Search

Objective: We aimed to investigate the relationship between advanced glycation end product (AGE) levels in patients with saphenous vein graft (SVG) failure and in patients without SVG failure., Subjects and Methods: In our study, 55 patients with a history of previous coronary artery bypass grafting (CABG) surgery, who subsequently underwent coronary angiography for any reason and were found to have either SVG occlusion or significant lesions, were included as study patients. Additionally, 55 patients who have had CABG surgery without SVG failure for at least 1 year served as the control group. AGE values of the patients were measured using the skin autofluorescence method., Results: In our study results, we observed a significant difference in AGE levels between the two groups of patients with similar demographic characteristics (SVG failure groups AGE 3.2 [2.8-3.6] vs. control groups AGE 2.4 [2.1-2.7] p < 0.001). In the receiver operating characteristic curve analysis, we determined the ability of AGE levels to detect SVG failure with an area under the curve of 0.869. We found that in patients with AGE >3, it could detect SVG failure with a sensitivity of 70.9% and a specificity of 87.3%., Conclusions: Our results demonstrate that AGE levels can predict SVG failure risk inexpensively, easily, and quickly., Objective: We aimed to investigate the relationship between advanced glycation end product (AGE) levels in patients with saphenous vein graft (SVG) failure and in patients without SVG failure., Subjects and Methods: In our study, 55 patients with a history of previous coronary artery bypass grafting (CABG) surgery, who subsequently underwent coronary angiography for any reason and were found to have either SVG occlusion or significant lesions, were included as study patients. Additionally, 55 patients who have had CABG surgery without SVG failure for at least 1 year served as the control group. AGE values of the patients were measured using the skin autofluorescence method., Results: In our study results, we observed a significant difference in AGE levels between the two groups of patients with similar demographic characteristics (SVG failure groups AGE 3.2 [2.8-3.6] vs. control groups AGE 2.4 [2.1-2.7] p < 0.001). In the receiver operating characteristic curve analysis, we determined the ability of AGE levels to detect SVG failure with an area under the curve of 0.869. We found that in patients with AGE >3, it could detect SVG failure with a sensitivity of 70.9% and a specificity of 87.3%., Conclusions: Our results demonstrate that AGE levels can predict SVG failure risk inexpensively, easily, and quickly., (© 2024 The Author(s). Published by S. Karger AG, Basel.)

Vein graft disease is the process by which saphenous vein grafts, utilised for revascularisation during coronary artery bypass graft surgery, undergo an inflammation-driven intimal hyperplasia and accelerated atherosclerosis process in subsequent years after implantation. The role of the arterial circulation, particularly the haemodynamic properties' impact on graft patency, have been investigated but have not to date been explored in depth at the transcriptomic level. We have undertaken the first-in-man spatial transcriptomic analysis of the long saphenous vein in response to ex vivo acute arterial haemodynamic stimulation, utilising a combination of a custom 3D-printed perfusion bioreactor and the 10X Genomics Visium Spatial Gene Expression technology. We identify a total of 413 significant genes (372 upregulated and 41 downregulated) differentially expressed in response to arterial haemodynamic conditions. These genes were associated with pathways including NFkB , TNF , MAPK , and PI3K/Akt , among others. These are established pathways involved in the initiation of an early pro-inflammatory response, leukocyte activation and adhesion signalling, tissue remodelling, and cellular differentiation. Utilising unsupervised clustering analysis, we have been able to classify subsets of the expression based on cell type and with spatial resolution. These findings allow for further characterisation of the early saphenous vein graft transcriptional landscape during the earliest stage of implantation that contributes to vein graft disease, in particular validation of pathways and druggable targets that could contribute towards the therapeutic inhibition of processes underpinning vein graft disease.

To better understand mechanisms of serotonin- (5-HT) mediated vasorelaxation, isolated lateral saphenous veins from cattle were assessed for vasoactivity using myography in response to increasing concentrations of 5-HT or selective 5-HT receptor agonists. Vessels were pre-contracted with 1 × 10 -4  M phenylephrine and exposed to increasing concentrations of 5-HT or 5-HT receptor agonists that were selective for 5-HT 1B , 5-HT 2B , 5-HT 4 , and 5-HT 7 . Vasoactive response data were normalized as a percentage of the maximum contractile response induced by the phenylephrine pre-contraction. At 1 × 10 -7  M 5-HT, a relaxation was observed with an 88.7% decrease (p < 0.01) from the phenylephrine maximum. At 1 × 10 -4  M 5-HT, a contraction was observed with a 165% increase (p < 0.01) from the phenylephrine maximum. Increasing concentrations of agonists selective for 5-HT 2B , 5-HT 4 , or 5-HT 7 resulted in a 27%, 92%, or 44% (p < 0.01) decrease from the phenylephrine maximum, respectively. Of these 5-HT receptor agonists, the selective 5-HT 4 receptor agonist resulted in the greatest potency (-log EC 50 ) value (6.30) compared with 5-HT 2B and 5-HT 7 receptor agonists (4.21 and 4.66, respectively). To confirm the involvement of 5-HT 4 in 5-HT-mediated vasorelaxation, blood vessels were exposed to either DMSO (solvent control) or a selective 5-HT 4 antagonist (1 × 10 -5  M) for 5-min prior to the phenylephrine pre-contraction and 5-HT additions. Antagonism of the 5-HT 4 receptor attenuated the vasorelaxation caused by 5-HT. Approximately 94% of the vasorelaxation occurring in response to 5-HT could be accounted for through 5-HT 4 , providing strong evidence that 5-HT-mediated vasorelaxation occurs through 5-HT 4 activation in bovine peripheral vasculature., (© 2024 The Author(s). Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

Vascular pathologies show locational predisposition throughout the body; further insights into the transcriptomics basis of this vascular heterogeneity are needed. We analyzed transcriptomes from cultured endothelial cells and vascular smooth muscle cells from nine adult canine macrovessels: the aorta, coronary artery, vena cava, portal vein, femoral artery, femoral vein, saphenous vein, pulmonary vein, and pulmonary artery. We observed that organ-specific expression patterns persist in vitro, indicating that these genes are not regulated by blood flow or surrounding cell types but are likely fixed in the epigenetic memory. We further demonstrated the preserved location-specific expression of GATA4 protein in cultured cells and in the primary adult vessel. On a functional level, arterial and venous endothelial cells differed in vascular network morphology as the arterial networks maintained a higher complexity. Our findings prompt the rethinking of the extrapolation of results from single-origin endothelial cell systems., (© 2023. Springer Nature Limited.)

Saphenous vein bypass grafting is an effective technique used to treat peripheral arterial disease (PAD). However, restenosis is the major clinical challenge for the graft vessel among people with PAD postoperation. We hypothesize that there is a common culprit behind arterial occlusion and graft restenosis. To investigate this hypothesis, we found TGF-β, a gene specifically upregulated in PAD arteries, by bioinformatics analysis. TGF-β has a wide range of biological activities and plays an important role in vascular remodeling. We discuss the molecular pathway of TGF-β and elucidate its mechanism in vascular remodeling and intimal hyperplasia, including EMT, extracellular matrix deposition, and fibrosis, which are the important pathways contributing to stenosis. Additionally, we present a case report of a patient with graft restenosis linked to the TGF-β pathway. Finally, we discuss the potential applications of targeting the TGF-β pathway in the clinic to improve the long-term patency of vein grafts.

Background: Long noncoding RNAs (lncRNAs) have been widely suggested to bind with the microRNA (miRNA) sites and play roles of competing endogenous RNAs (ceRNAs), which can thus affect and regulate target gene and mRNA expression. Such lncRNA-related ceRNAs are identified to exert vital parts in vascular disease. Nonetheless, it remains unknown about how the lncRNA-miRNA-mRNA network functions in the varicose great saphenous veins., Methods: This study acquired the lncRNA and mRNA expression patterns from the GEO database and identifies the differentially expressed mRNAs and lncRNAs by adopting the R software "limma" package. Then, miRcode, miRDB, miRTarbase, and TargetScan were used to establish the miRNA-mRNA pairs and lncRNA-miRNA pairs. In addition, the lncRNA-miRNA-mRNA ceRNA network was constructed by using Cytoscape. Protein-protein interaction, Gene Ontology functional annotations, and Kyoto Encyclopedia of Genes and Genomes enrichment were carried out to examine the candidate hub genes, the functions of genes, and the corresponding pathways., Results: In line with the preset theory, we constructed ceRNA network comprising 12 lncRNAs, 38 miRNAs, and 149 mRNAs. Kyoto Encyclopedia of Genes and Genomes analysis indicated that the PI3K/Akt signaling pathway played a vital part in the development of varicose great saphenous veins. AC114730, AC002127, and AC073342 were significant biomarkers. At the same time, we predicted the potential miRNA, which may exert a significant influence on the varicose great saphenous veins, namely, miR-17-5p, miR-129-5p, miR-1297, miR-20b-5p, and miR-33a-3p., Conclusion: By performing ceRNA network analysis, our study detects new lncRNAs, miRNAs, and mRNAs, which can be applied as underlying biomarkers of varicose great saphenous veins and as therapeutic targets for the treatment of varicose great saphenous veins., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2022 Jiabo Wei et al.)

Atherosclerosis is one of the leading non-communicable diseases in Sri Lanka. Analysis of fatty acid composition in blood vessels is important in understanding the development of atherosclerosis. Here, analyses of fatty acid profiles in major arteries which are commonly used in Coronary Artery Bypass Graft surgery (CABG) were subjected to investigation. Patients (n = 27) undergoing elective CABG were enrolled in the study. A small biopsy segment of the saphenous vein (SV), radial artery (RA), and left internal mammary artery (LIMA) of patients was obtained during the surgery. The fatty acid (FA) profile of tissue samples was analyzed using Gas Chromatography-Mass Spectroscopy (GCMS). Among the different arteries tested, palmitic acid and stearic acid were the predominant fatty acids. As far as monounsaturated FA (MUFA) are concerned, oleic acid was found to be the most abundant MUFA in vessels. The FA profile of LIMA samples had a higher SFA percentage and lower unsaturated FA percentage compared to other vessels. Furthermore, the vessel samples of RA indicated the highest percentage of pro-inflammatory ω -6 polyunsaturated fatty acids (PUFA) as well as a higher percentage ratio between ω -6: ω -3 PUFA. The fatty acid composition and ω -6: ω -3 PUFA ratio suggests that LIMA graft is preferred for CABG over RA and SV., Competing Interests: The authors have declared that no competing interests exist.

