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.

Chitinase-3-like protein 1 is an independent risk factor for the early failure of forearm autologous arteriovenous fistulas in uremic patients.

Chitinase-3-like protein 1 (CHI3L1) has been introduced as a marker of inflammation in different diseases, which can promote cell proliferation and differentiation. It has also been demonstrated that elevated serum CHI3L1 concentration can independently predict all-cause mortality in uremic patients. However, the impact of CHI3L1 on the early failure of autologous arteriovenous fistulas (AVFs) in uremic patients remains unknown. We conducted a prospective observational cohort study of 109 uremic patients (mean age 53.2 ± 14.7 years, 67.9% males), who received forearm AVF surgery, and were consecutively enrolled with a median follow-up time of 15 months. The early failure was defined as a fistula that never developed adequately for dialysis or that failed within the first 3 months of use. Serum CHI3L1 concentration was determined by the ELISA method. Among 109 uremic patients, 24 patients had AVF failure. The optimal cutoff value based on the receiver operating characteristics analysis of CHI3L1 was 122.6 ng/mL, with the area under the curve of 0.73 (P = 0.001). The Kaplan-Meier survival analysis demonstrated that patients with CHI3L1 < 122.6 ng/mL had better AVF patency than patients with CHI3L1 ≥ 122.6 ng/mL (Log-rank test, P = 0.001). Multivariable Cox proportional hazards regression analysis showed that baseline CHI3L1 level (≥ 122.6 ng/mL vs. < 122.6 ng/mL) was significantly associated with AVF failure after adjustment for confounders (adjusted hazard ratio [HR], 3.67; 95% CI, 1.44-9.36). The study demonstrated that Increased baseline serum level of CHI3L1 is independently associated with higher risk of the early failure of forearm AVFs.

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.

Upregulation of miR­423 improves autologous vein graft restenosis via targeting ADAMTS­7.

Coronary artery bypass graft (CABG) is one of the primary methods of treating coronary heart disease (CHD); however, vein graft restenosis is a major limiting factor of the effectiveness of CABG. Emerging evidence has indicated that miR­423 is associated with vascular diseases. Additionally, upregulation of a disintegrin and metalloproteinase with thrombospondin motifs­7 (ADAMTS­7) contributes to neointima formation by promoting the proliferation and migration of vascular smooth muscle cells and inhibiting the proliferation and migration of endothelial cells. The aim of the present study was to examine the effects of miR­423 target, ADAMTS­7, on regulating vein graft disease and identify novel biomarkers for use in therapy of vein graft failure (VGF). Aberrant expression of miR­423 in plasma of patients with CHD prior to and following CABG confirms that miR­423 may be a suitable target for preventing VGF. Furthermore, a dual­luciferase reporter gene assay indicated that miR­423 directly interacted with ADAMTS­7 and suppressed its expression. Ectopic expression of miR­423 suppressed ADAMTS­7, resulting in decreased proliferation and migration rates of human umbilical vein smooth muscle cells by targeting ADAMTS­7, but resulted in increased proliferation and migration of human umbilical vein endothelial cells in vitro. Overexpression of miR­423 also enhanced re­endothelialization and decreased neointimal formation in a rat vein graft model. In conclusion, the results of the present study demonstrated that the miR­423/ADAMTS­7 axis may possess potential clinical value for the prevention and treatment of restenosis in patients with CHD following CABG.

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.

Reduction-Responsive Nucleic Acid Delivery Systems To Prevent In-Stent Restenosis in Rabbits.

Cardiovascular and cerebrovascular ischemic diseases seriously affect human health. Endovascular stent placement is an effective treatment but always leads to in-stent restenosis (ISR). Gene-eluting stent, which combines gene therapy with stent implantation, is a potential method to prevent ISR. In this study, an efficient gene-eluting stent was designed on the basis of one new nucleic acid delivery system to decrease the possibility of ISR. The reduction-responsive branched nucleic acid vector (SKP) with low cytotoxicity was first synthesized via ring-opening reaction. The impressive in vitro transfection performances of SKP were proved using luciferase reporter, enhanced green fluorescent protein plasmid, and vascular endothelial growth factor plasmid (pVEGF). Subsequently, SKP/pVEGF complexes were coated on the surfaces of pretreated clinical stents to construct gene-eluting stents (S-SKP/pVEGF). Antirestenosis performance of S-SKP/pVEGF was evaluated via implanting stents into rabbit aortas. S-SKP/pVEGF could lead to the localized upregulation of VEGF proteins, improve the progress of re-endothelialization, and inhibit the development of ISR in vivo. Such efficient pVEGF-eluting stent with responsive nucleic acid delivery systems is very promising to prevent in-stent restenosis of cerebrovascular diseases.

