascular-access failure is caused by hypoxia-mediated fibroblast-to-myofibroblast differentiation accompanied by proliferation and migration, and that diabetic patients have worse clinical outcomes. The aim of this study was to determine the functional and gene expression outcomes of matrix metalloproteinase-2 (Mmp-2) silencing in fibroblasts cultured under hyperglycemia and euglycemia with hypoxic and normoxic stimuli. MATERIALS AND METHODS: AKR-2B fibroblasts were stably transduced using lentivirus-mediated shRNA-Mmp-2 or scrambled controls and subjected to hypoxia or normoxia under hyperglycemic or euglycemic conditions for 24 and 72 h. Gene expression of vascular endothelial growth factor-A (Vegf-A), Vegfr-1, Mmp-2, Mmp-9 and tissue inhibitors of matrix metalloproteinases (Timps) were determined by RT-PCR. Collagen I and IV secretion and cellular proliferation and migration were determined. RESULTS: Under hyperglycemic conditions, there is a significant reduction in the average gene expression of Vegf-A and Mmp-9, with an increase in Timp-1 at 24 h of hypoxia (p < 0.05) in Mmp-2-silenced fibroblasts when compared to controls. In addition, there is a decrease in collagen I and IV secretion and cellular migration. The euglycemic cells were able to reverse these findings. CONCLUSION: These findings demonstrate the rationale for using anti-Mmp-2 therapy in dialysis patients with hemodialysis vascular access in helping to reduce stenosis formation.

Soluble Jagged-1 inhibits restenosis of vein graft by attenuating Notch signaling.

The excessive proliferation of vascular smooth muscle cells was key factor in the restenosis of vein graft. And the Notch signaling was demonstrated to regulate vSMC proliferation and differentiation. Soluble Jagged-1 (sJag1) can inhibit Notch signaling in vitro and in vivo; however, its capacity to suppress restenosis of vein graft remains unknown. Under the microscope, the left jugular vein of these rats was interposed into the left common carotid artery, followed without any treatment (control), or with Ad-Jag1 (treatment) or placebo (DMSO) post operation. We showed that Ad-Jag1 can attenuate restenosis of vein graft by inducing decreased proliferation and increased apoptosis in vivo. Notch1-Hey2 signaling is critical for the development of intima thickening by controlling vSMC-fate determination. By blocking Notch signaling, Ad-Jag1 can significantly inhibit intima thickening. These studies identify that Ad-Jag1 can restore the vSMC phenotype and inhibit the vSMC proliferation by suppression of Notch1 signaling, and thus open a new avenue for the treatment of restenosis in vein graft.

microRNAs Distinctively Regulate Vascular Smooth Muscle and Endothelial Cells: Functional Implications in Angiogenesis, Atherosclerosis, and In-Stent Restenosis.

Endothelial cells (EC) and vascular smooth muscle cells (VSMC) are the main cell types within the vasculature. We describe here how microRNAs (miRs)--noncoding RNAs that can regulate gene expression via translational repression and/or post-transcriptional degradation--distinctively modulate EC and VSMC function in physiology and disease. In particular, the specific roles of miR-126 and miR-143/145, master regulators of EC and VSMC function, respectively, are deeply explored. We also describe the mechanistic role of miRs in the regulation of the pathophysiology of key cardiovascular processes including angiogenesis, atherosclerosis, and in-stent restenosis post-angioplasty. Drawbacks of currently available therapeutic options are discussed, pointing at the challenges and potential clinical opportunities provided by miR-based treatments.

Genetic variants associated with vein graft stenosis after coronary artery bypass grafting.

BACKGROUND: Vein graft stenosis after coronary artery bypass grafting (CABG) is common. Identifying genes associated with vein graft stenosis after CABG could reveal novel mechanisms of disease and discriminate patients at risk for graft failure. We hypothesized that genome-wide association would identify these genes. METHODS: We performed a genome-wide association study on a subset of patients presenting for cardiac catheterization for concern of ischemic heart disease, who also underwent CABG and subsequent coronary angiography after CABG for clinical indications (n = 521). Cases were defined as individuals with ≥50% stenosis in any vein graft on any cardiac catheterization, and controls were defined as those who did not have vein graft stenosis on any subsequent cardiac catheterization. Multivariable logistic regression was used to assess the association between single nucleotide polymorphisms (SNPs) and vein graft stenosis. RESULTS: Sixty-nine percent of patients had vein graft failure after CABG. Seven SNPs were significantly associated with vein graft stenosis, including intronic SNPs in the genes PALLD (Rs6854137, P = 3.77 × 10(-6)), ARID1B (Rs184074, P = 5.97 × 10(-6)), and TMEM123 (Rs11225247, P = 8.25 × 10(-6)); and intergenic SNPs near the genes ABCA13 (Rs10232860, P = 4.54 × 10(-6)), RMI2 (Rs9921338, P = 6.15 × 10(-6)), PRM2 (Rs7198849, P = 7.27 × 10(-6)), and TNFSF4 (Rs17346536, P = 9.33 × 10(-6)). CONCLUSIONS: We have identified novel genetic variants that may predispose to risk of vein graft failure after CABG, many within biologically plausible pathways. These polymorphisms merit further investigation, as they could assist in stratifying patients with multi-vessel coronary artery disease, which could lead to alterations in management and revascularization strategy.