Chronic venous insufficiency (CVI), in which blood return to the heart is impaired, is a prevalent condition worldwide. Valve incompetence is a complication of CVI that results in blood reflux, thereby aggravating venous hypertension. While CVI has a complex course and is known to produce alterations in the vein wall, the underlying pathological mechanisms remain unclear. This study examined the presence of DNA damage, pro-inflammatory cytokines and extracellular matrix remodelling in CVI-related valve incompetence. One hundred and ten patients with CVI were reviewed and divided into four groups according to age (<50 and ≥50 years) and a clinical diagnosis of venous reflux indicating venous system valve incompetence (R) (n = 81) or no reflux (NR) (n = 29). In vein specimens (greater saphenous vein) from each group, PARP, IL-17, COL-I, COL-III, MMP-2 and TIMP-2 expression levels were determined by RT-qPCR and immunohistochemistry. The younger patients with valve incompetence showed significantly higher PARP, IL-17, COL-I, COL-III, MMP-2 and reduced TIMP-2 expression levels and a higher COL-I/III ratio. Young CVI patients with venous reflux suffer chronic DNA damage, with consequences at both the local tissue and systemic levels, possibly associated with ageing., (© 2021 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

The rate of the remodeling of the arterialized saphenous vein conduit limits the outcomes of coronary artery bypass graft surgery (CABG), which may be influenced by endothelial dysfunction. We tested the hypothesis that high stretch (HS) induces human saphenous vein endothelial cell (hSVEC) dysfunction and examined candidate underlying mechanisms. Our results showed that in vitro HS reduces NO bioavailability, increases inflammatory adhesion molecule expression (E-selectin and VCAM1) and THP-1 cell adhesion. HS decreases F-actin in hSVECs, but not in human arterial endothelial cells, and is accompanied by G-actin and cofilin's nuclear shuttling and increased reactive oxidative species (ROS). Pre-treatment with the broad-acting antioxidant N-acetylcysteine (NAC) supported this observation and diminished stretch-induced actin remodeling and inflammatory adhesive molecule expression. Altogether, we provide evidence that increased oxidative stress and actin cytoskeleton remodeling play a role in HS-induced saphenous vein endothelial cell dysfunction, which may contribute to predisposing saphenous vein graft to failure.

Introduction: Several treatment modalities have been postulated to improve the efficacy of varicose vein treatment. Addition of glycerine to the sclerosing material has been documented to increase its viscosity and subsequently prolong the duration of stability, in addition to the direct sclerosing effect of glycerine. This histological and immunohistochemical study investigated the efficacy of addition of glycerine 72% to sclerotherapy on the human varicose vein., Methods: After surgical stripping of great saphenous veins, three equal segments were resected between two clamps. Specimen 1 was injected with saline only, specimen 2 was exposed to foam sclerosant 2%, and specimen 3 was exposed to a mixture of foam sclerosant 2% and glycerine 72%. All segments were left for 5min. Vein segments were then processed for histological and immunohistochemical study., Results: Microscopically, haematoxylin and eosin-stained specimen 1 showed endothelial swelling, cytoplasmic eosinophilia and pyknotic nuclei. The media showed sarcoplasm vacuolisation and necrosis. Specimen 3 showed hypereosinophilic sarcoplasm of the smooth muscle fibres. Oedema was less evident, with a relative decrease in the thickness of the wall compared with specimen 2. Immunohistochemically, the expression of smooth muscle actin was weak in specimen 3 compared with specimens 1 and 2. Expression of CD31 antibody was much reduced in specimen 2 which showed conserved islands of endothelial cells. By contrast, there was a complete loss of endothelial cells in specimen 3., Conclusion: Addition of glycerine 72% to foam sclerosant has a more damaging effect on human vein wall.

Objectives: We examined the expression, distribution, and contribution to vasodilatation of the calcium-activated potassium (K Ca ) channel family in the commonly used coronary artery bypass graft internal thoracic artery (ITA) and saphenous vein (SV) to understand the role of large conductance K Ca (BK Ca ), intermediate-conductance K Ca (IK Ca ), and small-conductance K Ca (SK Ca ) channel subtypes in graft dilating properties determined by endothelium-smooth muscle interaction that is essential to the postoperative performance of the graft., Methods: Real-time polymerase chain reaction and western blot were employed to detect the messenger RNA and protein level of K Ca channel subtypes. Distribution of K Ca channel subtypes was examined by immunohistochemistry. K Ca subtype-mediated vasorelaxation was studied using wire myography., Results: Both ITA and SV express all K Ca channel subtypes with each subtype distributed in both endothelium and smooth muscle. ITA and SV do not differ in the overall expression level of each K Ca channel subtype, corresponding to comparable relaxant responses to respective subtype activators. In ITA, BK Ca is more abundantly expressed in smooth muscle than in endothelium, whereas SK Ca exhibits more abundance in the endothelium. In comparison, SV shows even distribution of K Ca channel subtypes in the 2 layers. The BK Ca subtype in the K Ca family plays a significant role in vasodilatation of ITA, whereas its contribution in SV is quite limited., Conclusions: K Ca family is abundantly expressed in ITA and SV. There are differences between these 2 grafts in the abundance of K Ca channel subtypes in the endothelium and the smooth muscle. The significance of the BK Ca subtype in vasodilatation of ITA may suggest the potential of development of BK Ca modulators for the prevention and treatment of ITA spasm during/after coronary artery bypass graft surgery., (Copyright © 2019 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.)

Chronic venous disease (CVeD) has a remarkable prevalence, with an estimated annual incidence of 2%. It has been demonstrated how the loss of homeostatic mechanisms in the vein wall can take part in the physiopathology of CVeD. In this regard, it has been described how different axis, such as IGF-1/PAPP-A/STC-2 axis, may play an essential role in tissue homeostasis. The aim of this research is to study both genetic and protein expressions of the IGF-1/PAPP-A/STC-2 axis in CVeD patients. It is a cross-sectional study in which genetic (RT-qPCR) and protein (immunohistochemistry) expression analysis techniques were accomplished in saphenous veins from CVeD patients ( n = 35) in comparison to individuals without vascular pathology (HV). Results show a significant increase in both genetic and protein expressions of PAPP-A and IGF-1, and a decrement STC-2 expression at the same time in CVeD patients. Our study is a pioneer for demonstrating that the expression of the different components of the IGF-1/PAPP-A/STC-2 axis is altered in CVeD patients. This fact can be a part of the loss of homeostatic mechanisms of the venous tissue. Further research should be destined to deepen into alterations of this axis, as well as to evaluate the usage of these components as therapeutic targets for CVeD., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2020 Miguel A. Ortega et al.)

The long saphenous vein (LSV) is commonly used as a conduit in coronary artery bypass grafting. However, long term patency remains limited by the development of vascular inflammation, intimal hyperplasia and accelerated atherosclerosis. The impact of acute exposure of venous endothelial cells (ECs) to acute arterial wall shear stress (WSS) in the arterial circulation, and the subsequent activation of inflammatory pathways, remain poorly defined. Here, we tested the hypothesis that acute exposure of venous ECs to high shear stress is associated with inflammatory responses that are regulated by NF-κB both in-vitro and ex-vivo. Analysis of the LSV endothelium revealed that activation of NF-κB occurred within 30 min after exposure to arterial rates of shear stress. Activation of NF-κB was associated with increased levels of CCL2 production and enhanced binding of monocytes in LSVECs exposed to 6 h acute arterial WSS. Consistent with this, ex vivo exposure of LSVs to acute arterial WSS promoted monocyte interactions with the vessel lumen. Inhibition of the NF-κB pathway prevented acute arterial WSS-induced CCL2 production and reduced monocyte adhesion, both in vitro and in human LSV ex vivo, demonstrating that this pathway is necessary for the induction of the acute arterial WSS-induced pro-inflammatory response. We have identified NF-κB as a critical regulator of acute endothelial inflammation in saphenous vein in response to acute arterial WSS. Localised endothelial-specific inhibition of the NF-κB pathway may be beneficial to prevent vein graft inflammation and consequent failure.

Chronic venous disease (CVD) is the most common reported chronic condition in the United States, affecting more than 25 million Americans. Regardless of its high occurrence, current therapeutic options are far from ideal due to their palliative nature. For best treatment outcomes, challenging cases of chronic venous insufficiency (CVI) are treated by repair or replacement of venous valves. Regrettably, the success of venous valve transplant is dependent on the availability of autologous venous valves and hindered by the possibility of donor site complications and increased patient morbidity. Therefore, the use of alternative tissue sources to provide off-the-shelf venous valve replacements has potential to be extremely beneficial to the field of CVI. This manuscript demonstrates the capability of producing off-the-shelf fully functional venous valved extracellular matrix (ECM) scaffold conduits from bovine saphenous vein (SV), using an antigen removal (AR) method. AR ECM scaffolds maintained native SV structure-function relationships and associated venous valves function. Conversely, SDS decellularization caused significant changes to the collagen and elastin macromolecular structures, resulting in collagen fibril merging, elimination of fibril crimp, amalgaming collagen fibers and fragmentation of the inner elastic lamina. ECM changes induced by SDS decellularization resulted in significant venous valve dysfunction. Venous valved conduits generated using the AR approach have potential to serve as off-the-shelf venous valve replacements for CVI. STATEMENT OF SIGNIFICANCE: Retention of the structure and composition of extracellular matrix (ECM) proteins within xenogeneic scaffolds for tissue engineering is of crucial importance, due to the undeniable effect ECM proteins can impose on repopulating cells and function of the resultant biomaterial. This manuscript demonstrates that alteration or elimination of ECM proteins via commonly utilized decellularization approach results in complete disruption of venous valve function. Conversely, retention of the delicate ECM structure and composition of native venous tissue, using an antigen removal tissue processing method, results in preservation of native venous valve function., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier Ltd.)

