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Introduction: Hemostasis is a rapid response by the body to stop bleeding at sites of vessel injury. Both platelets and fibrin are important for the formation of a hemostatic plug. Mice have been used to uncover the molecular mechanisms that regulate the activation of platelets and coagulation under physiologic conditions. However, measurements of hemostasis in mice are quite variable, and current methods do not quantify platelet adhesion or fibrin formation at the site of injury., Methods: We describe a novel hemostasis model that uses intravital fluorescence microscopy to quantify platelet adhesion, fibrin formation and time to hemostatic plug formation in real time. Repeated vessel injuries of ~ 50-100 μm in diameter were induced with laser ablation technology in the saphenous vein of mice., Results: Hemostasis in this model was strongly impaired in mice deficient in glycoprotein Ibα or talin-1, which are important regulators of platelet adhesiveness. In contrast, the time to hemostatic plug formation was only minimally affected in mice deficient in the extrinsic tissue factor (TF(low)) or the intrinsic factor IX coagulation pathways, even though platelet adhesion was significantly reduced. A partial reduction in platelet adhesiveness obtained with clopidogrel led to instability within the hemostatic plug, especially when combined with impaired coagulation in TF(low) mice., Conclusions: In summary, we present a novel, highly sensitive method to quantify hemostatic plug formation in mice. On the basis of its sensitivity to platelet adhesion defects and its real-time imaging capability, we propose this model as an ideal tool with which to study the efficacy and safety of antiplatelet agents., (© 2014 International Society on Thrombosis and Haemostasis.)

Saphenous vein graft disease is a timely problem in coronary artery bypass grafting. Indeed, after exposure of the vein to arterial blood flow, a progressive modification in the wall begins, due to proliferation of smooth muscle cells in the intima. As a consequence, the graft progressively occludes and this leads to recurrent ischemia. In the present study we employed a novel ex vivo culture system to assess the biological effects of arterial-like pressure on the human saphenous vein structure and physiology, and to compare the results to those achieved in the presence of a constant low pressure and flow mimicking the physiologic vein perfusion. While under both conditions we found an activation of Matrix Metallo-Proteases 2/9 and of microRNAs-21/146a/221, a specific effect of the arterial-like pressure was observed. This consisted in a marked geometrical remodeling, in the suppression of Tissue Inhibitor of Metallo-Protease-1, in the enhanced expression of TGF-β1 and BMP-2 mRNAs and, finally, in the upregulation of microRNAs-138/200b/200c. In addition, the veins exposed to arterial-like pressure showed an increase in the density of the adventitial vasa vasorum and of cells co-expressing NG2, CD44 and SM22α markers in the adventitia. Cells with nuclear expression of Sox-10, a transcription factor characterizing multipotent vascular stem cells, were finally found in adventitial vessels. Our findings suggest, for the first time, a role of arterial-like wall strain in the activation of pro-pathologic pathways resulting in adventitial vessels growth, activation of vasa vasorum cells, and upregulation of specific gene products associated to vascular remodeling and inflammation.

Vascular smooth muscle cell (VSMC) migration and proliferation is central to neointima formation in vein graft failure following coronary artery bypass. However, there are currently no pharmacological interventions that prevent vein graft failure through intimal occlusion. It is hence a therapeutic target. Here, we investigated the contribution of GPR35 to human VSMC and endothelial cell (EC) migration, using a scratch-wound assay, and also the contribution to proliferation, using MTS and BrdU assays, in in vitro models using recently characterized human GPR35 ortholog-selective small-molecule agonists and antagonists. Real-time PCR studies showed GPR35 to be robustly expressed in human VSMCs and ECs. Stimulation of GPR35, with either the human-selective agonist pamoic acid or the reference agonist zaprinast, promoted VSMC migration in the scratch-wound assay. These effects were blocked by coincubation with either of the human GPR35-specific antagonists, CID-2745687 or ML-145. These GPR35-mediated effects were produced by inducing alterations in the actin cytoskeleton via the Rho A/Rho kinase signaling axis. Additionally, the agonist ligands stimulated a proliferative response in ECs. These studies highlight the potential that small molecules that stimulate or block GPR35 activity can modulate vascular proliferation and migration. These data propose GPR35 as a translational therapeutic target in vascular remodeling., (© 2016 The Author(s) Published by S. Karger AG, Basel.)

Type 2 diabetes (T2DM) confers increased risk of endothelial dysfunction, coronary heart disease, and vulnerability to vein graft failure after bypass grafting, despite glycaemic control. This study explored the concept that endothelial cells (EC) cultured from T2DM and nondiabetic (ND) patients are phenotypically and functionally distinct. Cultured human saphenous vein- (SV-) EC were compared between T2DM and ND patients in parallel. Proliferation, migration, and in vitro angiogenesis assays were performed; western blotting was used to quantify phosphorylation of Akt, ERK, and eNOS. The ability of diabetic stimuli (hyperglycaemia, TNF-α, and palmitate) to modulate angiogenic potential of ND-EC was also explored. T2DM-EC displayed reduced migration (~30%) and angiogenesis (~40%) compared with ND-EC and a modest, nonsignificant trend to reduced proliferation. Significant inhibition of Akt and eNOS, but not ERK phosphorylation, was observed in T2DM cells. Hyperglycaemia did not modify ND-EC function, but TNF-α and palmitate significantly reduced angiogenic capacity (by 27% and 43%, resp.), effects mimicked by Akt inhibition. Aberrancies of EC function may help to explain the increased risk of SV graft failure in T2DM patients. This study highlights the importance of other potentially contributing factors in addition to hyperglycaemia that may inflict injury and long-term dysfunction to the homeostatic capacity of the endothelium.

Background: Diabetes mellitus predisposes to thrombotic and proliferative vascular remodeling, to which thrombin contributes via activation of protease-activated receptor (PAR) 1. However, the use of PAR-1 inhibitors to suppress remodeling may be limited by severe bleeding. We recently reported upregulation of an additional thrombin receptor, PAR-4, in human vascular smooth muscle cells exposed to high glucose and have now examined PAR-4 as a novel mediator linking hyperglycemia, hypercoagulation, and vascular remodeling in diabetes mellitus., Methods and Results: PAR-4 expression was increased in carotid atherectomies and saphenous vein specimens from diabetic versus nondiabetic patients and in aorta and carotid arteries from streptozotocin-diabetic versus nondiabetic C57BL/6 mice. Vascular PAR-1 mRNA was not increased in diabetic mice. Ligated carotid arteries from diabetic mice developed more extensive neointimal hyperplasia and showed greater proliferation than arteries from nondiabetic mice. The augmented remodeling response was absent in diabetic mice deficient in PAR-4. At the cellular level, PAR-4 expression was controlled via the mRNA stabilizing actions of human antigen R, which accounted for the stimulatory actions of high glucose, angiotensin II, and H2O2 on PAR-4 expression, whereas cicaprost via protein kinase A activation counteracted this effect., Conclusions: PAR-4 appears to play a hitherto unsuspected role in diabetic vasculopathy. The development of PAR-4 inhibitors might serve to limit mainly proliferative processes in restenosis-prone diabetic patients, particularly those patients in whom severe bleeding attributed to selective PAR-1 blockade or complete thrombin inhibition must be avoided or those who do not require anticoagulation., (© 2014 American Heart Association, Inc.)

Polycystic ovary syndrome (PCOS) causes vascular damage to arteries; however, there are no data for its effect on veins. Our aim was to clarify the effects of dihydrotestosterone (DHT)-induced PCOS both on venous biomechanics and on pharmacological reactivity in a rat model and to test the possible modulatory role of vitamin D3 (vitD). PCOS was induced in female Wistar rats by DHT treatment (83 μg/day, subcutaneous pellet). After 10 wk, the venous biomechanics, norepinephrine (NE)-induced contractility, and acetylcholine-induced relaxation were tested in saphenous veins from control animals and from animals treated with DHT or DHT with vitD using pressure angiography. Additionally, the expression levels of endothelial nitric oxide synthase (eNOS) and cyclooxygenase (COX-2) were measured using immunohistochemistry. Increased diameter, wall thickness, and distensibility as well as decreased vasoconstriction were detected after the DHT treatment. Concomitant vitD treatment lowered the mechanical load on the veins, reduced distensibility, and resulted in vessels that were more relaxed. Although there was no difference in the endothelial dilation tested using acetylcholine (ACh), the blocking effect of N(G)-nitro-l-arginine methyl ester (l-NAME) was lower and was accompanied by lower COX-2 expression in the endothelium after the DHT treatment. Supplementation with vitD prevented these alterations. eNOS expression did not differ among the three groups. We conclude that the hyperandrogenic state resulted in thicker vein walls. These veins showed early remodeling and altered vasorelaxant mechanisms similar to those of varicose veins. Alterations caused by the chronic DHT treatment were prevented partially by concomitant vitD administration., (Copyright © 2014 the American Physiological Society.)

