The expression of the α1C-adrenoceptor subtype in human and rabbit blood vessels has been analyzed using the reverse transcriptase/polymerase chain reaction technique (RT/PCR). The 20 bp primers employed were designed from the bovine α1C-adrenoceptor and flank a least conserved region - the putative third cytoplasmic loop. RT/PCR products generated from rabbit and human brain mRNA both had 93% homology to the bovine α1C-adrenoceptor and were used as species and subtype specific probes in Southern blot analysis of vascular RT/PCR products. Poly A+ RNA was purified from the human saphenous vein and rabbit aorta, renal, pulmonary and central ear arteries and amplified by RT/PCR. Size analysis by agarose gel electrophoresis, together with Southern hybridization of the resulting cDNA products confirm the expression of the α1C-adrenoceptor in these vessels.

No / The effects of a conjugated linoleic acid (CLA) mixture of single isomers (50:50, w/w, cis9,trans11:trans10,cis12) and the individual isomers on (a) the production of resting and calcium ionophore stimulated 14C-eicosanoids and (b) the incorporation of 14C-arachidonic acid (AA) into membrane phospholipids of human saphenous vein endothelial cells were investigated. The CLA mixture and the individual isomers were found to inhibit resting production of 14C-prostaglandin F2a by 50, 43 and 40%, respectively. A dose dependent inhibition of stimulated 14C-prostaglandins was observed with the CLA mixture (IC50 100 ¿M). The cis9,trans11 and trans10,cis12 (50 ¿M) isomers individually inhibited the overall production of stimulated 14C-prostaglandins (between 35 and 55% and 23 and 42%, respectively). When tested at a high concentration (100 ¿M), cis9,trans11 was found to inhibit eicosanoid production in contrast to trans10,cis12 that caused stimulation. The overall degree of 14C-AA incorporation into membrane phospholipids of the CLA (mixture and individual isomers) treated cells was found to be lower than that of control cells and the cis9,trans11 isomer was found to increase the incorporation of 14C-AA into phosphatidylcholine. Docosahexaenoic acid, eicosapentaenoic acid and linoleic acid did not alter the overall degree of incorporation of 14C-AA. The results of this study suggest that both isomers inhibit eicosanoid production, and although trans10,cis12 exhibits pro-inflammatory activity at high concentrations, the CLA mixture maintains its beneficial anti-inflammatory action that contributes to its anti-carcinogenic and anti-atherogenic properties.

Yes / 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.

Yes / 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.

Yes / Background/purpose Coronary heart disease is the leading cause of morbidity in patients with type 2 diabetes mellitus (T2DM), frequently resulting in a requirement for coronary revascularization using the internal mammary artery (IMA) or saphenous vein (SV). Patency rates of SV grafts are inferior to IMA and further impaired by T2DM whilst IMA patencies appear similar in both populations. Smooth muscle cells (SMC) play a pivotal role in graft integration; we therefore examined the phenotype and proliferative function of IMA- and SV-SMC isolated from non-diabetic (ND) patients or those diagnosed with T2DM. Methods/materials SMC were cultured from fragments of SV or IMA. Morphology was analyzed under light microscopy (spread cell area measurements) and confocal microscopy (F-actin staining). Proliferation was analyzed by cell counting. Levels of RhoA mRNA, protein and activity were measured by real-time RT-PCR, western blotting and G-LISA respectively. Results IMA-SMC from T2DM and ND patients were indistinguishable in both morphology and function. By comparison, SV-SMC from T2DM patients exhibited significantly larger spread cell areas (1.5-fold increase, P < 0.05), truncated F-actin fibers and reduced proliferation (33% reduction, P < 0.05). Furthermore, lower expression and activity of RhoA were observed in SV-SMC of T2DM patients (37% reduction in expression, P < 0.05 and 43% reduction in activity, P < 0.01). Conclusions IMA-SMC appear impervious to phenotypic modulation by T2DM. In contrast, SV-SMC from T2DM patients exhibit phenotypic and functional changes accompanied by reduced RhoA activity. These aberrancies may be epigenetic in nature, compromising SMC plasticity and SV graft adaptation in T2DM patients.