Coronary artery disease (CAD) remains one of the most important causes of morbidity and mortality worldwide, and the availability of percutaneous or surgical revascularization procedures significantly improves survival. However, both strategies are daunted by complications which limit long-term effectiveness. In-stent restenosis (ISR) is a major drawback for intracoronary stenting, while graft failure is the limiting factor for coronary artery bypass graft surgery (CABG), especially using veins. Conversely, internal thoracic artery (ITA) is known to maintain long-term patency in CABG. Understanding the biology and pathophysiology of ISR and vein graft failure (VGF) and mechanisms behind ITA resistance to failure is crucial to combat these complications in CAD treatment. This review intends to provide an overview of the biological mechanisms underlying stent and VGF and of the potential therapeutic strategy to prevent these complications. Interestingly, despite being different modalities of revascularization, mechanisms of failure of stent and saphenous vein grafts are very similar from the biological standpoint., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2019. For permissions, please email: journals.permissions@oup.com.)

Objectives: In varicose veins, vascular smooth muscle cells (VSMCs) often shows phenotypic transition and abnormal proliferation and migration. Evidence suggests the FOXC2-Notch pathway may be involved in the pathogenesis of varicose veins. Here, this study aimed to explore the role of long non-coding RNA FOXC2-AS1 (FOXC2 antisense RNA 1) in phenotypic transition, proliferation, and migration of varicose vein-derived VSMCs and to explore whether the FOXC2-Notch pathway was involved in this process., Methods: The effect of FOXC2-AS1 on the proliferation and migration of human great saphenous vein smooth muscle cells (SV-SMCs) was analyzed using MTT assay and Transwell migration assay, respectively. The levels of contractile marker SM22α and synthetic marker osteopontin were measured by immunohistochemistry and Western blot to assess the phenotypic transition., Results: The human varicose veins showed thickened intima, media and adventitia layers, increased synthetic VSMCs, as well as upregulated FOXC2-AS1 and FOXC2 expression. In vitro assays showed that FOXC2-AS1 overexpression promoted phenotypic transition, proliferation, and migration of SV-SMCs. However, the effect of FOXC2-AS1 overexpression could be abrogated by both FOXC2 silencing and the Notch signaling inhibitor FLI-06. Furthermore, FOXC2-AS1 overexpression activated the Notch pathway by upregulating FOXC2., Conclusion: FOXC2-AS1 overexpression promotes phenotypic transition, proliferation, and migration of SV-SMCs, at least partially, by activating the FOXC2-Notch pathway.

Coronary artery bypass grafting (CABG) is one of the most efficient procedures for patients with advanced coronary artery disease. From all the blood vessels with the potential to be used in this procedure, the internal thoracic artery (ITA) and the saphenous vein (SV) are the most commonly applied as aortocoronary conduits. Nevertheless, in order to evaluate the graft patency and efficiency effectively, basic knowledge should be constantly expanding at the molecular level as well, as the understanding of predictive factors is still limited. In this study, we have employed the expressive microarray approach, validated with Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR), to analyze the transcriptome of both venous and arterial grafts. Searching for potential molecular factors, we analyzed differentially expressed gene ontologies involved in bone development and morphogenesis, for the possibility of discovery of new markers for the evaluation of ITA and SV segment quality. Among three ontological groups of interest-"endochondral bone morphogenesis", "ossification", and "skeletal system development"-we found six genes common to all of them. BMP6 , SHOX2 , COL13A1 , CSGALNACT1 , RUNX2, and STC1 showed differential expression patterns in both analyzed vessels. STC1 and COL13A1 were upregulated in ITA samples, whereas others were upregulated in SV. With regard to the Runx2 protein function in osteogenic phenotype regulation, the RUNX2 gene seems to be of paramount importance in assessing the potential of ITA, SV, and other vessels used in the CABG procedure. Overall, the presented study provided valuable insight into the molecular background of conduit characterization, and thus indicated genes that may be the target of subsequent studies, also at the protein level. Moreover, it has been suggested that RUNX2 may be recognized as a molecular marker of osteogenic changes in human blood vessels.

Rationale: In response to blood vessel wall injury, aberrant proliferation of vascular smooth muscle cells (SMCs) causes pathological remodeling. However, the controlling mechanisms are not completely understood., Objective: We recently showed that the human long noncoding RNA, SMILR, promotes vascular SMCs proliferation by a hitherto unknown mechanism. Here, we assess the therapeutic potential of SMILR inhibition and detail the molecular mechanism of action., Methods and Results: We used deep RNA-sequencing of human saphenous vein SMCs stimulated with IL (interleukin)-1α and PDGF (platelet-derived growth factor)-BB with SMILR knockdown (siRNA) or overexpression (lentivirus), to identify SMILR-regulated genes. This revealed a SMILR-dependent network essential for cell cycle progression. In particular, we found using the fluorescent ubiquitination-based cell cycle indicator viral system that SMILR regulates the late mitotic phase of the cell cycle and cytokinesis with SMILR knockdown resulting in ≈10% increase in binucleated cells. SMILR pulldowns further revealed its potential molecular mechanism, which involves an interaction with the mRNA of the late mitotic protein CENPF (centromere protein F) and the regulatory Staufen1 RNA-binding protein. SMILR and this downstream axis were also found to be activated in the human ex vivo vein graft pathological model and in primary human coronary artery SMCs and atherosclerotic plaques obtained at carotid endarterectomy. Finally, to assess the therapeutic potential of SMILR, we used a novel siRNA approach in the ex vivo vein graft model (within the 30 minutes clinical time frame that would occur between harvest and implant) to assess the reduction of proliferation by EdU incorporation. SMILR knockdown led to a marked decrease in proliferation from ≈29% in controls to ≈5% with SMILR depletion., Conclusions: Collectively, we demonstrate that SMILR is a critical mediator of vascular SMC proliferation via direct regulation of mitotic progression. Our data further reveal a potential SMILR-targeting intervention to limit atherogenesis and adverse vascular remodeling.

Resuscitation with 0.9% Normal Saline (NS), a non-buffered acidic solution, leads to increased morbidity and mortality in the critically ill. The goal of this study was to determine the molecular mechanisms of endothelial injury after exposure to NS. The hypothesis of this investigation is that exposure of endothelium to NS would lead to loss of cell membrane integrity, resulting in release of ATP, activation of the purinergic receptor (P2X7R), and subsequent activation of stress activated signaling pathways and inflammation. Human saphenous vein endothelial cells (HSVEC) incubated in NS, but not buffered electrolyte solution (Plasma-Lyte, PL), exhibited abnormal morphology and increased release of lactate dehydrogenase (LDH), adenosine triphosphate (ATP), and decreased transendothelial resistance (TEER), suggesting loss of membrane integrity. Incubation of intact rat aorta (RA) or human saphenous vein in NS but not PL led to impaired endothelial-dependent relaxation which was ameliorated by apyrase (hydrolyzes ATP) or SB203580 (p38 MAPK inhibitor). Exposure of HSVEC to NS but not PL led to activation of p38 MAPK and its downstream substrate, MAPKAP kinase 2 (MK2). Treatment of HSVEC with exogenous ATP led to interleukin 1β (IL-1β) release and increased vascular cell adhesion molecule (VCAM) expression. Treatment of RA with IL-1β led to impaired endothelial relaxation. IL-1β treatment of HSVEC led to increases in p38 MAPK and MK2 phosphorylation, and increased levels of arginase II. Incubation of porcine saphenous vein (PSV) in PL with pH adjusted to 6.0 or less also led to impaired endothelial function, suggesting that the acidic nature of NS is what contributes to endothelial dysfunction. Volume overload resuscitation in a porcine model after hemorrhage with NS, but not PL, led to acidosis and impaired endothelial function. These data suggest that endothelial dysfunction caused by exposure to acidic, non-buffered NS is associated with loss of membrane integrity, release of ATP, and is modulated by P2X7R-mediated inflammatory responses., Competing Interests: The authors have declared that no competing interests exist.

Objective- To determine the role of the oncofetal protein TPBG (trophoblast glycoprotein) in normal vascular function and reparative vascularization. Approach and Results- Immunohistochemistry of human veins was used to show TPBG expression in vascular smooth muscle cells and adventitial pericyte-like cells (APCs). ELISA, Western blot, immunocytochemistry, and proximity ligation assays evidenced a hypoxia-dependent upregulation of TPBG in APCs not found in vascular smooth muscle cells or endothelial cells. This involves the transcriptional modulator CITED2 (Atypical chemokine receptor 3 CBP/p300-interacting transactivator with glutamic acid (E)/aspartic acid (D)-rich tail) and downstream activation of CXCL12 (chemokine [C-X-C motif] ligand-12) signaling through the CXCR7 (C-X-C chemokine receptor type 7) receptor and ERK1/2 (extracellular signal-regulated kinases 1/2). TPBG silencing by siRNA transfection downregulated CXCL12, CXCR7, and pERK (phospho Thr202/Tyr204 ERK1/2) and reduced the APC migratory and proangiogenic capacities. TPBG forced expression induced opposite effects, which were associated with the formation of CXCR7/CXCR4 (C-X-C chemokine receptor type 4) heterodimers and could be contrasted by CXCL12 and CXCR7 neutralization. In vivo Matrigel plug assays using APCs with or without TPBG silencing evidenced TPBG is essential for angiogenesis. Finally, in immunosuppressed mice with limb ischemia, intramuscular injection of TPBG-overexpressing APCs surpassed naïve APCs in enhancing perfusion recovery and reducing the rate of toe necrosis. Conclusions- TPBG orchestrates the migratory and angiogenic activities of pericytes through the activation of the CXCL12/CXCR7/pERK axis. This novel mechanism could be a relevant target for therapeutic improvement of reparative angiogenesis.