Vein graft adaptation to the arterial environment is characterized by loss of venous identity, with reduced Ephrin type-B receptor 4 (Eph-B4) expression but without increased Ephrin-B2 expression. We examined changes of vessel identity of human saphenous veins in a flow circuit in which shear stress could be precisely controlled. Medium circulated at arterial or venous magnitudes of laminar shear stress for 24 hours; histologic, protein, and RNA analyses of vein segments were performed. Vein endothelium remained viable and functional, with platelet endothelial cell adhesion molecule (PECAM)-expressing cells on the luminal surface. Venous Eph-B4 expression diminished (p = .002), Ephrin-B2 expression was not induced (p = .268), and expression of osteopontin (p = .002) was increased with exposure to arterial magnitudes of shear stress. Similar changes were not found in veins placed under venous flow or static conditions. These data show that human saphenous veins remain viable during ex vivo application of shear stress in a bioreactor, without loss of the venous endothelium. Arterial magnitudes of shear stress cause loss of venous identity without gain of arterial identity in human veins perfused ex vivo. Shear stress alone, without immunologic or hormonal influence, is capable of inducing changes in vessel identity and, specifically, loss of venous identity.

Objective: To test the hypothesis that the venous graft when implanted in an arterial bypass might endure a significantly elevated water filtration rate, leading to fast infiltration/accumulation of low-density lipoproteins (LDLs) within the graft wall and hence the accelerated atherogenesis., Methods: We measured filtration rates of the swine femoral artery and lateral saphenous vein under venous and arterial hemodynamic conditions in vitro. Based on the measured filtration rates, we numerically simulated LDL transport in an arterial bypass model with a venous graft. Moreover, in order to validate the theoretical prediction, we measured DiI-LDLs uptake by both arteries and veins under arterial condition., Results: The experimental results showed that when subjected to arterial hemodynamic condition, the filtration rate of the venous wall was increased sharply, and significantly higher than that of the artery. Moreover, different from the hydraulic conductivity of artery, the venous one would increase with increasing pressure. The numerical results showed that for the venous graft, both luminal surface LDL concentration and LDL accumulation within the vessel wall were significantly higher than those for the host artery. In addition, the experiment, which was used to confirm theoretical prediction, showed that the amount of DiI-LDLs uptake by the venous wall was much higher compared with arterial wall., Conclusion: The present study suggests that the accelerated atherogenesis of the venous graft is attributable to its elevated filtration rate that may lead to both aggravated concentration polarization of LDLs and enhanced convective transport of LDLs into the graft wall., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Type 2 diabetes (T2DM) promotes premature atherosclerosis and inferior prognosis after arterial reconstruction. Vascular smooth muscle cells (SMC) respond to patho/physiological stimuli, switching between quiescent contractile and activated synthetic phenotypes under the control of microRNAs (miRs) that regulate multiple genes critical to SMC plasticity. The importance of miRs to SMC function specifically in T2DM is unknown. This study was performed to evaluate phenotype and function in SMC cultured from non-diabetic and T2DM patients, to explore any aberrancies and investigate underlying mechanisms. Saphenous vein SMC cultured from T2DM patients (T2DM-SMC) exhibited increased spread cell area, disorganised cytoskeleton and impaired proliferation relative to cells from non-diabetic patients (ND-SMC), accompanied by a persistent, selective up-regulation of miR-143 and miR-145. Transfection of premiR-143/145 into ND-SMC induced morphological and functional characteristics similar to native T2DM-SMC; modulating miR-143/145 targets Kruppel-like factor 4, alpha smooth muscle actin and myosin VI. Conversely, transfection of antimiR-143/145 into T2DM-SMC conferred characteristics of the ND phenotype. Exposure of ND-SMC to transforming growth factor beta (TGFβ) induced a diabetes-like phenotype; elevated miR-143/145, increased cell area and reduced proliferation. Furthermore, these effects were dependent on miR-143/145. In conclusion, aberrant expression of miR-143/145 induces a distinct saphenous vein SMC phenotype that may contribute to vascular complications in patients with T2DM, and is potentially amenable to therapeutic manipulation., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Active smoking upregulated matrix metalloproteinase-2 (MMP-2) and MMP-9 gene expression in saphenous veins (SVs) before coronary operation. However, little is known about the effects of secondhand smoke (SHS), a smoking status that is more widely harmful to the general population, on MMP gene expression in SVs. Health effects of SHS were investigated mainly in nonsmokers married to smokers. Because the vast majority of women are nonsmokers in China, we had an opportunity to evaluate the relations between SHS and MMP gene expression in SVs of women patients before operation., Methods: A total of 258 woman patients were divided into three groups: control group, nonsmokers; SHS group, patients exposed to SHS until operation; active smoking group, patients who smoked a minimum of 1 package of cigarettes every day for more than 20 years. Messenger RNA and protein levels of MMP-2 and MMP-9 were analyzed. Saphenous vein graft patency after coronary operation was evaluated., Results: The clinical backgrounds in the three groups were comparable. Compared with the control group, MMP-2 and MMP-9 gene expression was significantly increased in the SHS and active smoking groups (p<0.05). The degree of MMP gene expression changed by SHS or active smoking was comparable. A significant difference of SV graft patency was found among the three groups. An association of increased MMP gene expression with lowered SV graft patency was found in follow-up., Conclusions: Our data revealed that SHS had similar power as active smoking to affect MMP gene expression in SVs before operation and SV graft patency after coronary operation in women nonsmokers., (Copyright © 2014 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

Proliferation and migration of smooth muscle cells and the resultant intimal hyperplasia cause coronary artery bypass graft failure. Both internal mammary artery and saphenous vein are the most commonly used bypass conduits. Although an internal mammary artery graft is immune to restenosis, a saphenous vein graft is prone to develop restenosis. We found significantly higher activity of phosphatase and tensin homolog (PTEN) in the smooth muscle cells of the internal mammary artery than in the saphenous vein. In this article, we critically review the pathophysiology of vein-graft failure with detailed discussion of the involvement of various factors, including PTEN, matrix metalloproteinases, and tissue inhibitor of metalloproteinases, in uncontrolled proliferation and migration of smooth muscle cells towards the lumen, and invasion of the graft conduit. We identified potential target sites that could be useful in preventing and (or) reversing unwanted consequences following coronary artery bypass graft using saphenous vein.

Introduction: Human saphenous vein (HSV) is the most widely used bypass conduit for peripheral and coronary vascular reconstructions. However, outcomes are limited by a high rate of intimal hyperplasia (IH). HSV undergoes a series of ex vivo surgical manipulations prior to implantation, including hydrostatic distension, marking, and warm ischemia in solution. We investigated the impact of surgical preparation on HSV cellular function and development of IH in organ culture. We hypothesized that oxidative stress is a mediator of HSV dysfunction., Methods: HSV was collected from patients undergoing vascular bypass before and after surgical preparation. Smooth muscle and endothelial function were measured using a muscle bath. Endothelial preservation was assessed with immunohistochemical staining. An organ culture model was used to investigate the influence of surgical preparation injury on the development of IH. Superoxide levels were measured using a high-performance liquid chromatography-based assay. The influence of oxidative stress on HSV physiologic responses was investigated by exposing HSV to hydrogen peroxide (H2O2)., Results: Surgical vein graft preparation resulted in smooth muscle and endothelial dysfunction, endothelial denudation, diminished endothelial nitric oxide synthase staining, development of increased IH, and increased levels of reactive oxygen species. Experimental induction of oxidative stress in unmanipulated HSV by treatment with H2O2 promoted endothelial dysfunction. Duration of storage time in solution did not contribute to smooth muscle or endothelial dysfunction., Conclusions: Surgical vein graft preparation causes dysfunction of the smooth muscle and endothelium, endothelial denudation, reduced endothelial nitric oxide synthase expression, and promotes IH in organ culture. Moreover, increased levels of reactive oxygen species are produced and may promote further vein graft dysfunction. These results argue for less injurious means of preparing HSV prior to autologous transplantation into the arterial circulation., (Copyright © 2014 Society for Vascular Surgery. All rights reserved.)