Adventitial transduction of lentivirus-shRNA-VEGF-A in arteriovenous fistula reduces venous stenosis formation.

Venous neointimal hyperplasia (VNH) causes hemodialysis vascular access failure. Here we tested whether VNH formation occurs in part due to local vessel hypoxia caused by surgical trauma to the vasa vasorum of the outflow vein at the time of arteriovenous fistula placement. Selective targeting of the adventitia of the outflow vein at the time of fistula creation was performed using a lentivirus-delivered small-hairpin RNA that inhibits VEGF-A expression. This resulted in significant increase in mean lumen vessel area, decreased media/adventitia area, and decreased constrictive remodeling with a significant increase in apoptosis (increase in caspase 3 activity and TUNEL staining) accompanied with decreased cellular proliferation and hypoxia-inducible factor-1α at the outflow vein. There was significant decrease in cells staining positive for α-smooth muscle actin (a myofibroblast marker) and VEGFR-1 expression with a decrease in MMP-2 and MMP-9. These results were confirmed in animals that were treated with humanized monoclonal antibody to VEGF-A with similar results. Since hypoxia can cause fibroblast to differentiate into myofibroblasts, we silenced VEGF-A gene expression in fibroblasts and subjected them to hypoxia. This decreased myofibroblast production, cellular proliferation, cell invasion, MMP-2 activity, and increased caspase 3. Thus, VEGF-A reduction at the time of arteriovenous fistula placement results in increased positive vascular remodeling.

Role of Girdin in intimal hyperplasia in vein grafts and efficacy of atelocollagen-mediated application of small interfering RNA for vein graft failure.

OBJECTIVE: Intimal hyperplasia is a major obstacle to patency in grafted veins. Although migration and proliferation of vascular smooth muscle cells (SMCs) pivotally affect the vascular remodeling process, no therapy has been established to prevent intimal hyperplasia of vein grafts. We previously reported that the actin-binding protein Girdin crucially affects arterial remodeling. In this study, we investigated the role of Girdin in venous SMCs and evaluated a therapeutic strategy for vein graft failure in vivo using small interfering RNA (siRNA) that targets Girdin. METHODS: We investigated the relationship between Girdin expression and intimal hyperplasia using a rabbit vein graft model. Vein grafts under low-flow conditions were performed in Japanese White rabbits. For in vitro analyses, we isolated primary venous SMCs from vein graft neointima. siRNA that targets Girdin was mixed with atelocollagen, which stabilizes and releases nucleic acid reagents slowly and is applied perivascularly to the vein grafts at operation. Intimal hyperplasia was evaluated 4 weeks later. RESULTS: In the rabbit model, increased Girdin expression was seen in the neointima after the grafting operation. Using primary venous SMCs, we showed that Girdin is required for rearrangement of the actin cytoskeleton in venous SMCs and that siRNA-mediated Girdin knockdown significantly reduced venous SMC migration and proliferation. Girdin knockdown via perivascular application of siRNA using atelocollagen markedly reduced intimal thickening after the grafting operation. CONCLUSIONS: Depletion of Girdin attenuated venous SMCs migration and proliferation in vitro and intimal hyperplasia in vein grafts in vivo. Our findings suggest that Girdin affects migration and proliferation of vascular SMCs in vein grafts and that controlled release of Girdin siRNA using atelocollagen could be a novel therapeutic strategy for vein graft failure.

Patients carrying CYP2C19 loss of function alleles have a reduced response to clopidogrel therapy and a greater risk of in-stent restenosis after endovascular treatment of lower extremity peripheral arterial disease.