Background: Cardiopulmonary bypass (CPB) applied during coronary artery bypass grafting (CABG), promotes inflammation, generation of reactive oxygen species (ROS) and up-regulation of matrix metalloproteinases (MMPs). All these complications may lead to contractile dysfunction, restenosis and early graft failure, restricting long-term efficacy of bypass grafts. Low-dose doxycycline is a potent MMP inhibitor and ROS scavenger. In this study, we aimed to investigate the effects of doxycycline on ROS generation, MMP regulation and contractile dysfunction induced by H 2 O 2 in human saphenous vein (HSV) grafts. Methods: HSV grafts (n=7) were divided into four groups after removing endothelial layer by mechanical scratching and incubated with 10 µM H 2 O 2 and/or 10 µM doxycycline for 16 hrs. Untreated segments served as control. Concentration-response curves to noradrenaline (NA), potassium chloride (KCl), serotonin (5-HT) and papaverine were performed. Superoxide anion and other ROS levels were determined by using lucigenin- and luminol-enhanced chemiluminescence assays, respectively. Expression/activity of gelatinases (MMP-2/MMP-9) was examined by gelatin zymography. MMP-13 expression was evaluated by immunostaining/immunoscoring. Results: H 2 O 2 incubation increased superoxide anion and other ROS levels. Doxycycline prevented these increments. H 2 O 2 suppressed contractile responses to NA, KCl and 5-HT. Doxycycline ameliorated contractions to NA and KCl but not to 5-HT. H 2 O 2 or doxycycline did not altered relaxation to papaverine. MMP-2 and MMP-13 expression increased with H 2 O 2 , but doxycycline inhibited MMP-2 up-regulation/activation. Conclusion: Low-dose doxycycline may have beneficial effects on increased oxidative stress, MMP up-regulation/activation and contractile dysfunction in HSV grafts., Competing Interests: The authors report no conflicts of interest in this work.

Objective: The study aimed to investigate the biologic effects of the 1470-nm endovenous laser (EVL), with a jacketed fiber and a radial fiber, during EVL ablation of an ex vivo dominant extrafascial tributary of the great saphenous vein in our in vitro model by histology and immunohistochemistry., Methods: Ten segments of the dominant extrafascial tributary of the great saphenous vein were harvested by a consultant vascular surgeon from patients during routine varicose vein surgery. Six segments were treated using an ex vivo model of our design by a 1470-nm EVL with a jacketed fiber. The other four segments were also treated by a 1470-nm EVL but with a radial-firing fiber. Each segment was split into five sections and treated at five different linear endovenous energy densities (LEEDs) at 10 W: 0, 20, 40, 60, and 80 J/cm. The veins were incubated and subsections collected at 6 and 24 hours after treatment. Subsections were immersed in buffered formalin and taken for histologic and immunohistochemical analysis. Histopathologic analysis was then performed., Results: Treatment with the radial fiber led to a pattern of damage that was more homogeneous than with the jacketed fiber, with no carbonization of tissue present. Significant transmural damage and necrosis were observed at LEEDs of 60 and 80 J/cm in both treatment groups. At the same LEEDs, p53 and caspase 3 analysis showed that transmural cell wall vein death (necrosis or apoptosis) occurred by 6 hours after treatment with both fibers., Conclusions: There was a significant difference in the effects of treatment with a jacketed fiber and a radial fiber in EVL ablation in vitro. Although both fibers caused transmural vein wall cell death at similar LEEDs, the pattern of damage with the radial fiber was more homogeneous. There was no overtreatment of tissue in terms of carbonization after treatment with the radial fiber. Treatment with the jacketed fiber showed carbonization of tissue at the same LEEDs., (Copyright © 2019. Published by Elsevier Inc.)

The role of cytokines in the pathogenesis of chronic venous disease (CVD) remains obscure. It has been postulated that oscillatory flow present in incompetent veins causes proinflammatory changes. Our earlier study confirmed this hypothesis. This study is aimed at assessing chemokines and growth factors (GFs) released by lymphocytes in patients with great saphenous vein (GSV) incompetence. In 34 patients exhibiting reflux in GSV, blood was derived from the cubital vein and from the incompetent saphenofemoral junction. In 12 healthy controls, blood was derived from the cubital vein. Lymphocyte culture with and without stimulation by phytohemagglutinin (PHA) was performed. Eotaxin, interleukin 8 (IL-8), macrophage inflammatory protein 1 A and 1B (MIP-1A and MIP-1B), interferon gamma-induced protein (IP-10), monocyte chemoattractant protein-1 (MCP-1), interleukin 5 (IL-5), fibroblast growth factor (FGF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), platelet-derived growth factor-BB (PDGF-BB), and vascular endothelial growth factor (VEGF) were assessed in culture supernatants by a Bio-Plex assay. Higher concentrations of eotaxin and G-CSF were revealed in the incompetent GSV, compared with the concentrations in the patients' upper limbs. The concentrations of MIP-1A and MIP-1B were higher in the CVD group while the concentration of VEGF was lower. In the stimulated cultures, the concentration of G-CSF proved higher in the incompetent GSV, as compared with the patients' upper limbs. Between the groups, the concentration of eotaxin was higher in the CVD group, while the IL-5 and MCP-1 concentrations were lower. IL-8, IP-10, FGF, GM-CSF, and PDGF-BB did not reveal any significant differences in concentrations between the samples. These observations suggest that the concentrations of chemokines and GFs are different in the blood of CVD patients. The oscillatory flow present in incompetent veins may play a role in these changes. However, the role of cytokines in CVD requires further study.

Objectives: The best preservation solution for a free vascular graft prior to coronary artery bypass grafting (CABG) remains controversial. The aim of this investigation was to evaluate the microenvironment of the human saphenous vein graft when preserved in normal saline (NS) solution or autologous heparinized whole blood (AWB)., Methods: Between January 2014 and December 2014, 21 patients who underwent CABG were enrolled and a total of 162 saphenous vein graft rings were collected. NS and AWB were used to investigate the influence of the microenvironment. The hypoxia, oxidative stress and vascular apoptosis were assayed by western blot, and endothelial integrity was assessed by immunohistochemical analysis., Results: The level of PaO2 in AWB was lower than that in NS (median: 100.5 mmHg vs 185.8 mmHg, P = 0.004). This hypoxic condition led to the production of more hypoxia-inducible factor-1 (median: 60.1% vs 15.1%, P = 0.008) and endothelial nitric oxide synthase (median: 52.6% vs 25%, P = 0.046) within 30 min of preservation time. The fact that higher levels of glutathione peroxidase resulted in the preservation of AWB suggests that it is beneficial to boost the vascular antioxidant defense with lower levels of NOX2. AWB led to increased Bcl-2, reduced cytochrome c and cleaved 85 kDa poly ADP-ribose polymerase apoptotic fragments., Conclusions: We concluded that AWB possesses a microenvironment that is superior to that of NS for saphenous vein graft preservation prior to CABG.

Background: To obtain a saphenous vein graft (SVG) for coronary artery bypass grafting (CABG), the benefit of using a no-touch (NT) technique in vascular function has not been fully investigated., Methods and results: The pathological and physiological functions of human SVGs with a NT technique to preserve the perivascular adipose tissue (PVAT) and ones obtained by using a conventional (CON) technique removing PVAT, were examined. Immunohistochemistry of the section of SVGs showed that the phosphorylation of endothelial nitric oxide synthase in the endothelium of the NT group was more responsive to vascular endothelial growth factor. A myograph of SVGs showed greater contraction with phenylephrine in the NT group. However, the strong contraction was eliminated in SVGs taken by electrocautery. In the 10 patients whose SVGs were taken without electrocautery, endothelial-dependent relaxation with bradykinin was apparently increased in the CON group more than in the NT group. Smooth muscle relaxation with nitroprusside was higher in the CON group at the lower concentrations; however, the relaxation became greater in the NT group at the high concentrations. Therefore, the effect of neutralizing PVAT-released factors in the both groups was further examined. After medium of NT and CON were exchanged in half, relaxation of SVGs was immediately restored in the NT group., Conclusions: The results suggest that the NT technique preserves the functions of vasoconstriction and relaxation. Also, the presence of PVAT-released vasoconstrictive factors was suspected.

Objective: When an autogenous vein is harvested and used for arterial bypass, it suffers physical and biologic injuries that may set in motion the cellular processes that lead to wall thickening, fibrosis, stenosis, and ultimately graft failure. Whereas the injurious effects of surgical preparation of the vein conduit have been extensively studied, little is known about the influence of the clinical environment of the donor leg from which the vein is obtained., Methods: We studied the cellular responses of fresh saphenous vein samples obtained before implantation in 46 patients undergoing elective lower extremity bypass surgery. Using an ex vivo model of response to injury, we quantified the outgrowth of cells from explants of the adventitial and medial layers of the vein. We correlated this cellular outgrowth with the clinical characteristics of the patients, including the Wound, Ischemia, and foot Infection classification of the donor leg for ischemia, wounds, and infection as well as smoking and diabetes., Results: Cellular outgrowth was significantly faster and more robust from the adventitial layer than from the medial layer. The factors of leg ischemia (P < .001), smoking (P = .042), and leg infection (P = .045) were associated with impaired overall outgrowth from the adventitial tissue on multivariable analysis. Only ischemia (P = .046) was associated with impaired outgrowth of smooth muscle cells (SMCs) from the medial tissue. Co-culture of adventitial cells and SMCs propagated from vein explants revealed that adventitial cells significantly inhibited the growth of SMCs, whereas SMCs promoted the growth of adventitial cells. The AA genotype of the -838C>A p27 polymorphism (previously associated with superior graft patency) enhanced these effects, whereas the factor of smoking attenuated adventitial cell inhibition of SMC growth. Comparing gene expression, the cells cultured from the media overexpress Kyoto Encyclopedia of Genes and Genomes pathways associated with inflammation and infection, whereas those from the adventitia overexpress gene families associated with development and stem/progenitor cell maintenance., Conclusions: The adverse clinical environment of the leg may influence the biologic behavior of the cells in the vein wall, especially the adventitial cells. Chronic ischemia was the most significant factor that retards adventitial cell outgrowth. The cells arising from the vein adventitia may be key players in determining a healthy adaptive or a pathologic response to the injuries associated with vein grafting., (Copyright © 2018 Society for Vascular Surgery. All rights reserved.)