Cardiovascular disease is a leading cause of morbidity and mortality in United States, including Nebraska. Neointimal hyperplasia leading to restenosis is a major public health problem. Identification of key signaling molecules in biochemical pathways is an attractive strategy for development of predictive biomarkers in occlusive vascular diseases (OVD). Our pilot study aimed to identify the role of Wnt-frizzled signaling in restenosis in a North American cohort. North American patients (n = 9) undergoing coronary artery bypass graft surgery at Nebraska Heart Institute, Lincoln, were enrolled. Human saphenous veins (SV) (n = 9) and left internal mammary arteries (LIMA) (n = 9) received post-surgery at Creighton University, Omaha, were harvested using Collagenase-IV digestion method. Isolated primary VSMCs were cultured for 3-4 weeks, and passages P3-P7, were used for molecular biology experiments. The study was approved by the Institutional Review Board. RNA was extracted using trizol method and mRNA transcripts were identified using reverse transcriptase-polymerase chain reaction followed by 2% agarose gel electrophoresis. Mean age of surgery patients (n = 9) was 60.3 years (SD ± 6.5 years). Wnt2 and Wnt5a mRNA transcripts were expressed in human VSMCs; however, Wnt1, Wnt4 and Wnt11 mRNA were not expressed; beta-actin was used as an internal control. Receptor studies demonstrated the expression of Fzd1, Fzd2 and Fzd5 mRNA in hVSMCs. Our preliminary data implicates the public health significance of Wnt signaling in bypass graft patients in Nebraska. Future molecular biology approach-based community health studies targeting Wnt pathway may aid in the development of cost-effective predictive biomarkers for OVD susceptible populations.

Objective: The present study attempts to compare the immunohistochemistry (IHC) of von Willebrand factor (vWf) , endothelial cadherin, Caveolin and endothelial Nitric Oxide Synthase (eNOS) in VasoView Endoscopic Vein Harvesting (EVH) versus traditional Open Vein Harvesting (OVH) techniques for Coronary Artery Bypass Graft (CABG) Surgery performed in Javad al Aemeh Hospital of Mashhad, Iran in 2013,., Methods and Materials: Forty-seven patients were scheduled for CABG (30 EVH and 17 OVH) among whom patients with relatively same gender and similar age were selected. Three separate two cm vein samples were harvested from each patient's saphenous vein. Each portion was collected from distal, middle and proximal zones of the saphenous vein. The tissues were deparaffinized, and antigen retrieval was done using EZ-retriever followed by an immunohistochemistry evaluation with vWf, e-cadherin, Caveolin and eNOS. In addition, demographic questioner as of Lipid profile, FBS, BMI, and cardiovascular risk factors were collected. Data analyses, including parametric and nonparametric tests were undertaken using the SPSS 16 software. A P value < 0.05 was regarded as statistically significant., Results: The mean age of the EVH and OVH groups were 63.76 ± 9.51 and 63.63 ± 8.31 years respectively with no significant difference between them (p = 0.989). In addition, there was no great difference between the EVH and OVH groups in lipid profile, DM, HTN, smoking history, CVA, and valvular dysfunction (P > 0.05). Qualitative report of vWf, e-cadherin, Caveolin and eNOS reveals no significant difference between the EVH and OVH (P > 0.05)., Conclusion: This study indicates that VasoView EVH technique causes no endothelial damage in comparison with OVH. This study could be a molecular confirmation for the innocuous of EVH technique.

Background: Advanced glycation endproducts (AGEs) and its receptors (RAGEs) are heterogeneous signaling proteins associated to diabetes and responsible of endothelial alterations leading to atherosclerosis progression and graft failure. The aim of this study was to investigate the role of statin in reducing AGEs related endothelial damage., Methods: Endothelial cell(EC) obtained from leftovers of saphenous vein grafts of non-diabetic patients were incubated with AGEs (2 and 20 μM) and subsequently treated with Simvastatin. Neutrophils (PNM) adherence, ROS production and RAGE and peroxisome proliferator-activated receptors-gamma (PPAR-γ) expression were analyzed. As clinical validation of the in vitro findings, ECs of diabetic patients in optimized glycaemic control administered with a 3 weeks Simvastatin regimen were similarly processed., Results: Simvastatin blunted the rise in PMN adhesion and ROS generation following stimulation of saphenous vein EC culture with AGEs in vitro. This effect was time dependent and was associated to an increase in PPAR-γ induction paralleled by a decrease in RAGEs expression. Parallely, data from diabetic patients administered with Simvastatin showed a similar significant reduction in PNM adhesion and ROS generation. Simvastatin treatment significantly decreased RAGEs expression in ECs from diabetic patients and determined a slight increase in PPAR-γ expression but the latter failed to reach statistical significance. Interference in the function of these two crucial pathways might be at the root of the statin antinflammatory and antithrombotic effect in the context of AGEs-associated damage., Conclusions: Despite the recently raised warning on the use of statins in the diabetic population, this study elucidates their cornerstone position in endothelial homeostasis of saphenous grafts in patients with controlled diabetes., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Aims: The chemokine receptor CCR5 and its inflammatory ligands have been linked to atherosclerosis, an accelerated form of which occurs in saphenous vein graft disease. We investigated the function of vascular smooth muscle CCR5 in human coronary artery and saphenous vein, vascular tissues susceptible to atherosclerosis, and vasospasm., Methods and Results: CCR5 ligands were vasoconstrictors in saphenous vein and coronary artery. In vein, constrictor responses to CCL4 were completely blocked by CCR5 antagonists, including maraviroc. CCR5 antagonists prevented the development of a neointima after 14 days in cultured saphenous vein. CCR5 and its ligands were expressed in normal and diseased coronary artery and saphenous vein and localized to medial and intimal smooth muscle, endothelial, and inflammatory cells. [(125)I]-CCL4 bound to venous smooth muscle with KD = 1.15 ± 0.26 nmol/L and density of 22 ± 9 fmol mg(-1) protein., Conclusions: Our data support a potential role for CCR5 in vasoconstriction and neointimal formation in vitro and imply that CCR5 chemokines may contribute to vascular remodelling and augmented vascular tone in human coronary artery and vein graft disease. The repurposing of maraviroc for the treatment of cardiovascular disease warrants further investigation.

Unlabelled: Varicose veins are elongated and dilated saphenous veins. Despite the high prevalence of this disease, its pathogenesis remains unclear., Aims: In this study, we investigated the control of matrix metalloproteinases (MMPs) expression by prostaglandin (PG)E₂ during the vascular wall remodeling of human varicose veins., Methods and Results: Varicose (small (SDv) and large diameter (LDv)) and healthy saphenous veins (SV) were obtained after surgery. Microsomal and cytosolic PGE-synthases (mPGES and cPGES) protein and mRNA responsible for PGE₂ metabolism were analyzed in all veins. cPGES protein was absent while its mRNA was weakly expressed. mPGES-2 expression was similar in the different saphenous veins. mPGES-1 mRNA and protein were detected in healthy veins and a significant decrease was found in LDv. Additionally, 15-hydroxyprostaglandin dehydrogenase (15-PGDH), responsible for PGE₂ degradation, was over-expressed in varicose veins. These variations in mPGES-1 and 15-PGDH density account for the decreased PGE₂ level observed in varicose veins. Furthermore, a significant decrease in PGE₂ receptor (EP4) levels was also found in SDv and LDv. Active MMP-1 and total MMP-2 concentrations were significantly decreased in varicose veins while the tissue inhibitors of metalloproteinases (TIMP -1 and -2), were significantly increased, probably explaining the increased collagen content found in LDv. Finally, the MMP/TIMP ratio is restored by exogenous PGE₂ in varicose veins and reduced in presence of an EP4 receptor antagonist in healthy veins., Conclusions: In conclusion, PGE₂ could be responsible for the vascular wall thickening in human varicose veins. This mechanism could be protective, strengthening the vascular wall in order to counteract venous stasis.

Objectives: The aim of this study was to investigate the effects of the vasodilating β-blocker nebivolol and the cardioselective β-blocker metoprolol on nitric oxide (NO) levels at vascular graft endothelium and vasa vasorum compared to controls in patients undergoing coronary artery bypass graft surgery., Methods: This was a prospective study. Fifty-five patients were divided into three groups: nebivolol group (group N, n = 23), metoprolol group (group M, n = 16), and control group (group A, n = 16). Group N received nebivolol 5 mg once daily, and group M received metoprolol 50 mg once daily for 15 days in the preoperative period. Control patients did not use β-blocker therapy. Tissue samples of both left internal mammary artery (LIMA) and saphenous vein grafts were investigated for NO activity using immunohistochemical methods., Results: Demographic characteristics and risk factors were similar between groups. We observed the highest NO activity in group N in both endothelial and vasa vasorum samples of LIMA and saphenous veins. NO activity of metoprolol group was similar to controls., Conclusions: According to our results, we think that nebivolol may be safer and preferable in order to diminish graft spasm in patients undergoing coronary artery bypass graft surgery due to the NO-mediated vasodilating effect., (Copyright © 2013. Published by Elsevier Ltd.)