The association of genotype polymorphisms with vascular access patency in hemodialysis patients.

Some hemodialysis patients suffer from repeat dysfunction of dialysis vascular access and need procedures of angioplasty, thrombectomy, and even temporary catheter use. Why these patients are vulnerable to vascular access dysfunction and how to improve its patency are imperative to be discovered. Traditional risk factors for vascular access function had been widely investigated but could not fully explain this question. Several genotype polymorphisms were demonstrated to increase the incidence of cardiovascular disease and might also be linked to higher risk of vascular access dysfunction. As the major causes of arteriovenous access thrombosis are hypercoagulable status and arteriovenous access stenosis, the investigated genes mainly focus on the mediators of the coagulation cascade, inflammatory process, and endothelial dysfunction. The reported polymorphisms of genes significantly associated with arteriovenous access dysfunction included genes encoding methylene tetrahydrofolate reductase, coagulation factors, heme oxygenase-1, matrix metalloproteinase, transforming growth factor-ß1, tumor necrosis factor-α, vascular endothelial growth factor-A, renin-angiotensin-aldosterone system, and protein methyl transferase. However, further prospective study is indispensable to elucidate the association between the genotype polymorphisms and the outcome of vascular access. More and more therapeutic options that focus on genotype polymorphisms may generate a great benefit to the patency of vascular access of uremic patients.

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.

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.

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.

Preventing graft restenosis after coronary artery bypass grafting with tissue-type plasminogen activator.

OBJECTIVE: To explore the feasibility and safety of using tissue-type plasminogen activator (t-PA) to prevent graft restenosis after coronary artery bypass grafting (CABG). METHODS: In this prospective observational study, 37 patients underwent CABG between June 2009 and May 2013. These patients were grouped according to the anti-coagulation strategy after surgery: t-PA (n = 12) and conventional treatments (n = 25). In the t-PA group, the patients received acetylsalicylic acid (ASA) and clopidogrel plus intravenous infusion of t-PA (0.25 mg/kg/day) starting at 24 h after surgery and that lasted for 3 days. In the conventional group, the patients received only ASA and clopidogrel. 64-row spiral computed tomographic coronary angiography was performed at 1 week, 1, and 3 months after surgery to evaluate the patency of the graft vessel. RESULTS: The mean stenosis severity of the saphenous vein grafts was lower in the t-PA group compared with the conventional group at 3 months after surgery (p < 0.05), but there was no significant difference at 1 week and 1 month (p > 0.05). The patency rate of the grafts was not significantly different between the two groups at 1 week, 1, and 3 months after surgery (p > 0.05). CONCLUSION: Early application of t-PA after CABG was feasible and safe, and might help prevent early restenosis of SV grafts. Additional clinical randomized trials are necessary to address this issue.

The Anti-Apoptotic Properties of APEX1 in the Endothelium Require the First 20 Amino Acids and Converge on Thioredoxin-1.

The APEX nuclease (multifunctional DNA repair enzyme) 1 (APEX1) has a disordered N-terminus, a redox, and a DNA repair domain. APEX1 has anti-apoptotic properties, which have been linked to both domains depending on cell type and experimental conditions. AIMS: As protection against apoptosis is a hallmark of vessel integrity, we wanted to elucidate whether APEX1 acts anti-apoptotic in primary human endothelial cells and, if so, what the underlying mechanisms are. RESULTS: APEX1 inhibits apoptosis in endothelial cells by reducing Cathepsin D (CatD) cleavage, potentially by binding to the unprocessed form. Diminished CatD activation results in increased Thioredoxin-1 protein levels leading to reduced Caspase 3 activation. Consequently, apoptosis rates are decreased. This depends on the first twenty amino acids in APEX1, because APEX1 (21-318) induces CatD activity, decreases Thioredoxin-1 protein levels, and, thus, increases Caspase 3 activity and apoptosis. Along the same lines, APEX1 (1-20) inhibits Caspase 3 cleavage and apoptosis. Furthermore, re-expression of Thioredoxin-1 via lentiviral transduction rescues endothelial cells from APEX1 (21-318)-induced apoptosis. In an in vivo model of restenosis, which is characterized by oxidative stress, endothelial activation, and smooth muscle cell proliferation, Thioredoxin-1 protein levels are reduced in the endothelium of the carotids. INNOVATION: APEX1 acts anti-apoptotic in endothelial cells. This anti-apoptotic effect depends on the first 20 amino acids of APEX1. CONCLUSION: As proper function of the endothelium during life span is a hallmark for individual health span, a detailed characterization of the functions of the APEX1N-terminus is required to understand all its cellular properties. Antioxid. Redox Signal. 26, 616-629.