Changes in extracellular matrix proteins may contribute significantly to the adaptation of vein grafts to the arterial circulation. We examined the production and distribution of versican and hyaluronan in intact human vein rings cultured ex vivo, veins perfused ex vivo, and cultured venous adventitial and smooth muscle cells. Immunohistochemistry revealed higher levels of versican in the intima/media compared to the adventitia, and no differences in hyaluronan. In the vasa vasorum, versican and hyaluronan associated with CD34+ progenitor cells. Culturing the vein rings for 14 days revealed increased versican immunostaining of 30-40% in all layers, with no changes in hyaluronan. Changes in versican accumulation appear to result from increased synthesis in the intima/media and decreased degradation in the adventitia as versican transcripts were increased in the intima/media, but unchanged in the adventitia, and versikine (the ADAMTS-mediated cleavage product of versican) was increased in the intima/media, but decreased in the adventitia. In perfused human veins, versican was specifically increased in the intima/media in the presence of venous pressure, but not with arterial pressure. Unexpectedly, cultured adventitial cells express and accumulate more versican and hyaluronan than smooth muscle cells. These data demonstrate a differential regulation of versican and hyaluronan in human venous adventitia vs. intima/media and suggest distinct functions for these extracellular matrix macromolecules in these venous wall compartments during the adaptive response of vein grafts to the arterial circulation., Competing Interests: The authors have declared that no competing interests exist.

Objective: Venous valves are essential but are prone to injury, thrombosis, and fibrosis. We compared the behavior and gene expression of smooth muscle cells (SMCs) in the valve sinus vs nonvalve sites to elucidate biologic differences associated with vein valves., Methods: Tissue explants of fresh human saphenous veins were prepared, and the migration of SMCs from explants of valve sinus vs nonvalve sinus areas was measured. Proliferation and death of SMCs were determined by staining for Ki67 and terminal deoxynucleotidyl transferase dUTP nick end labeling. Proliferation and migration of passaged valve vs nonvalve SMCs were determined by cell counts and using microchemotaxis chambers. Global gene expression in valve vs nonvalve intima-media was determined by RNA sequencing., Results: Valve SMCs demonstrated greater proliferation in tissue explants compared with nonvalve SMCs (19.3% ± 5.4% vs 6.8% ± 2.0% Ki67-positive nuclei at 4 days, respectively; mean ± standard error of the mean, five veins; P < .05). This was also true for migration (18.2 ± 2.7 vs 7.5 ± 3.0 migrated SMCs/explant at 6 days, respectively; 24 veins, 15 explants/vein; P < .0001). Cell death was not different (39.6% ± 16.1% vs 41.5% ± 16.0% terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells, respectively, at 4 days, five veins). Cultured valve SMCs also proliferated faster than nonvalve SMCs in response to platelet-derived growth factor subunit BB (2.9 ± 0.2-fold vs 2.1 ± 0.2-fold of control, respectively; P < .001; n = 5 pairs of cells). This was also true for migration (6.5 ± 1.2-fold vs 4.4 ± 0.8-fold of control, respectively; P < .001; n = 7 pairs of cells). Blockade of fibroblast growth factor 2 (FGF2) inhibited the increased responses of valve SMCs but had no effect on nonvalve SMCs. Exogenous FGF2 increased migration of valve but not of nonvalve SMCs. Unlike in the isolated, cultured cells, blockade of FGF2 in the tissue explants did not block migration of valve or nonvalve SMCs from the explants. Thirty-seven genes were differentially expressed by valve compared with nonvalve intimal-medial tissue (11 veins). Peptide-mediated inhibition of SEMA3A, one of the differentially expressed genes, increased the number of migrated SMCs of valve but not of nonvalve explants., Conclusions: Valve compared with nonvalve SMCs have greater rates of migration and proliferation, which may in part explain the propensity for pathologic lesion formation in valves. Whereas FGF2 mediates these effects in cultured SMCs, the mediators of these stimulatory effects in the valve wall tissue remain unclear but may be among the differentially expressed genes discovered in this study. One of these genes, SEMA3A, mediates a valve-specific inhibitory effect on the injury response of valve SMCs., (Copyright © 2017 Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved.)

Atherosclerosis is the underlying pathology of many cardiovascular diseases. The formation and rupture of atherosclerotic plaques in the coronary arteries results in angina and myocardial infarction. Venous coronary artery bypass grafts are designed to reduce the consequences of atherosclerosis in the coronary arteries by diverting blood flow around the atherosclerotic plaques. However, vein grafts suffer a high failure rate due to intimal thickening that occurs as a result of vascular cell injury and activation and can act as 'a soil' for subsequent atherosclerotic plaque formation. A clinically-proven method for the reduction of vein graft intimal thickening and subsequent major adverse clinical events is currently not available. Consequently, a greater understanding of the underlying mechanisms of intimal thickening may be beneficial for the design of future therapies for vein graft failure. Vein grafting induces inflammation and endothelial cell damage and dysfunction, that promotes vascular smooth muscle cell (VSMC) migration, and proliferation. Injury to the wall of the vein as a result of grafting leads to the production of chemoattractants, remodelling of the extracellular matrix and cell-cell contacts; which all contribute to the induction of VSMC migration and proliferation. This review focuses on the role of altered behaviour of VSMCs in the vein graft and some of the factors which critically lead to intimal thickening that pre-disposes the vein graft to further atherosclerosis and re-occurrence of symptoms in the patient.

Human saphenous vein (HSV) is harvested and prepared prior to implantation as an arterial bypass graft. Injury and the response to injury from surgical harvest and preparation trigger cascades of molecular events and contribute to graft remodeling and intimal hyperplasia. Apoptosis is an early response after implantation that contributes the development of neointimal lesions. Here, we showed that surgical harvest and preparation of HSV leads to vasomotor dysfunction, increased apoptosis and downregulation of the phosphorylation of the anti-apoptotic protein, Niban. A model of subfailure overstretch injury in rat aorta (RA) was used to demonstrate impaired vasomotor function, increased extracellular ATP (eATP) release, and increased apoptosis following pathological vascular injury. The subfailure overstretch injury was associated with activation of p38 MAPK stress pathway and decreases in the phosphorylation of the anti-apoptotic protein Niban. Treatment of RA after overstretch injury with antagonists to purinergic P2X7 receptor (P2X7R) antagonists or P2X7R/pannexin (PanX1) complex, but not PanX1 alone, restored vasomotor function. Inhibitors to P2X7R and PanX1 reduced stretch-induced eATP release. P2X7R/PanX1 antagonism led to decrease in p38 MAPK phosphorylation, restoration of Niban phosphorylation and increases in the phosphorylation of the anti-apoptotic protein Akt in RA and reduced TNFα-stimulated caspase 3/7 activity in cultured rat vascular smooth muscle cells. In conclusion, inhibition of P2X7R after overstretch injury restored vasomotor function and inhibited apoptosis. Treatment with P2X7R/PanX1 complex inhibitors after harvest and preparation injury of blood vessels used for bypass conduits may prevent the subsequent response to injury that lead to apoptosis and represents a novel therapeutic approach to prevent graft failure.

Background and Purpose: The side effects of cyclooxygenase-2 (COX-2) inhibitors on the cardiovascular system could be associated with reduced prostaglandin (PG)I 2 synthesis. Microsomal PGE synthase-1 (mPGES-1) catalyses the formation of PGE 2 from COX-derived PGH 2 . This enzyme is induced under inflammatory conditions and constitutes an attractive target for novel anti-inflammatory drugs. However, it is not known whether mPGES-1 inhibitors could be devoid of cardiovascular side effects. The aim of this study was to compare, in vitro, the effects of mPGES-1 and COX-2 inhibitors on vascular tone in human blood vessels., Experimental Approach: The vascular tone and prostanoid release from internal mammary artery (IMA) and saphenous vein (SV) incubated for 30 min with inhibitors of mPGES-1 or COX-2 were investigated under normal and inflammatory conditions., Key Results: In inflammatory conditions, mPGES-1 and COX-2 proteins were more expressed, and increased levels of PGE 2 and PGI 2 were released. COX-2 and NOS inhibitors increased noradrenaline induced vascular contractions in IMA under inflammatory conditions while no effect was observed in SV. Interestingly, the mPGES-1 inhibitor significantly reduced (30-40%) noradrenaline-induced contractions in both vessels. This effect was reversed by an IP (PGI 2 receptor) antagonist but not modified by NOS inhibition. Moreover, PGI 2 release was increased with the mPGES-1 inhibitor and decreased with the COX-2 inhibitor, while both inhibitors reduced PGE 2 release., Conclusions and Implications: In contrast to COX-2 inhibition, inhibition of mPGES-1 reduced vasoconstriction by increasing PGI 2 synthesis. Targeting mPGES-1 could provide a lower risk of cardiovascular side effects, compared with those of the COX-2 inhibitors., Linked Articles: This article is part of a themed section on Targeting Inflammation to Reduce Cardiovascular Disease Risk. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.22/issuetoc and http://onlinelibrary.wiley.com/doi/10.1111/bcp.v82.4/issuetoc., (© 2017 The British Pharmacological Society.)