Long non-coding RNAs (lncRNAs) are key regulatory molecules involved in a variety of biological processes and human diseases. However, the pathological effects of lncRNAs on primary varicose great saphenous veins (GSVs) remain unclear. The purpose of the present study was to identify aberrantly expressed lncRNAs involved in the prevalence of GSV varicosities and predict their potential functions. Using microarray with 33,045 lncRNA and 30,215 mRNA probes, 557 lncRNAs and 980 mRNAs that differed significantly in expression between the varicose great saphenous veins and control veins were identified in six pairs of samples. These lncRNAs were sub-grouped and mRNAs expressed at different levels were clustered into several pathways with six focused on metabolic pathways. Quantitative real-time PCR replication of nine lncRNAs was performed in 32 subjects, validating six lncRNAs (AF119885, AK021444, NR&#95;027830, G36810, NR&#95;027927, uc.345-). A coding-non-coding gene co-expression network revealed that four of these six lncRNAs may be correlated with 11 mRNAs and pathway analysis revealed that they may be correlated with another 8 mRNAs associated with metabolic pathways. In conclusion, aberrantly expressed lncRNAs for GSV varicosities were here systematically screened and validated and their functions were predicted. These findings provide novel insight into the physiology of lncRNAs and the pathogenesis of varicose veins for further investigation. These aberrantly expressed lncRNAs may serve as new therapeutic targets for varicose veins. The Human Ethnics Committee of Shanghai East Hospital, Tongji University School of Medicine approved the study (NO.: 2011-DF-53).

Diabetes accelerates saphenous vein grafts calcification after years of coronary artery bypass grafting (CABG) surgery. Vascular smooth muscle cells (VSMC) undergoing a phenotypic switch to osteoblast-like cells play a key role in this process. The receptor for advanced glycation and products (RAGE) and toll-like receptors (TLRs) are all involved in various cardiovascular calcification processes. Therefore, the role of their common ligand, high mobility group box 1 (HMGB1), in high-glucose-induced calcification in VSMC of saphenous vein was investigated. In this study, VSMC were cultured from saphenous vein of patients arranged for CABG. We first demonstrated high-glucose-induced HMGB1 translocation from nucleus to cytosol, and this translocation was induced through a NADPH oxidase and PKC-dependent pathway. We next found high glucose also increased TLR2, TLR4, and RAGE expression. Then, we revealed downregulating HMGB1 expression abolished high-glucose-induced calcification accompanied by NFκB inactivation and low expression of bone morphogenetic protein-2 (BMP-2). We further demonstrated NFκB activation was necessary in high-glucose-induced BMP-2 expression and calcification. Finally, by using a chromatin immunoprecipitation assay, we demonstrated NFκB transcriptional regulation of BMP-2 promoter was induced by NFκB binding to its κB element on the BMP-2 promoter. Our findings thus suggest HMGB1 plays an important role in mediating the calcification process induced by high glucose through NFκB activation and BMP-2 expression in VSMC of saphenous vein.

Background: Atherosclerosis is a chronic progressive inflammatory disease of blood vessels particularly the arteries. The development of atherosclerotic plaques or atherogenesis is a complex process that is influenced by cardiovascular risk factors such as vascular inflammation and dyslipidemia. This study demonstrates the ability of tumor necrosis factor-alpha (TNF-α) and low density lipoproteins (LDL) to induce atherosclerotic plaque in human saphenous vein (HSV) organ culture., Methods: Normal HSV segments, from male patients who had coronary bypass graft, were cultured in DMEM containing 5% heat inactivated fetal bovine serum. TNF-α (5 ng/ml) was applied in combination with native LDL (nLDL) or oxidized LDL (oxLDL) at the dose of 50 μg/ml for 14 days. The phenotypic changes of the organ cultures characteristic of initial atherosclerotic plaques were evaluated. The effect of anti-atherogenic agent, 17-β estradiol (E2), was also determined., Results: Histologic, histomorphometric, and immunohistochemical examinations revealed that HSV rings stimulated with TNF-α + nLDL or TNF-α + oxLDL can exhibit the essential morphological features of atherogenesis, including fibrous cap formation, cholesterol clefts, evident thickening of the intimal layer, increased proliferation of smooth muscle cells (SMC) and migration to the subendothelial layer, significant SMC foam cell formation, and increased expression of adhesion molecules in the vascular wall. Addition of E2 (50 nM) to the culture significantly modulated the critical changes. Consistently, mRNA profiling of the HSV model revealed that 50 of 84 genes of atherosclerosis were up-regulated., Conclusions: Phenotypic changes characteristic of the initial development of atherosclerotic plaques can be induced in HSV organ culture.

Pharmacologic profiling of serotonin (5HT) receptors of bovine lateral saphenous vein has shown that cattle grazing endophyte-infected (Neotyphodium coenophialum) tall fescue (Lolium arundinaceum) have altered responses to ergovaline, 5HT, 5HT2A, and 5HT7 agonists. To determine if 5HT receptor activity of tall fescue alkaloids is affected by grazing endophyte-free (EF), wild-type [Kentucky-31 (KY31)], novel endophyte AR542-infected (MAXQ), or novel endophyte AR584-infected (AR584) tall fescue, contractile responses of lateral saphenous veins biopsied from cattle grazing these different fescue-endophyte combinations were evaluated in presence or absence of antagonists for 5HT2A (ketanserin) or 5HT7 (SB-269970) receptors. Biopsies were conducted over 2 yr on 35 mixed-breed steers (361.5 ± 6.3 kg) grazing EF (n = 12), KY31 (n = 12), MAXQ (n = 6), or AR584 (n = 5) pasture treatments (3 ha) between 84 and 98 d (Yr 1) or 108 to 124 d (Yr 2). Segments (2 to 3 cm) of vein were surgically biopsied, sliced into 2- to 3-mm cross-sections, and suspended in a myograph chamber containing 5 mL of oxygenated Krebs-Henseleit buffer (95% O2/5% CO2; pH = 7.4; 37°C). Veins were exposed to increasing concentrations of 5HT, ergovaline, and ergovaline + 1 × 10(-5) M ketanserin or + 1 × 10(-6) M SB-269970 in Yr 1. In Yr 2, ergotamine and ergocornine were evaluated in presence or absence of 1 × 10(-5) M ketanserin. Contractile response data were normalized to a reference addition of 1 × 10(-4) M norepinephrine. In Yr 1, contractile response to 5HT and ergovaline were least (P < 0.05) in KY31 pastures and the presence of ketanserin greatly reduced (P < 0.05) the response to ergovaline in all pastures. However, presence of SB-269970 did not (P = 0.91) alter contractile response to ergovaline. In Yr 2, there was no difference in contractile response to ergotamine (P = 0.13) or ergocornine (P = 0.99) across pasture treatments, but ketanserin reduced (P < 0.05) the contractile response to both alkaloids. The 5HT2A receptor is involved in alkaloid-induced vascular contraction and alkaloid binding may be affected by exposure to different endophyte-fescue combinations.

Surgical specimens of vein were obtained from the tertiary venous network and/or saphenous vein from patients (n=20) affected by chronic venous disease (CVD). Into the venous segments, which subsequently were surgically ablated, the following hemodynamic parameters were assessed by echo-color-doppler (ECD): peak systolic velocity, end diastolic velocity, whose combination allowed the calculation of the resistance index (RI) and the reflux time (RT). Highly purified venous endothelial cell (VEC) cultures derived from venous segments of these CVD patients were then characterized for the profile of cytokines and chemokines released in the culture supernatants. Among the 27 cytokines and chemokines examined, we found a positive and significant correlation (R=0.5; p=0.03) only between the spontaneous release of PDGF-BB by VEC cultures and the RT values of the patients from which the VEC were isolates. In addition, the release of PDGF-BB in the VEC culture supernatants was significantly (p<0.01) increased upon in vitro treatment with recombinant TNF-α. By using pharmacological inhibitors, specific for the main pathways, NF-kB, ERK1/2 and p38 MAPK, activated by exposure of endothelial cells to TNF-α, we found that only NF-kB appeared to be significantly involved in mediating the PDGF-BB induction by TNF-α. Of interest, the release of PDGF-BB in response to the in vitro inflammatory stimulation, maintained a positive and significant correlation with RT (R=0.6; p=0.01), while showing a negative correlation with RI (R=-0.5; p=0.03). The potential implications of our findings for the pathophysiology of CVD are discussed., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Atherosclerosis commonly affects the arteries harvested from patients 70 years of age or older. Saphenous vein grafts appear to maintain a higher patency rate after coronary artery bypass grafting in these subjects. The infiltration of macrophages is an early step in saphenous vein graft atherosclerosis; however, little is known regarding the underlying mechanisms of infiltration. The objective of the present report is to evaluate the presence of CD68-positive cells in the saphenous vein wall and correlate initial CD68-positive infiltration to specific clinical and biochemical parameters and the graft patency rate as estimated in patients undergoing coronary artery bypass grafting. A total of 309 patients were allocated into two groups: A1 patients, who were between 50 and 70 years of age, and A2 patients, who were 70 years or older at the time of vein harvesting. CD68 expression was evaluated by immunohistochemistry. There were no significant differences between A1 and A2 patients regarding macrophage expression within any of the analyzed vascular regions. Saphenous vein macrophages were never present in the tunica intima unless they were also expressed in the media or the adventitia. The patients with CD68-positive cells in the tunica intima had a significantly higher number of bypass stenoses when compared with the subjects who did not have CD68-positive cells in this layer. These findings suggest that the CD68-positive cells (those that have not yet developed into foam cells) present in the intima of saphenous vein grafts might serve as a very early marker of graft occlusion.