OBJECTIVE: This study evaluated the relationship between the cytochrome P450 (CYP) 2C19 genotype and the antiplatelet effect of clopidogrel therapy and investigated whether genotyping can predict the risk of ischemic events after endovascular treatment (ET) of lower extremity peripheral arterial disease. METHODS: From January 2011 to July 2012, 120 consecutive patients with arteriosclerosis obliterans (TransAtlantic Inter-Society Consensus for the Management of Peripheral Arterial Disease [TASC II] A-C) in the superficial femoral artery were included in a prospectively maintained database. Patients received 75 mg clopidogrel and 100 mg aspirin daily for at least 5 days before TaqMan (Life Technologies, Grand Island, NY) of CYP2C19 single-nucleotide polymorphisms and thromboelastography of the clopidogrel response. ET was subsequently performed, and follow-up evaluations, including duplex ultrasound imaging and ankle-brachial index assessment, were performed at 1, 3, 6, and 12 months after ET. During the follow-up, stent patency was assessed by ultrasound imaging, computed tomography angiography, or digital subtraction angiography. RESULTS: A total of 74 ET procedures were performed. Fifty of the enrolled patients (41.7%) completed the follow-up examinations and were included in the analysis. The mean duration of follow-up was 9.8 ± 2.1 months (range, 1-30 months). Carriers of at least one CYP2C19 loss-of-function (LOF) allele had a diminished pharmacodynamic response to clopidogrel (51.6 ± 20.1 vs. 39.8 ± 15.2 for patients without and with LOF alleles, respectively; P = .022). Carriers of one LOF allele had an increased incidence of ischemic events compared with patients without any LOF alleles (59.0% vs. 20.8%, respectively; P = .008). This trend was even more evident in patients with two LOF alleles compared with patients with no LOF alleles (100% vs. 20.8% ischemic events; P = .002). The cumulative primary patency rate at 12 months was 56.0%, with significant differences between groups (73.1% vs. 34.6% in patients without and with LOF alleles, respectively; P = .0.006). CYP2C19 LOF carrier status was associated with an increased rate of primary end points (P = .007). On the basis of their adenosine diphosphate-induced platelet aggregation, patients with high platelet reactivity had a significantly higher risk of ischemic events (P = .012). CYP2C19 genotypic classification (adjusted hazard ratio, 2.688; 95% confidence interval, 1.366-5.288; P = .004) and history of smoking (adjusted hazard ratio, 2.430; 95% confidence interval, 1.024-5.765; P = .044) were independent risk factors for ischemic events. CONCLUSIONS: CYP2C19 LOF alleles were associated with a diminished platelet response to clopidogrel treatment. Patients carrying CYP2C19 LOF alleles who are treated with clopidogrel may trend toward a poor prognosis after ET.

Upregulation of let-7a inhibits vascular smooth muscle cell proliferation in vitro and in vein graft intimal hyperplasia in rats.

BACKGROUND: Proliferation of vascular smooth muscle cells (VSMCs) is a crucial event in the pathogenesis of intimal hyperplasia, which is the main cause of restenosis after vascular reconstruction. In this study, we assessed the impact of let-7a microRNA (miRNA) on the proliferation of VSMCs. METHODS: Using miRNA microarrays analysis for miRNA expression in the vein graft model. Lentiviral vector-mediated let-7a was transfected into the vein grafts. In situ hybridization was performed to detect let-7a. Cultured rat VSMCs were transfected with let-7a mimics for different periods of time. Cell proliferation, migration and cell cycle activity were monitored following transfection of the let-7a mimics. Immunohistochemical and Western blotting analysis the expression levels of c-myc and K-ras. RESULTS: We found that let-7a was the most downregulated miRNA in the vein graft model. In vivo proliferation of VSMCs was assessed in a rat model of venous graft intimal hyperplasia. Let-7a was found to localize mainly to the VSMCs. Let-7a miRNA expression was increased in VSMCs in the neointima of the let-7a treated group. Intimal hyperplasia was suppressed by upregulation of let-7a via lentiviral vector-mediated mimics. In cultured VSMCs, the expression of let-7a increased upon starving, and the upregulation of let-7a miRNA significantly decreased cell proliferation and migration. Immunohistochemical and Western blotting analysis demonstrated that treatment with let-7a mimics resulted in decreased expression levels of c-myc and K-ras. CONCLUSIONS: The results indicate that let-7a miRNA is a novel regulator of VSMC proliferation in intimal hyperplasia. These findings suggest that let-7a miRNA is a promising therapeutic target for the prevention of intimal hyperplasia.