Aims: Experimental evidence suggests that telomere length (TL) is shortened by oxidative DNA damage, reflecting biological aging. We explore the value of blood (BTL) and vascular TL (VTL) as biomarkers of systemic/vascular oxidative stress in humans and test the clinical predictive value of BTL in acute myocardial infarction (AMI)., Methods and Results: In a prospective cohort of 290 patients surviving recent AMI, BTL measured on admission was a strong predictor of all-cause [hazard ratio (HR) [95% confidence interval (CI)]: 3.21 [1.46-7.06], P = 0.004] and cardiovascular mortality (HR [95% CI]: 3.96 [1.65-9.53], P = 0.002) 1 year after AMI (for comparisons of short vs. long BTL, as defined by a T/S ratio cut-off of 0.916, calculated using receiver operating characteristic analysis; P adjusted for age and other predictors). To explore the biological meaning of these findings, BTL was quantified in 727 consecutive patients undergoing coronary artery bypass grafting (CABG), and superoxide (O2.-) was measured in peripheral blood mononuclear cells (PBMNC). VTL/vascular O2.- were quantified in saphenous vein (SV) and mammary artery (IMA) segments. Patients were genotyped for functional genetic polymorphisms in P22ph°x (activating NADPH-oxidases) and vascular smooth muscle cells (VSMC) selected by genotype were cultured from vascular tissue. Short BTL was associated with high O2.- in PBMNC (P = 0.04) but not in vessels, whereas VTL was related to O2.- in IMA (ρ = -0.49, P = 0.004) and SV (ρ = -0.52, P = 0.01). Angiotensin II (AngII) incubation of VSMC (30 days), as a means of stimulating NADPH-oxidases, increased O2.- and reduced TL in carriers of the high-responsiveness P22ph°x alleles (P = 0.007)., Conclusion: BTL predicts cardiovascular outcomes post-AMI, independently of age, whereas VTL is a tissue-specific (rather than a global) biomarker of vascular oxidative stress. The lack of a strong association between BTL and VTL reveals the importance of systemic vs. vascular factors in determining clinical outcomes after AMI., (© The Author 2017. Published by Oxford University Press on behalf of the European Society of Cardiology)

Background: Neointimal hyperplasia following angioplasty occurs via vascular smooth muscle cell proliferation. The mechanisms involved are not fully understood but include mitogen-activated protein kinases ERK1/2 (extracellular signal-regulated kinases 1 and 2). We recently identified the intracellular mediator PEA-15 (phosphoprotein enriched in astrocytes 15) in vascular smooth muscle cells as a regulator of ERK1/2-dependent proliferation in vitro. PEA-15 acts as a cytoplasmic anchor for ERK1/2, preventing nuclear localization and thereby reducing ERK1/2-dependent gene expression. The aim of the current study was to examine the role of PEA-15 in neointimal hyperplasia in vivo., Method and Results: Mice deficient in PEA-15 or wild-type mice were subjected to wire injury of the carotid artery. In uninjured arteries from PEA-15-deficient mice, ERK1/2 had increased nuclear translocation and increased basal ERK1/2-dependent transcription. Following wire injury, arteries from PEA-15-deficient mice developed neointimal hyperplasia at an increased rate compared with wild-type mice. This occurred in parallel with an increase in a proliferative marker and vascular smooth muscle cell proliferation. In wild-type mice, PEA-15 expression was decreased in vascular smooth muscle cells at an early stage before any increase in intima:media ratio. This regulation of PEA-15 expression following injury was also observed in an ex vivo human model of hyperplasia., Conclusions: These results indicate, for the first time, a novel protective role for PEA-15 against inappropriate vascular proliferation. PEA-15 expression may also be repressed during vascular injury, suggesting that maintenance of PEA-15 expression is a novel therapeutic target in vascular disease., (© 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.)

Objective: Varicose veins (VVs) are a common disorder of venous dilation and tortuosity, but the underlying mechanism is unclear. The functional integrity and phenotypic differences of VVs are also unclear. This study tested the hypothesis that phenotypic and functional differences exist between smooth muscle cells (SMCs) derived from VVs and normal veins., Methods: SMCs were isolated from 28 samples of varicose great saphenous veins (VGSVs) and normal great saphenous (NGSVs) and cultured. Proliferation, migration, adhesion, and aging capacity in SMCs were compared in the two veins. Bas, Bcl-2, caspase-3, matrix metalloproteinase (MMP)-2 MMP-9, tissue inhibitor of metalloproteinases (TIMP)-1, and TIMP-2 messenger (m)RNA expression and protein content were detected by fluorescence quantitative polymerase chain reaction and immunoblotting., Results: The microfilament structure of the framework was increased in SMCs in the VGSV group. Proliferation, migration, adhesion, and the aging cell count in SMCs in the VGSV group were significantly higher than the corresponding regions in the NGSV group (P < .05). Bas and caspase-3 mRNA expression and protein content were decreased, whereas Bcl-2 mRNA expression and protein content were increased in the VGSV group compared with the NGSV group (P < .05). MMP-2, MMP-9, TIMP-1, and TIMP-2 mRNA expression and protein content in the VGSV group were increased compared with the NGSV group (P < .05)., Conclusions: SMCs derived from VGSVs are more dedifferentiated and demonstrate increased proliferative and synthetic capacity. These results suggest the presence of phenotypic and functional differences between SMCs derived from VGSVs and NGSVs. The phenotypic and functional abnormalities in SMCs may be associated with the pathogenesis in VGSVs., (Copyright © 2017 Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved.)

Type 2 diabetes mellitus prevalence is growing globally, and the leading cause of mortality in these patients is cardiovascular disease. Epigenetic mechanisms such as microRNAs (miRs) and DNA methylation may contribute to complications of type 2 diabetes mellitus. We discovered an aberrant type 2 diabetes mellitus-smooth muscle cell phenotype driven by persistent up-regulation of miR-145. This study aimed to determine whether elevated expression was due to changes in methylation at the miR-145 promoter. Smooth muscle cells were cultured from saphenous veins of 22 non-diabetic and 22 type 2 diabetes mellitus donors. DNA was extracted, bisulphite treated and pyrosequencing used to interrogate methylation at 11 CpG sites within the miR-145 promoter. Inter-patient variation was high irrespective of type 2 diabetes mellitus. Differential methylation trends were apparent between non-diabetic and type 2 diabetes mellitus-smooth muscle cells at most sites but were not statistically significant. Methylation at CpGs -112 and -106 was consistently lower than all other sites explored in non-diabetic and type 2 diabetes mellitus-smooth muscle cells. Finally, miR-145 expression per se was not correlated with methylation levels observed at any site. The persistent up-regulation of miR-145 observed in type 2 diabetes mellitus-smooth muscle cells is not related to methylation at the miR-145 promoter. Crucially, miR-145 methylation is highly variable between patients, serving as a cautionary note for future studies of this region in primary human cell types., Competing Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship and/or publication of this article.

Background: Endothelial dysfunction contributes significantly to the development of vascular diseases. However, a therapy able to reduce this derangement still needs to be identified. We evaluated the effects of pharmacological inhibition of Rac1, a small GTPase protein promoting oxidative stress, in human endothelial dysfunction., Methods and Results: We performed vascular reactivity studies to test the effects of NSC23766, a Rac1 inhibitor, on endothelium-dependent vasorelaxation of saphenous vein segments collected from 85 subjects who had undergone surgery for venous insufficiency and from 11 patients who had undergone peripheral vascular surgery. The endothelium-dependent vasorelaxation of the varicose segments of saphenous veins collected from patients with venous insufficiency was markedly impaired and was also significantly lower than that observed in control nonvaricose vein tracts from the same veins. Rac1 activity, reactive oxygen species levels, and reduced nicotine adenine dinucleotide phosphate (NADPH) oxidase activity were significantly increased in varicose veins, and NSC23766 was able to significantly improve endothelium-dependent vasorelaxation of dysfunctional saphenous vein portions in a nitric oxide-dependent manner. These effects were paralleled by a significant reduction of NADPH oxidase activity and activation of endothelial nitric oxide synthase. Finally, we further corroborated this data by demonstrating that Rac1 inhibition significantly improves venous endothelial function and reduces NADPH oxidase activity in saphenous vein grafts harvested from patients with vascular diseases undergoing peripheral bypass surgery., Conclusions: Rac1 pharmacological inhibition rescues endothelial function and reduces oxidative stress in dysfunctional veins. Rac1 inhibition may represent a potential therapeutic intervention to reduce human endothelial dysfunction and subsequently vascular diseases in various clinical settings., (© 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.)

Background: Intimal hyperplasia remains the primary cause of vein graft failure for the 1 million yearly bypass procedures performed using human saphenous vein (HSV) grafts. This response to injury is caused in part by the harvest and preparation of the conduit. The use of Brilliant Blue FCF (FCF) restores injury-induced loss of function in vascular tissues possibly via inhibition of purinergic receptor signaling. This study investigated whether pretreatment of the vein graft with FCF prevents intimal hyperplasia., Methods: Cultured rat aortic smooth muscle cells (A7r5) were used to determine the effect of FCF on platelet-derived growth factor-mediated migration and proliferation, cellular processes that contribute to intimal hyperplasia. The effectiveness of FCF treatment during the time of explantation on preventing intimal hyperplasia was evaluated in a rabbit jugular-carotid interposition model and in an organ culture model using HSV., Results: FCF inhibited platelet-derived growth factor-induced migration and proliferation of A7r5 cells. Treatment with FCF at the time of vein graft explantation inhibited the subsequent development of intimal thickening in the rabbit model. Pretreatment with FCF also prevented intimal thickening of HSV in organ culture., Conclusions: Incorporation of FCF as a component of vein graft preparation at the time of explantation represents a potential therapeutic approach to mitigate intimal hyperplasia, reduce vein graft failure, and improve outcome of the autologous transplantation of HSV., (Copyright © 2016. Published by Elsevier Inc.)

Hydrogen sulfide (H2S) is a mediator with demonstrated protective effects for the cardiovascular system. On the other hand, prostaglandin (PG)E2 is involved in vascular wall remodeling by regulating matrix metalloproteinase (MMP) activities. We tested the hypothesis that endogenous H2S may modulate PGE2, MMP-1 activity and endogenous tissue inhibitors of MMPs (TIMP-1/-2). This regulatory pathway could be involved in thinning of abdominal aortic aneurysm (AAA) and thickening of saphenous vein (SV) varicosities. The expression of the enzyme responsible for H2S synthesis, cystathionine-γ-lyase (CSE) and its activity, were significantly higher in varicose vein as compared to SV. On the contrary, the endogenous H2S level and CSE expression were lower in AAA as compared to healthy aorta (HA). Endogenous H2S was responsible for inhibition of PGE2 synthesis mostly in varicose veins and HA. A similar effect was observed with exogenous H2S and consequently decreasing active MMP-1/TIMP ratios in SV and varicose veins. In contrast, in AAA, higher levels of PGE2 and active MMP-1/TIMP ratios were found versus HA. These findings suggest that differences in H2S content in AAA and varicose veins modulate endogenous PGE2 production and consequently the MMP/TIMP ratio. This mechanism may be crucial in vascular wall remodeling observed in different vascular pathologies (aneurysm, varicosities, atherosclerosis and pulmonary hypertension).