Experiments on isolated preparations of varicose human great saphenous vein revealed different sensitivity of the distal and proximal portions to P2X receptor agonist α,β-methylene-ATP, but not to norepinephrine and histamine. It is suggested that restructuring of the P2 receptor system plays an important role in the pathogenesis of varicose veins, which affects the trunk of the great saphenous vein in varying degrees.

Background: Adiponectin is an adipokine with potentially important roles in human cardiovascular disease states. We studied the role of adiponectin in the cross-talk between adipose tissue and vascular redox state in patients with atherosclerosis., Methods and Results: The study included 677 patients undergoing coronary artery bypass graft surgery. Endothelial function was evaluated by flow-mediated dilation of the brachial artery in vivo and by vasomotor studies in saphenous vein segments ex vivo. Vascular superoxide (O2(-)) and endothelial nitric oxide synthase (eNOS) uncoupling were quantified in saphenous vein and internal mammary artery segments. Local adiponectin gene expression and ex vivo release were quantified in perivascular (saphenous vein and internal mammary artery) subcutaneous and mesothoracic adipose tissue from 248 patients. Circulating adiponectin was independently associated with nitric oxide bioavailability and O2(-) production/eNOS uncoupling in both arteries and veins. These findings were supported by a similar association between functional polymorphisms in the adiponectin gene and vascular redox state. In contrast, local adiponectin gene expression/release in perivascular adipose tissue was positively correlated with O2(-) and eNOS uncoupling in the underlying vessels. In ex vivo experiments with human saphenous veins and internal mammary arteries, adiponectin induced Akt-mediated eNOS phosphorylation and increased tetrahydrobiopterin bioavailability, improving eNOS coupling. In ex vivo experiments with human saphenous veins/internal mammary arteries and adipose tissue, we demonstrated that peroxidation products produced in the vascular wall (ie, 4-hydroxynonenal) upregulate adiponectin gene expression in perivascular adipose tissue via a peroxisome proliferator-activated receptor-γ-dependent mechanism., Conclusions: We demonstrate for the first time that adiponectin improves the redox state in human vessels by restoring eNOS coupling, and we identify a novel role of vascular oxidative stress in the regulation of adiponectin expression in human perivascular adipose tissue.

Objective: Patients with congenital and acquired heart diseases or arteriopathy require small-diameter vascular grafts for arterial reconstruction. Autologous veins are the most suitable graft, but when absent, an alternative is necessary. This work addresses the issue., Background: Tissue-engineering efforts to create such grafts by modifications of acellular natural scaffolds are considered a promising area., Methods: Homologous saphenous veins harvested from cadavers and organ donors were processed by decellularization with detergent and enzymatic digestion, followed by crosslinking by dye-mediated photooxidation. They were validated for acellularity, mechanical strength, and crosslink stability. In-vitro and in-vivo cytotoxicity and hemocompatibility studies were conducted. Collagen conformity was studied by Fourier transform infrared spectroscopy, and heat stability by differential scanning calorimetry. A limited large animal study was performed., Results: The processing method delivered biocompatible, hemocompatible, effectively crosslinked grafts, with high heat stability of 126 , an enthalpy value of 183.5 J·g(-1), and collagen conformity close to that of the native vein. The mechanical strength was 250% better than the native vein. The presence of extracellular matrix proteins allowed the acellular vein to become a triple-layered vascular structure in the sheep venous system., Conclusion: Crosslinking after decellularization by the dye-mediated photooxidation method could be reproduced in any human vein to obtain a small-diameter vascular grafts.

Aims: The long-term failure of autologous saphenous vein bypass grafts due to neointimal thickening is a major clinical burden. Identifying novel strategies to prevent neointimal thickening is important. Thus, this study aimed to identify microRNAs (miRNAs) that are dysregulated during neointimal formation and determine their pathophysiological relevance following miRNA manipulation., Methods and Results: We undertook a microarray approach to identify dysregulated miRNAs following engraftment in an interpositional porcine graft model. These profiling experiments identified a number of miRNAs which were dysregulated following engraftment. miR-21 levels were substantially elevated following engraftment and these results were confirmed by quantitative real-time PCR in mouse, pig, and human models of vein graft neointimal formation. Genetic ablation of miR-21 in mice or grafted veins dramatically reduced neointimal formation in a mouse model of vein grafting. Furthermore, pharmacological knockdown of miR-21 in human veins resulted in target gene de-repression and a significant reduction in neointimal formation., Conclusion: This is the first report demonstrating that miR-21 plays a pathological role in vein graft failure. Furthermore, we also provided evidence that knockdown of miR-21 has therapeutic potential for the prevention of pathological vein graft remodelling.

Objective: This study was carried out to determine high pressure and pulsatile flow perfusion effects on human saphenous vein (HSV) segments obtained from diabetic and non-diabetic patients., Methods: The veins were perfused with oxygenated Krebs solution for 3 h, with a pulsatile flow rate of 100 mL/min and pressures of 250 × 200 or 300 × 250 mmHg. After perfusion, veins were studied by light microscopy; nitric oxide synthase (NOS) isoforms, CD34 and nitrotyrosine immunohistochemistry and tissue nitrite/nitrate (NO(x)) and malondialdehyde (MDA) quantification., Results: Light microscopy revealed endothelial denuding areas in all HSV segments subjected to 300 × 250 mmHg perfusion pressure, but the luminal area was similar. The percentage of luminal perimeter covered by endothelium decreased as perfusion pressures increased, and significant differences were observed between groups. The endothelial nitric oxide synthase (eNOS) isoform immunostaining decreased significantly in diabetic patients' veins independent of the perfusion pressure levels. The inducible NOS (iNOS), neuronal NOS (nNOS) and nitrotyrosine immunostaining were similar. Significant CD34 differences were observed between the diabetic 300 × 250 mmHg perfusion pressure group and the non-diabetic control group. Tissue nitrite/nitrate and MDA were not different among groups., Conclusions: Pulsatile flow and elevated pressures for 3 h caused morphological changes and decreased the eNOS expression in the diabetic patients' veins.

Background: Human saphenous vein grafts are one of the salvage bypass conduits when endovascular procedures are not feasible or fail. Understanding the remodeling process that venous grafts undergo during exposure to arterial conditions is crucial to improve their patency, which is often compromised by intimal hyperplasia. The precise role of hemodynamic forces such as shear stress and arterial pressure in this remodeling is not fully characterized. The aim of this study was to determine the involvement of arterial shear stress and pressure on vein wall remodeling and to unravel the underlying molecular mechanisms., Methods: An ex vivo vein support system was modified for chronic (up to 1 week), pulsatile perfusion of human saphenous veins under controlled conditions that permitted the separate control of arterial shear stress and different arterial pressure (7 mm Hg or 70 mm Hg)., Results: Veins perfused for 7 days under high pressure (70 mm Hg) underwent significant development of a neointima compared with veins exposed to low pressure (7 mm Hg). These structural changes were associated with altered expression of several molecular markers. Exposure to an arterial shear stress under low pressure increased the expression of matrix metalloproteinase (MMP)-2 and MMP-9 and tissue inhibitor of metalloproteinase (TIMP)-1 at the transcript, protein, and activity levels. This increase was enhanced by high pressure, which also increased TIMP-2 protein expression despite decreased levels of the cognate transcript. In contrast, the expression of plasminogen activator inhibitor-1 increased with shear stress but was not modified by pressure. Levels of the venous marker Eph-B4 were decreased under arterial shear stress, and levels of the arterial marker Ephrin-B2 were downregulated under high-pressure conditions., Conclusions: This model is a valuable tool to identify the role of hemodynamic forces and to decipher the molecular mechanisms leading to failure of human saphenous vein grafts. Under ex vivo conditions, arterial perfusion is sufficient to activate the remodeling of human veins, a change that is associated with the loss of specific vein markers. Elevation of pressure generates intimal hyperplasia, even though veins do not acquire arterial markers., Clinical Relevance: The pathological remodeling of the venous wall, which leads to stenosis and ultimately graft failure, is the main limiting factor of human saphenous vein graft bypass. This remodeling is due to the hemodynamic adaptation of the vein to the arterial environment and cannot be prevented by conventional therapy. To develop a more targeted therapy, a better understanding of the molecular mechanisms involved in intimal hyperplasia is essential, which requires the development of ex vivo models of chronic perfusion of human veins., (Copyright © 2013 Society for Vascular Surgery. Published by Mosby, Inc. All rights reserved.)