Surface-mediated transfection of a pDNA vector encoding short hairpin RNA to downregulate TGF-ß1 expression for the prevention of in-stent restenosis.

In-stent restenosis is one of the most serious modes of failure of cardiovascular stent implant. Although drug-eluting stents have been proven to reduce in-stent restenosis, the nonspecific inhibitory effects of anti-proliferative drugs, such as rapamycin, result in delayed re-endothelialization and fatal late stent thrombosis. Although many studies have focused on promoting rapid re-endothelialization, a feasible method of reducing excessive extracellular matrix (ECM) production and cell proliferation might provide a promising way to efficiently inhibit the restenosis in vivo. In this study, we constructed a surface-mediated gene delivery system through a layer-by-layer assembly of protamine sulfate (PrS) and a functional plasmid DNA (pDNA) encoding short hairpin RNA to downregulate the expression of transforming growth factor-ß1 (TGF-ß1), aiming to inhibit cell proliferation and reduce excessive ECM production. We demonstrated that (PrS/pDNA) films were successfully constructed with good stability under physiological conditions. The (PrS/pDNA) films were able to transfect fibroblasts, thus reducing the secretion of fibronectin and collagen and inhibiting cell proliferation in vitro. Further in vivo experiments showed that the transfection of arterial tissue led to significant local downregulation of TGF-ß1 and ECM proteins and inhibited neointimal hyperplasia. These functional gene delivery films avoid the use of non-specific drugs and may serve as part of a new strategy for targeting in-stent restenosis in the field of cardiovascular disease.

Inhibitory Effect of TLR4 Gene Silencing on Intimal Hyperplasia of Vein Grafting.

PURPOSE: The present study aimed to explore the regulating effect of Toll-like receptor 4 (TLR4) on intimal hyperplasia in rat vein grafts. METHODS: Rat models of external jugular vein carotid artery bypass grafting were established. Afterward, TLR4 small interfering RNA (siRNA) recombinant plasmids were constructed, which were transfected into rat vein graft bypass to study the effect of TLR4 silencing on intimal hyperplasia and to explore the underlying mechanisms. Real-time polymerase chain reaction and Western blot were used to detect the expression levels of TLR4 and inflammatory factors in TLR4 siRNA-transfected vein graft bypass. The intimal thickness was evaluated using hematoxylin-eosin staining. RESULTS: Compared with the scramble siRNA group, the intimal thickness of vein grafting was decreased significantly, while the inflammatory factors including interleukin (IL) 1ß, IL-6, and tumor necrosis factor α in grafted vein were dramatically downregulated in the TLR4 siRNA group. CONCLUSION: These results showed that local silencing of TLR4 in the vein grafts could inhibit intimal hyperplasia by downregulating the expression of inflammatory factors in the vein grafts, suggesting that TLR4 can be used as a new target for therapy of vascular intimal hyperplasia.

Genetic polymorphisms influence on the response to clopidogrel in peripheral artery disease patients following percutaneous transluminal angioplasty.

AIM: To study the association of ABCB1 and CYP2C19 polymorphisms and the clopidogrel response in Spanish peripheral artery disease patients following percutaneous transluminal angioplasty (PTA) and to perform a meta-analysis. MATERIALS & METHODS: 72 patients were recruited and 122 patients included in the meta-analysis. We evaluated the effect of ABCB1 3435 C>T, CYP2C19*2 and CYP2C19*3 and primary end point (restenosis/occlusion of the treated lesions) during 12 months after PTA. RESULTS: CYP2C19*2 and/or ABCB1 TT patients were associated with primary end point (OR: 5.00; 95% CI: 1.75-14.27). The meta-analysis confirmed the association of CYP2C19*2 and new atherothrombotic ischemic events (OR: 5.40; 95% CI: 2.30-12.70). CONCLUSION: The CYP2C19 and ABCB1 polymorphisms could be genetic markers of cardiovascular events in peripheral artery disease patients following PTA treated with clopidogrel.

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.

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.

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.