Aims: Extracellular matrix remodelling has been implicated in a number of vascular conditions, including venous hypertension and varicose veins. However, to date, no systematic analysis of matrix remodelling in human veins has been performed., Methods and Results: To understand the consequences of venous hypertension, normal and varicose veins were evaluated using proteomics approaches targeting the extracellular matrix. Varicose saphenous veins removed during phlebectomy and normal saphenous veins obtained during coronary artery bypass surgery were collected for proteomics analysis. Extracellular matrix proteins were enriched from venous tissues. The proteomics analysis revealed the presence of >150 extracellular matrix proteins, of which 48 had not been previously detected in venous tissue. Extracellular matrix remodelling in varicose veins was characterized by a loss of aggrecan and several small leucine-rich proteoglycans and a compensatory increase in collagen I and laminins. Gene expression analysis of the same tissues suggested that the remodelling process associated with venous hypertension predominantly occurs at the protein rather than the transcript level. The loss of aggrecan in varicose veins was paralleled by a reduced expression of aggrecanases. Chymase and tryptase β1 were among the up-regulated proteases. The effect of these serine proteases on the venous extracellular matrix was further explored by incubating normal saphenous veins with recombinant enzymes. Proteomics analysis revealed extensive extracellular matrix degradation after digestion with tryptase β1. In comparison, chymase was less potent and degraded predominantly basement membrane-associated proteins., Conclusion: The present proteomics study provides unprecedented insights into the expression and degradation of structural and regulatory components of the vascular extracellular matrix in varicosis., (© The Author 2016. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Objective: Papaverine is used to prevent vasospasm in human saphenous veins (HSV) during vein graft preparation prior to implantation as a bypass conduit. Papaverine is a nonspecific inhibitor of phosphodiesterases, leading to increases in both intracellular cGMP and cAMP. We hypothesized that papaverine reduces force by decreasing intracellular calcium concentrations ([Ca2+]i) and myosin light chain phosphorylation, and increasing actin depolymerization via regulation of actin regulatory protein phosphorylation., Approach and Results: HSV was equilibrated in a muscle bath, pre-treated with 1 mM papaverine followed by 5 μM norepinephrine, and force along with [Ca2+]i levels were concurrently measured. Filamentous actin (F-actin) level was measured by an in vitro actin assay. Tissue was snap frozen to measure myosin light chain and actin regulatory protein phosphorylation. Pre-treatment with papaverine completely inhibited norepinephrine-induced force generation, blocked increases in [Ca2+]i and led to a decrease in the phosphorylation of myosin light chain. Papaverine pre-treatment also led to increased phosphorylation of the heat shock-related protein 20 (HSPB6) and the vasodilator stimulated phosphoprotein (VASP), as well as decreased filamentous actin (F-actin) levels suggesting depolymerization of actin., Conclusions: These results suggest that papaverine-induced force inhibition of HSV involves [Ca2+]i-mediated inhibition of myosin light chain phosphorylation and actin regulatory protein phosphorylation-mediated actin depolymerization. Thus, papaverine induces sustained inhibition of contraction of HSV by the modulation of both myosin cross-bridge formation and actin cytoskeletal dynamics and is a pharmacological alternative to high pressure distention to prevent vasospasm.

Objective: Markers containing dyes such as crystal violet (CAS 548-62-9) are routinely used on the adventitia of vein bypass grafts to avoid twisting during placement. Because little is known about how these dyes affect vein graft healing and function, we determined the effect of crystal violet on cell migration and proliferation, which are responses to injury after grafting., Methods: Fresh human saphenous veins were obtained as residual specimens from leg bypass surgeries. Portions of the vein that had been surgically marked with crystal violet were analyzed separately from those that had no dye marking. In the laboratory, they were split into easily dissected inner and outer layers after removal of endothelium. This cleavage plane was within the circular muscle layer of the media. Cell migration from explants was measured daily as either (1) percentage of migration-positive explants, which exclusively measures migration, or (2) number of cells on the plastic surrounding each explant, which measures migration plus proliferation. Cell proliferation and apoptosis (Ki67 and TUNEL staining, respectively) were determined in dye-marked and unmarked areas of cultured vein rings. The dose-dependent effects of crystal violet were measured for cell migration from explants as well as for proliferation, migration, and death of cultured outer layer cells. Dye was extracted from explants with ethanol and quantified by spectrophotometry., Results: There was significantly less cell migration from visibly blue compared with unstained outer layer explants by both methods. There was no significant difference in migration from inner layer explants adjacent to blue-stained or unstained sections of vein because dye did not penetrate to the inner layer. Ki67 staining of vein in organ culture, which is a measure of proliferation, progressively increased up to 6 days in nonblue outer layer and was abolished in the blue outer layer. Evidence of apoptosis (TUNEL staining) was present throughout the wall and not different in blue-stained and unstained vein wall segments. Blue outer layer explants had 65.9 ± 8.0 ng dye/explant compared with 2.1 ± 1.3 for nonblue outer layer explants. Dye applied in vitro to either outer or inner layer explants dose dependently inhibited migration (IC50∼10 ng/explant). The IC50s of crystal violet for outer layer cell proliferation and migration were 0.1 and 1.2 μg/mL, whereas the EC50 for death was between 1 and 10 μg/mL., Conclusions: Crystal violet inhibits venous cell migration and proliferation, indicating that alternative methods should be considered for marking vein grafts., (Copyright © 2016 Society for Vascular Surgery. All rights reserved.)

Varicose veins of lower extremities are a heritable common disorder. Mechanisms underlying its pathogenesis are still vague. Structural failures such as valve weakness and wall dilatation in saphenous vein result in venous retrograde flow in lower extremities of body. Reflux of blood leads to distal high venous pressure resulting in distended veins. In an earlier study, we observed a positive association between c.-512C>T FoxC2 gene polymorphism and upregulated FoxC2 expression in varicose vein specimens. FoxC2 overexpression in vitro in venous endothelial cells resulted in the elevated mRNA expression of arterial endothelial markers such as Delta-like ligand 4 (Dll4) and Hairy/enhancer-of-split related with YRPW motif protein 2 (Hey2). We hypothesized that an altered FoxC2-Dll4 signaling underlies saphenous vein wall remodeling in patients with varicose veins. Saphenous veins specimens were collected from 22 patients with varicose veins and 20 control subjects who underwent coronary artery bypass grafting. Tissues were processed for paraffin embedding and sections were immunostained for Dll4, Hey2, EphrinB2, α-SMA, Vimentin, and CD31 antigens and examined under microscope. These observations were confirmed by quantitative real-time PCR and western blot analysis. An examination of varicose vein tissue specimens by immunohistochemistry indicated an elevated expression of Notch pathway components, such as Dll4, Hey2, and EphrinB2, and smooth muscle markers, which was further confirmed by gene and protein expression analyses. We conclude that the molecular alterations in Dll4-Hey2 signaling are associated with smooth muscle cell hypertrophy and hyperplasia in varicose veins. Our observations substantiate a significant role for altered FoxC2-Dll4 signaling in structural alterations of saphenous veins in patients with varicose veins.

Background: Persistent thromboxane (TX) generation while receiving aspirin therapy is associated with an increased risk of cardiovascular events. The Reduction in Graft Occlusion Rates (RIGOR) study found that aspirin-insensitive TXA2 generation, indicated by elevated urine 11-dehydro-TXB2 (UTXB2) 6 months after coronary artery bypass graft surgery, was a potent risk factor for vein graft thrombosis and originated predominantly from nonplatelet sources. Our goal was to identify risks factors for nonplatelet TXA2 generation., Methods and Results: Multivariable modeling was performed by using clinical and laboratory variables obtained from 260 RIGOR subjects with verified aspirin-mediated inhibition of platelet TXA2 generation. The strongest variable associated with UTXB2 6 months after surgery, accounting for 47.2% of the modeled effect, was urine 8-iso-prostaglandin (PG)F2α, an arachidonic acid metabolite generated nonenzymatically by oxidative stress (standardized coefficient 0.442, P<0.001). Age, sex, race, lipid therapy, creatinine, left ventricular ejection fraction, and aspirin dose were also significantly associated with UTXB2 (P<0.03), although they accounted for only 4.8% to 10.2% of the modeled effect. Urine 8-iso-PGF2α correlated with risk of vein graft occlusion (odds ratio 1.67, P=0.001) but was not independent of UTXB2. In vitro studies revealed that endothelial cells generate TXA2 in response to oxidative stress and direct exposure to 8-iso-PGF2α., Conclusions: Oxidative stress-induced formation of 8-iso-PGF2α is strongly associated with nonplatelet thromboxane formation and early vein graft thrombosis after coronary artery bypass graft surgery. The endothelium is potentially an important source of oxidative stress-induced thromboxane generation. These findings suggest therapies that reduce oxidative stress could be useful in reducing cardiovascular risks associated with aspirin-insensitive thromboxane generation., (© 2016 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.)