To date the question of epigenetic mechanisms of gene regulation in the context of cardiovascular diseases is of a considerable interest. Here, for the first time DNA methylation profiles of vascular tissues of atherosclerotic patients have been analyzed with using the microarray Infinium HumanMethylation27 BeadChip ("Illumina", USA). As the result, within 286 genes 314 CpG-sites that varied significantly in the DNA methylation level between the tissue samples of carotid (in the area of atherosclerotic plaques and nearby macroscopically intact tissues of the vascular wall) and mammary arteries as well saphenous veins have been identified. The most pronounced differences in the methylation level were registered for CpG-sites of homeobox genes HOXA2 and HOXD4 as well as imprinted gene MEST. In particular, these genes were found to be hypomethylated in the carotid atherosclerotic plaques compared to their methylation patterns in intact tissues of internal mammary arteries and saphenous veins.

Background: Hyperglycemia-induced venous endothelial dysfunction accelerates the progression of vein graft failure in patients with diabetes undergoing surgical coronary revascularization. Recent studies suggest the importance of bone morphogenic protein-4 (BMP4)-induced arterial endothelial dysfunction in the development of hypertension and atherosclerosis. The present study investigated the potential role of BMP4 in the pathogenesis of venous endothelial dysfunction in the setting of diabetes., Methods: Segments of saphenous vein from pigs and from patients with diabetes or patients without diabetes, as well as human umbilical venous endothelial cells (HUVECs), were used. The changes of BMP4 expression in veins from patients and in HUVECs cultured under hyperglycemic conditions were evaluated by Western blot assay. The effects of BMP4 on the production of reactive oxygen species (ROS) and endothelium-dependent venous relaxation were assessed by using dihydroethidium fluorescence and isometric tension measurements, respectively., Results: The impaired venous endothelium-dependent relaxations (2.9%±4.8% versus control group 74.1%±10%; p<0.01) accompanied by markedly increased BMP4 expression were observed in the diabetic group. The level of BMP4 expression in HUVECs treated with high levels of glucose were elevated in a glucose concentration-dependent manner. Ex vivo treatment with the BMP4 antagonist noggin significantly improved endothelium-dependent relaxations and inhibited accumulation of ROS in saphenous veins from patients with diabetes. Noggin treatment had no effect on the venous endothelium-dependent relaxations in individuals without diabetes. Meanwhile, BMP4 inhibited acetylcholine-induced relaxation (control group, 90%±7.1% versus BMP4-treated group, 52%±12.6%; p<0.05) and enhanced ROS production in porcine saphenous veins. Such harmful effects were again reversed by noggin., Conclusions: The increased BMP4 expression and related ROS overproduction may play an important role in the development of hyperglycemia-induced venous endothelial dysfunction., (Copyright © 2013 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

Objectives: Cryopreserved blood vessels are being increasingly employed in vascular reconstruction procedures but freezing/thawing is associated with significant cell death that may lead to graft failure. Vascular cells express connexin proteins that form gap junction channels and hemichannels. Gap junction channels directly connect the cytoplasm of adjacent cells and may facilitate the passage of cell death messengers leading to bystander cell death. Two hemichannels form a gap junction channel but these channels are also present as free non-connected hemichannels. Hemichannels are normally closed but may open under stressful conditions and thereby promote cell death. We here investigated whether blocking gap junctions and hemichannels could prevent cell death after cryopreservation., Materials and Methods: Inclusion of Gap27, a connexin channel inhibitory peptide, during cryopreservation and thawing of human saphenous veins and femoral arteries was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assays and histological examination., Results: We report that Gap27 significantly reduces cell death in human femoral arteries and saphenous veins when present during cryopreservation/thawing. In particular, smooth muscle cell death was reduced by 73% in arteries and 71% in veins, while endothelial cell death was reduced by 32% in arteries and 51% in veins., Conclusions: We conclude that inhibiting connexin channels during cryopreservation strongly promotes vascular cell viability., (Copyright © 2012 European Society for Vascular Surgery. Published by Elsevier Ltd. All rights reserved.)

Intimal hyperplasia and thrombosis are responsible for the poor patency rates of small-diameter vascular grafts. These complications could be avoided by a rapid and strong adhesion of endothelial cells to the prosthetic surfaces, which typically consist of expanded polytetrafluoroethylene (PTFE) for small-diameter vessels. We have previously described two peptide micropatterning strategies that increase the endothelialization rates of PTFE. The micropatterns were generated either by inkjet printing 300 μm squares or by spraying 10.1 ± 0.1 μm diameter droplets of the CGRGDS cell adhesion peptide, while the remaining surface was functionalized using the CWQPPRARI cell migration peptide. We now directly compare these two micropatterning strategies and examine the effect of hydrodynamic stress on human saphenous vein endothelial cells grown on the patterned surfaces. No significant differences in cell adhesion were observed between the two micropatterning methods. When compared to unpatterned surfaces treated with a uniform mixture of the two peptides, the cell expansion was significantly higher on sprayed or printed surfaces after 9 days of static cell culture. In addition, after 6 h of exposure to hydrodynamic stress, the cell retention and cell cytoskeleton reorganization on the patterned surfaces was improved when compared to untreated or random treated surfaces. These results indicate that micropatterned surfaces lead to improved rates of PTFE endothelialization with higher resistance to hydrodynamic stress., (Copyright © 2012 Wiley Periodicals, Inc.)

Introduction: Varicose veins affect one-third of the adult population in western countries, but their pathogenesis is incompletely characterized. One of the most controversial issues is the role of inflammation. It is well known that inflammation involves an increased expression/activity of inflammatory mediators., Objective: The aim of this study was to investigate the presence or absence of mediators of inflammation in varicose as compared to healthy veins., Methods and Results: Using immunohistofluorescence on varicose and healthy veins, we investigated the presence of inflammatory cells. They were not detectable. Venous wall C-reactive protein (CRP), fibrinogen (EIA) and pentraxin-3 (Western blot) content were measured. CRP was significantly lower in varicose veins, but no difference was found for fibrinogen or pentraxin-3 between varicose and healthy veins. No difference was observed for enzymes involved in inflammation and responsible for arachidonic acid metabolism such as the acute phase reactant secreted phospholipase A₂-IIA and cyclooxygenase-2, as determined in varicose and healthy veins by Western blot and real-time qRT-PCR., Conclusions: Our experiments demonstrate no increase in the presence of mediators of inflammation in varicose as compared to healthy veins, suggesting that inflammation may not be an important contributor to the pathogenesis of varicose veins.

Introduction: The ultrastructure of venous valves and walls in chronic venous disease was investigated., Methods: Consecutive patients were categorised into one of three groups (group A: patients with C1 venous disease in accordance with CEAP (Clinical severity, Etiology, Anatomy, Pathophysiology); group B: C2 and C3; group C: C4, C5 and C6). The terminal or preterminal valve and adjacent vessel wall was harvested from the great saphenous vein. Sections were examined with a transmission electron microscope. The volumes of elastin and of collagen per unit surface area of valve were assessed, as well as the surface endothelium of valve and vessel wall., Results: The study population consisted of 17 patients. The elastin ratio was analysed by means of stereology. Mean values were: in group A, 0.45 μm3/m2; in group B, 0.67 μm3/m2; in group C, 0.97 μm3/m2. The ratio was similar for collagen (A, 15.7 μm3/m2; B, 26.8 μm3/m2; C, 30.1 μm3/m2). Surface analysis of the valve endothelium and the adjacent vessel wall endothelium showed a trend towards increasing damage with more severe disease., Conclusions: With progression of venous disease, the valve elastin content, assessed morphologically, seems to increase, and the endothelium of the venous valve and the vein wall tend to show more damage.

Venous valves play a crucial role in blood circulation, promoting the one-way movement of blood from superficial and deep veins towards the heart. By preventing retrograde flow, venous valves spare capillaries and venules from being subjected to damaging elevations in pressure, especially during skeletal muscle contraction. Pathologically, valvular incompetence or absence of valves are common features of venous disorders such as chronic venous insufficiency and varicose veins. The underlying causes of these conditions are not well understood, but congenital venous valve aplasia or agenesis may play a role in some cases. Despite progress in the study of cardiac and lymphatic valve morphogenesis, the molecular mechanisms controlling the development and maintenance of venous valves remain poorly understood. Here, we show that in valved veins of the mouse, three gap junction proteins (Connexins, Cxs), Cx37, Cx43, and Cx47, are expressed exclusively in the valves in a highly polarized fashion, with Cx43 on the upstream side of the valve leaflet and Cx37 on the downstream side. Surprisingly, Cx43 expression is strongly induced in the non-valve venous endothelium in superficial veins following wounding of the overlying skin. Moreover, we show that in Cx37-deficient mice, venous valves are entirely absent. Thus, Cx37, a protein involved in cell-cell communication, is one of only a few proteins identified so far as critical for the development or maintenance of venous valves. Because Cxs are necessary for the development of valves in lymphatic vessels as well, our results support the notion of common molecular pathways controlling valve development in veins and lymphatic vessels., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Advanced glycation end products (AGEs) seem to be involved in aging as well as in the development of cardiovascular diseases. During aging, AGEs accumulate in extracellular matrix proteins like collagen and contribute to vessel stiffness. Whether non-invasive measurement of AGE accumulation in the skin may reflect vessel function and vessel protein modification is unknown. Herein we set out to analyze the AGE-modifications in the collagens extracted from residual bypass graft material, the skin autofluorescence reflecting the accumulation of AGEs in the body as well as the pulse wave velocity reflecting vessel stiffness. Collagen types I and III (pepsin digestible collagen fraction) were isolated from the veins of 52 patients by proteolysis. The residual collagen fraction was further extracted by collagenase digestion. Collagen was quantified by hydroxyproline assay and AGEs by the AGE intrinsic fluorescence. Skin autofluorescence was measured with an autofluorescence reader; pulse wave velocity with the VICORDER. The collagen AGE autofluorescence in patient vein graft material increased with patient age. The pepsin digestible collagen fraction was significantly less modified in comparison to the collagenase digestible fraction. Decreasing amounts of extracted collagenase digestible collagen correspond with increasing AGE autofluorescence. Skin autofluorescence and vessel stiffness were significantly linked to the AGE autofluorescence of the collagenase digestible collagen fraction from graft material. In conclusion we have found that skin autofluorescence and pulse wave velocity as non-invasive parameters significantly correlate with the AGE contained in graft material and therefore are strong predictors of vessel AGE modifications in patients with coronary heart disease. Whether the analysis of the skin autofluorescence leads to an improvement of the risk stratification in patients suffering from cardiovascular disease has to be further tested., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Introduction: Migration of the smooth muscle cells (SMCs) to the tunica media in the saphenous vein (SV) transplants is facilitated by matrix metalloproteinases (MMPs). The aim of this study was to identify any associations between expression of MMP-2 or endogenous tissue inhibitors (TIMP-2 and TIMP-3) in the SV segments and late failure of the SV grafts., Methods: Two hundred consecutive patients with a mean age of 63.1 ± 8.9 years who underwent primary isolated venous CABG were examined. Patients were retrospectively split into two subgroups, with the SV graft disease (SVGD (+); n = 47) or without it (SVGD (-); n = 153). In the SV segments, immunohistochemical analysis of the expression of the MMP-2, TIMP-2, and -3 was performed., Results: In the SVGD (+) patients, tissue expression of MMP-2 was stronger, whereas that of both TIMPs was weaker than in the SVGD (-) patients. In majority of the SV segments obtained from the SVGD (-) individuals, a balance in MMP and TIMP expressions was found, whereas an upregulation of MMP-2 expression was usually noted in the SVGD (+) subjects., Conclusion: The strong expression of MMP-2 accompanied by reduced immunostaining of both TIMPs is associated with the development of the SV graft disease and unfavorable CABG outcomes.

Objective: Several studies have addressed the optimal storage conditions for vascular grafts during surgery. The results remain contradictionary. This may be attributed to the fact, that the various vascular beds have a different sensitivity to storage. We analyzed the impact of storage in isotonic saline solution (NaCl) or heparinized blood the vascular functions of human saphenous vein grafts. Special care was taken to choose storage conditions which are relevant for intraoperative storage of a saphenous vein graft in a setting of coronary artery bypass grafting with vein and internal mammary artery as grafts., Methods: Intraoperatively isolated V. saphena-segments (n = 36) were stored in NaCl or heparinized blood for approximately 30 minutes at room temperature. Subsequently, the segments were examined in a Mulvany-myograph. Following preconstriction with norepinephrine, concentration-relaxation curves were assessed for bradykinin and sodium-nitroprusside to assess developed vessel-wall tension as well as endothelium- and smooth-muscle-cell dependent vasorelaxation. The availability of adenosintriphosphate (energy charge) was determined based on liquid chromatography measurements of nucleotide tissue levels., Results: Mean storage time was 27.4 ± 2.4 min in NaCl- and 26.3 ± 2.7 min in blood-group, respectively. After this period, receptor-dependent and-independent maximum of developed vessel wall tension was significantly reduced in NaCl-group (p = 0.05 and p = 0.045, respectively). Furthermore, the energy charge was significantly (p = 0.046) better preserved after blood storage (74 ± 1%) in comparison to NaCl-group (68 ± 2%). Endothelium-induced vasodilatation in response to bradykinin reached only 12.3 ± 2.5% in NaCl-group, but 19.3 ± 5.2% in blood-group (p = 0.033). Alike, EC50-concentration of bradykinin for half-maximal relaxation was significantly lower in blood- than in NaCl-group (log EC50 -7.08 ± 0.3 and -5.91 ± 0.4; respectively; p = 0.046). Endothelium-independent smooth muscle relaxation in response to sodium-nitroprusside was not different between both groups., Conclusion: Heparinized blood better preserves vascular contractile and endothelial functions of the saphenous vein graft. Storage in NaCl rapidly compromises vascular functions and impaires cellular energy. NaCl should no longer be recommended for intraoperative storage of harvested V. saphena grafts.

Chronic venous insufficiency (CVI) is a common disease characterized by structural and functional abnormalities of the venous system. Until recently, the pathogenesis of CVI remains largely unknown. MicroRNAs (miRNAs) are a family of endogenous small non-coding RNAs emerged as post-transcriptional gene repressors and play essential roles in diverse pathological processes including vascular disease. However, their roles in CVI have not been elucidated. In this study, we employed oligonucleotide microarrays to perform a genome-wide miRNAs profiling in the great saphenous vein (GSV) tissues of patients with CVI. Our results revealed a total of 14 miRNAs that are expressed differentially in GSV tissues. Among them nine miRNAs were found significantly up-regulated, while five miRNAs were down-regulated significantly. Real-time RT-PCR verified statistically consistent expression of three selected miRNAs (miR-34a, miR-155 and miR-202) with microarrays analysis. These three miRNAs, which were described as crucial regulators in many biological processes and vascular diseases, might also play important roles in CVI. Functional annotation of target genes of differentially expressed miRNAs via bioinformatics approaches revealed that these predicted targets were significantly enriched and involved in several key signaling pathways important for CVI, including mitogen-activated protein kinase pathways, pathways in cancer, apoptosis, and cell cycle, and p53 signaling pathways. In summary, miRNAs might involve in multiple signaling pathways contributing to the pathological processes of CVI. These data may provide fundamental insights into the molecular basis of CVI, which may aid in designing novel approaches for prevention and treatment of this complex disease.

The saphenous vein remains the most widely used conduit for peripheral and coronary revascularization despite a high rate of vein graft failure. The most common cause of vein graft failure is intimal hyperplasia. No agents have been proven to be successful for the prevention of intimal hyperplasia in human subjects. The renin-angiotensin system is essential in the regulation of vascular tone and blood pressure in physiologic conditions. However, this system mediates cardiovascular remodeling in pathophysiologic states. Angiotensin II is becoming increasingly recognized as a potential mediator of intimal hyperplasia. Drugs modulating the renin-angiotensin system include angiotensin-converting enzyme inhibitors and angiotensin receptor blockers. These drugs are powerful inhibitors of atherosclerosis and cardiovascular remodeling, and they are first-line agents for management of several medical conditions based on class I evidence that they delay progression of cardiovascular disease and improve survival. Several experimental models have demonstrated that these agents are capable of inhibiting intimal hyperplasia. However, there are no data supporting their role in prevention of intimal hyperplasia in patients with vein grafts. This review summarizes the physiology of the renin-angiotensin system, the role of angiotensin II in the pathogenesis of cardiovascular remodeling, the medical indications for these agents, and the experimental data supporting an important role of the renin-angiotensin system in the pathogenesis of intimal hyperplasia., (Copyright © 2012 Annals of Vascular Surgery Inc. All rights reserved.)