Background: Vein bypass is an essential therapy for patients with advanced peripheral and coronary artery disease despite development of neointimal hyperplasia. We have shown that stimulation of the receptor tyrosine kinase ephrin type-B receptor 4 (Eph-B4) with its ligand ephrin-B2 prevents neointimal hyperplasia in murine vein grafts. This study determines whether Eph-B4 in adult human veins is capable of phosphorylation and activation of downstream signaling pathways, as well as functional to release nitric oxide (NO) and prevent neointimal hyperplasia in vitro., Methods: Discarded human saphenous veins were taken from the operating room and placed in organ culture without or with ephrin-B2/Fc (2 μg/mL) for 14 days, and the neointima/media ratio was measured in matched veins. Primary human umbilical vein endothelial cells were treated with ephrin-B2/Fc (2 μg/mL) and examined with quantitative polymerase chain reaction, Western blot, immunoassays, and for release of NO. Ephrin-B2/Fc (2 μg/mL) was placed on the adventitia of saphenous veins treated with arterial shear stress for 24 hours in a bioreactor and activated Eph-B4 examined with immunofluorescence., Results: The baseline intima/media ratio in saphenous vein rings was 0.456 ± 0.097, which increased to 0.726 ± 0.142 in untreated veins after 14 days in organ culture but only to 0.630 ± 0.132 in veins treated with ephrin-B2/Fc (n = 19, P = .017). Ephrin-B2/Fc stimulated Akt, endothelial NO synthase and caveolin-1 phosphorylation, and NO release (P = .007) from human umbilical vein endothelial cells (n = 6). Ephrin-B2/Fc delivered to the adventitia stimulated endothelial Eph-B4 phosphorylation after 24 hours of arterial stress in a bioreactor (n = 3)., Conclusions: Eph-B4 is present and functional in adult human saphenous veins, with intact downstream signaling pathways capable of NO release and prevention of neointimal hyperplasia in vitro. Adventitial delivery of ephrin-B2/Fc activates endothelial Eph-B4 in saphenous veins treated with arterial shear stress in vitro. These results suggest that stimulation of Eph-B4 function may be a candidate strategy for translation to human clinical trials designed to inhibit venous neointimal hyperplasia., (Published by Elsevier Inc.)

Purpose: To develop an ex vivo model for the analysis of macroscopic, histological and immunohistochemical changes after experimental endovenous laser ablation (EVLA) of the great saphenous vein (GSV)., Methods: We describe a model produced with glass tubes and introducer sheaths to mimic the physiological conditions of EVLA procedures, such as tumescence and blood flow. A pilot study was conducted to evaluate an ex vivo procedure of EVLA of an incompetent GSV segment using a 1470-nm radial fiber diode laser (7 W power) and an automatic pull-back device. The vein segment was analyzed macroscopically and by hematoxylin & eosin staining, elastic fiber histochemistry, Gomori's trichrome staining, and alpha-smooth muscle actin immunohistochemistry., Results: No perforations were observed macroscopically. No muscle cell adhesion was observed in the central part of the ablated vein, showing tissue disruption. There was low labeling for elastic fibers, disruption of muscle fibers, and a reduced expression of the specific marker for this cell type., Conclusion: This ex vivo endovenous laser ablation model is a low cost alternative to in vivo experiments, providing standardized experimental conditions.

Objective: Despite its large clinical impact, the underlying mechanisms for vein graft failure remain obscure and no effective therapeutic solutions are available. We tested the hypothesis that Notch signaling promotes vein graft disease., Approach and Results: We used 2 biotherapeutics for Delta-like ligand 4 (Dll4), a Notch ligand: (1) blocking antibody and (2) macrophage- or endothelial cell (EC)-targeted small-interfering RNA. Dll4 antibody administration for 28 days inhibited vein graft lesion development in low-density lipoprotein (LDL) receptor-deficient (Ldlr(-/-)) mice, and suppressed macrophage accumulation and macrophage expression of proinflammatory M1 genes. Dll4 antibody treatment for 7 days after grafting also reduced macrophage burden at day 28. Dll4 silencing via macrophage-targeted lipid nanoparticles reduced lesion development and macrophage accumulation, whereas EC-targeted Dll4 small-interfering RNA produced no effects. Gain-of-function and loss-of-function studies suggested in vitro that Dll4 induces proinflammatory molecules in macrophages. Macrophage Dll4 also stimulated smooth muscle cell proliferation and migration and suppressed their differentiation., Conclusions: These results suggest that macrophage Dll4 promotes lesion development in vein grafts via macrophage activation and crosstalk between macrophages and smooth muscle cells, supporting the Dll4-Notch axis as a novel therapeutic target., (© 2015 American Heart Association, Inc.)

Objectives: The excess proliferation of vascular smooth muscle cells (VSMCs) and the development of intimal hyperplasia is a hallmark of vein graft failure. This study aimed to verify that a single intraoperative transfection of early growth response gene-1 (Egr-1) decoy oligonucleotide (ODN) can suppress vein graft proliferation of VSMCs and intimal hyperplasia., Methods: In a rabbit model, jugular veins were treated with Egr-1 decoy ODN, scrambled decoy ODN, Fugene6, or were left untreated, then grafted to the carotid artery. The vein graft samples were obtained 48 h, 1, 2 or 3 weeks after surgery. The thickness of the intima and intima/media ratio in the grafts was analysed by haematoxylin-eosin (HE) staining. The expression of the Egr-1 decoy ODN transfected in the vein was analysed using fluorescent microscopy. Egr-1 mRNA was measured using reverse transcription-polymerase chain reaction. The expression of Egr-1 protein was analysed by Western blot and immunohistochemistry., Results: Transfection efficiency of the ODN was confirmed by 4', 6-diamidino-2-phenylindole staining. In the grafts treated with Egr-1 decoy ODN, our study achieved statistically significant inhibition of intimal hyperplasia by ∼58% at 3 weeks. Transfection of Egr-1 decoy ODNs decreased the protein expression of Egr-1 and Egr-1 mRNA., Conclusions: We confirmed that gene therapy using in vivo transfection of an Egr-1 decoy ODN significantly inhibits proliferation of VSMC and intimal hyperplasia of vein grafts in a rabbit model., (© The Author 2015. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.)

Epigenetic mechanisms of gene regulation in context of cardiovascular diseases are of considerable interest. So far, our current knowledge of the DNA methylation profiles for atherosclerosis affected and healthy human vascular tissues is still limited. Using the Illumina Infinium Human Methylation27 BeadChip, we performed a genome-wide analysis of DNA methylation in right coronary artery in the area of advanced atherosclerotic plaques, atherosclerotic-resistant internal mammary arteries, and great saphenous veins obtained from same patients with coronary heart disease. The resulting DNA methylation patterns were markedly different between all the vascular tissues. The genes hypomethylated in athero-prone arteries to compare with atherosclerotic-resistant arteries were predominately involved in regulation of inflammation and immune processes, as well as development. The great saphenous veins exhibited an increase of the DNA methylation age in comparison to the internal mammary arteries. Gene ontology analysis for genes harboring hypermethylated CpG-sites in veins revealed the enrichment for biological processes associated with the development. Four CpG-sites located within the MIR10B gene sequence and about 1 kb upstream of the HOXD4 gene were also confirmed as hypomethylated in the independent dataset of the right coronary arteries in the area of advanced atherosclerotic plaques in comparison with the other vascular tissues. The DNA methylation differences observed in vascular tissues of patients with coronary heart disease can provide new insights into the mechanisms underlying the development of pathology and explanation for the difference in graft patency after coronary artery bypass grafting surgery.

[Pyr(1)]apelin-13 is an endogenous vasodilator and inotrope but is downregulated in pulmonary hypertension and heart failure, making the apelin receptor an attractive therapeutic target. Agonists acting at the same G-protein-coupled receptor can be engineered to stabilize different conformational states and function as biased ligands, selectively stimulating either G-protein or β-arrestin pathways. We used molecular dynamics simulations of apelin/receptor interactions to design cyclic analogues and identified MM07 as a biased agonist. In β-arrestin and internalization assays (G-protein-independent), MM07 was 2 orders of magnitude less potent than [Pyr(1)]apelin-13. In a G-protein-dependent saphenous vein contraction assay, both peptides had comparable potency (pD2:[Pyr(1)]apelin-13 9.93±0.24; MM07 9.54±0.42) and maximum responses with a resulting bias for MM07 of ≈350- to 1300-fold for the G-protein pathway. In rats, systemic infusions of MM07 (10-100nmol) caused a dose-dependent increase in cardiac output that was significantly greater than the response to [Pyr(1)]apelin-13. Similarly, in human volunteers, MM07 produced a significant dose-dependent increase in forearm blood flow with a maximum dilatation double that is seen with [Pyr(1)]apelin-13. Additionally, repeated doses of MM07 produced reproducible increases in forearm blood flow. These responses are consistent with a more efficacious action of the biased agonist. In human hand vein, both peptides reversed an established norepinephrine constrictor response and significantly increased venous flow. Our results suggest that MM07 acting as a biased agonist at the apelin receptor can preferentially stimulate the G-protein pathway, which could translate to improved efficacy in the clinic by selectively stimulating vasodilatation and inotropic actions but avoiding activating detrimental β-arrestin-dependent pathways., (© 2015 The Authors.)

Saphenous veins used as arterial grafts are exposed to arterial levels of oxygen partial pressure (pO2), which are much greater than what they experience in their native environment. The object of this study is to determine the impact of exposing human saphenous veins to arterial pO2. Saphenous veins and left internal mammary arteries from consenting patients undergoing coronary artery bypass grafting were cultured ex vivo for 2 weeks in the presence of arterial or venous pO2 using an established organ culture model. Saphenous veins cultured with arterial pO2 developed intimal hyperplasia as evidenced by 2.8-fold greater intimal area and 5.8-fold increase in cell proliferation compared to those freshly isolated. Saphenous veins cultured at venous pO2 or internal mammary arteries cultured at arterial pO2 did not develop intimal hyperplasia. Intimal hyperplasia was accompanied by two markers of elevated reactive oxygen species (ROS): increased dihydroethidium associated fluorescence (4-fold, p<0.05) and increased levels of the lipid peroxidation product, 4-hydroxynonenal (10-fold, p<0.05). A functional role of the increased ROS saphenous veins exposed to arterial pO2 is suggested by the observation that chronic exposure to tiron, a ROS scavenger, during the two-week culture period, blocked intimal hyperplasia. Electron paramagnetic resonance based oximetry revealed that the pO2 in the wall of the vessel tracked that of the atmosphere with a ~30 mmHg offset, thus the cells in the vessel wall were directly exposed to variations in pO2. Monolayer cultures of smooth muscle cells isolated from saphenous veins exhibited increased proliferation when exposed to arterial pO2 relative to those cultured at venous pO2. This increased proliferation was blocked by tiron. Taken together, these data suggest that exposure of human SV to arterial pO2 stimulates IH via a ROS-dependent pathway.