Objective: Assessment of thrombus inflammation in vivo could provide new insights into deep vein thrombosis (DVT) resolution. Here, we develop and evaluate 2 integrated fluorescence molecular-structural imaging strategies to quantify DVT-related inflammation and architecture and to assess the effect of thrombus inflammation on subsequent DVT resolution in vivo., Methods and Results: Murine DVT were created with topical 5% FeCl(3) application to thigh or jugular veins (n=35). On day 3, mice received macrophage and matrix metalloproteinase activity fluorescence imaging agents. On day 4, integrated assessment of DVT inflammation and architecture was performed using confocal fluorescence intravital microscopy. Day 4 analyses showed robust relationships among in vivo thrombus macrophages, matrix metalloproteinase activity, and fluorescein isothiocyanate-dextran deposition (r>0.70; P<0.01). In a serial 2-time point study, mice with DVT underwent intravital microscopy at day 4 and day 6. Analyses revealed that the intensity of thrombus inflammation at day 4 predicted the magnitude of DVT resolution at day 6 (P<0.05). In a second approach, noninvasive fluorescence molecular tomography-computed tomography was used and detected macrophages within jugular DVT (P<0.05 versus sham controls)., Conclusions: Integrated fluorescence molecular-structural imaging demonstrates that the DVT-induced inflammatory response can be readily assessed in vivo and can inform the magnitude of thrombus resolution.

Aims: We have compared the endothelin receptor subtype affinity (K(D)) and selectivity of four structural classes of antagonists (peptide, sulphonamide-based, carboxylic acid-based, myceric acid-based) in human cardiovascular tissues to determine whether these are predicted by values reported for human cloned receptors. Additionally, affinities (K(B)) for these antagonists, determined in ET-1-mediated vasoconstriction assays in human blood vessels, were used to identify discrepancies between K(B) and K(D) determined in the same tissues., Main Methods: Competition binding experiments were carried out in sections of human left ventricle, coronary artery and homogenates of saphenous vein to determine K(D) values for structurally different ET(A)-selective (FR139317, BMS 182874, S97-139, sitaxentan, ambrisentan) and mixed (PD142893, Ro462005, bosentan, L-749329, SB209670) antagonists. Schild-derived values of antagonist affinity were obtained in vascular functional studies., Key Findings: When compared with previously reported data in human cloned endothelin receptors, those antagonists reported to be ET(A)-selective exhibited even greater ET(A) selectivity in human ventricle (BMS 182874, sitaxentan, ambrisentan) that expressed both receptor subtypes. Those antagonists reported to have <100 fold selectivity in cloned receptors (PD142893, Ro-462005, bosentan, SB209670, L-749329) did not distinguish between receptor subtypes in human left ventricle. For antagonists where we determined affinity in vascular functional and binding assays (Ro462005, bosentan, BMS 182874, L-749329, SB209670) there was no correlation between the degree of discrepancy in K(B) and K(D) and structural class., Significance: For an antagonist to retain ET(A)-selectivity in vivo it may be necessary to identify those compounds that have at least 1000 fold ET(A):ET(B) selectivity in in vitro assays., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Aims: Endothelin-1 (ET-1) has been shown to increase endothelial superoxide (O(2)(-)) production in experimental animal models. It is unclear whether ET-1 increases O(2)(-) production in humans. We sought to elucidate whether ET-1 increases O(2)(-) production in human vessels and to identify the mechanism behind this effect., Main Methods: Segments of internal mammary artery (IMA) and human saphenous vein (HSV) were harvested from 90 patients undergoing elective coronary artery bypass graft surgery. Paired vessel rings were incubated in the presence and absence of ET-1 (10(-10)M), the ET(A) receptor antagonist BQ123 alone, or in combination with the ET(B) receptor antagonist BQ788 (dual BQ) and known inhibitors of sources of O(2)(-) and further analysed for O(2)(-) production using lucigenin-enhanced chemiluminescence and DHE fluorescence., Key Findings: ET-1 increased O(2)(-) production in both IMA (2.6 ± 1.5 vs. 1.4 ± 0.8 relative light units/s/mg tissue (RLU); n=33; p < 0.0001) and HSV (1.4 ± 0.8 vs. 1.1 ± 0.6 RLU; n=24; p<0.05). The increase in O(2)(-)production induced by ET-1 in IMA was inhibited by co-incubation with dual BQ (p < 0.05; n=15) and BQ123 (p<0.05; n = 17). Of known O(2)(-) inhibitors, only incubation with Tiron and diphenyleneiodonium resulted in a significant reduction in ET-mediated O(2)(-) production., Significance: ET-1 increases O(2)(-) production especially in human arteries and less so in veins from patients with coronary artery disease via a receptor-dependent pathway involving a flavin dependent enzyme which is likely to be NADPH oxidase. Production of O(2)(-) may be an important factor underlying the negative effects of ET-1 on vascular function such as impairment of endothelium-dependent vasodilatation and pro-inflammatory effects., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Impaired venous drainage of the lower extremities determines a cascade of pathologic events leading to chronic venous disease (CVD). It is believed that the one cause of CVD is red blood cell extravasation and local iron overload that could generate free radicals and iron-dependent inflammation. The aim of this study was to investigate the relationship between: the intracellular iron deposits in varicose veins and tissue oxidative state measured by: the Proton Induced X-ray Emission Spectroscopy (Fe(PIXE)), (tSOD), (tGPx), (tTBARs) and (boxDNA). Patients with diagnosed CVD were qualified for surgical procedure. Entire trunk of the great saphenous vein (GSV) was extracted. Part located near medial ankle was considered competent (C) in duplex ultrasonography (USG) examination. The incompetent (I) part was extracted from GSV where USG showed incompetent valves and massive venous reflux. The difference between local tFe(PIXE), tTBARS, boxDNA, tGPx, tSOD in incompetent and competent part of vein tissue was statistically significant. Intima/media ratio directly correlated with Fe(PIXE) C/I concentration. Iron deposition in competent vs incompetent part of vein was also related to the oxidative stress parameters (boxDNA). The findings from this pilot study suggest that Fe(PIXE) measurement may be useful for explaining the progression of chronic venous disease., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: Doxycycline and micronized purified flavonoid fraction (MPFF) modulate vein wall remodeling that may be associated with hypoxia in varicose veins (VVs), vein graft stenosis, and deep venous thrombosis. We recently reported that in vitro exposure of non-VV (NVVs) and VVs to hypoxic conditions activates the hypoxia-inducible factor (HIF) pathway. This study investigated the in vitro effects of doxycycline and MPFF on the HIF pathway in hypoxic NVVs and VVs., Methods: Six NVVs and six VVs obtained from surgery were used to prepare vein organ cultures, which were exposed to hypoxia (1% O(2)), with and without MPFF (10(-5) mol/L) or doxycycline (5 μg/mL) for 16 hours. The veins were analyzed for HIF-1α, HIF-2α, and their target gene expression, with real-time polymerase chain reaction and Western blot. The differences between gene expressions were tested with one-way analysis of variance with repeated measures, followed by the Dunnett test for multiple comparisons. P < .05 was considered significant., Results: Treatment of NVV organ cultures exposed to hypoxia with doxycycline or MPFF did not significantly alter the expression of HIF-1α and HIF-2α messenger (m)RNA and protein compared with untreated. Doxycycline also did not significantly affect the expression of HIF-1α and HIF-2α mRNA and protein in VVs exposed to hypoxia compared with untreated VVs. However, MPFF significantly reduced the expression of HIF-1α but not HIF-2α mRNA in VVs exposed to hypoxia compared with untreated VVs. Interestingly, the reduction of the expression of HIF-1α mRNA in VVs by MPFF was not reflected at the protein level. The mRNA expression of HIF target genes, namely glucose transporter-1, carbonic anhydrase-9, vascular endothelial growth factor, B-cell lymphoma 2/adenovirus E1B 19-kDa protein-interacting protein 3, prolyl hydroxylase domain-2, and prolyl hydroxylase domain-3, was not significantly altered in NVVs and VVs exposed to hypoxia and treated with doxycycline or MPFF compared with those untreated., Conclusions: Doxycycline and MPFF at a concentration corresponding to a therapeutic dose do not alter the activation of the HIF pathway in NVV and VV organ cultures exposed to hypoxia. Our findings suggest vein wall remodeling actions in NVVs and VVs are likely not HIF-dependent., (Copyright © 2012 Society for Vascular Surgery. Published by Mosby, Inc. All rights reserved.)