Yes / 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.

Yes / Cardiovascular disease is a leading cause of morbidity and mortality. Smooth muscle cells (SMC) comprising the vascular wall can switch phenotypes from contractile to synthetic, which can promote the development of aberrant remodelling and intimal hyperplasia (IH). MicroRNA‐21 (miR‐21) is a short, non‐coding RNA that has been implicated in cardiovascular diseases including proliferative vascular disease and ischaemic heart disease. However, its involvement in the complex development of atherosclerosis has yet to be ascertained. Smooth muscle cells (SMC) were isolated from human saphenous veins (SV). miR‐21 was over‐expressed and the impact of this on morphology, proliferation, gene and protein expression related to synthetic SMC phenotypes monitored. Over‐expression of miR‐21 increased the spread cell area and proliferative capacity of SV‐SMC and expression of MMP‐1, whilst reducing RECK protein, indicating a switch to the synthetic phenotype. Furthermore, platelet‐derived growth factor BB (PDGF‐BB; a growth factor implicated in vasculoproliferative conditions) was able to induce miR‐21 expression via the PI3K and ERK signalling pathways. This study has revealed a mechanism whereby PDGF‐BB induces expression of miR‐21 in SV‐SMC, subsequently driving conversion to a synthetic SMC phenotype, propagating the development of IH. Thus, these signaling pathways may be attractive therapeutic targets to minimise progression of the disease. / King Saud University; College of Medicine , Riyadh, Saudi Arabia

Yes / Increased cardiovascular morbidity and mortality in individuals with type 2 diabetes (T2DM) is a significant clinical problem. Despite advancements in achieving good glycaemic control, this patient population remains susceptible to macrovascular complications. We previously discovered that vascular smooth muscle cells (SMC) cultured from T2DM patients exhibit persistent phenotypic aberrancies distinct from those of individuals without a diagnosis of T2DM. Notably, persistently elevated expression levels of microRNA-145 co-exist with characteristics consistent with aging, DNA damage and senescence. We hypothesised that increased expression of microRNA-145 plays a functional role in DNA damage signalling and subsequent cellular senescence specifically in SMC cultured from the vasculature of T2DM patients. In this study, markers of DNA damage and senescence were unambiguously and permanently elevated in native T2DM versus non-diabetic (ND)-SMC. Exposure of ND cells to the DNA-damaging agent etoposide inflicted a senescent phenotype, increased expression of apical kinases of the DNA damage pathway and elevated expression levels of microRNA-145. Overexpression of microRNA-145 in ND-SMC revealed evidence of functional links between them; notably increased secretion of senescence-associated cytokines and chronic activation of stress-activated intracellular signalling pathways, particularly the mitogen-activated protein kinase, p38a. Exposure to conditioned media from microRNA-145 overexpressing cells resulted in chronic p38a signalling in naïve cells, evidencing a paracrine induction and reinforcement of cell senescence. We conclude that targeting of microRNA-145 may provide a route to novel interventions to eliminate DNA-damaged and senescent cells in the vasculature and to this end further detailed studies are warranted.

A novel technique for saphenous vein (SV) graft harvesting, the No-touch technique (NT), has been developed at the Dept. of Cardiovascular surgery, Örebro University hospital. With NT the SV is harvested with a pedicle of surrounding tissue. This avoids graft spasm and eliminates the need for distension. The surrounding tissue acts as a structural support and is a rich source of vaso-dilating agents. A randomized controlled trial (RCT) has shown a significantly higher patency rate for NT SV grafts compared to SV grafts harvested with conventional technique (CT). This thesis evaluates some of the properties of the surrounding tissue and compares patency rates between NT SV and radial artery (RA) grafts and patency rates for internal thoracic artery (ITA) grafts harvested with and without surrounding tissue. Paper I investigated vasa vasorum (VV) in SV grafts and showed that the NT preserves an intact VV whereas CT does not. This could be one of the mechanisms underlying the improved patency for NT SV grafts. Paper II evaluated VV and associated nitric oxide (NO) in SV and arterial grafts. SV grafts showed a higher number and larger VV, which correlated with NO production, compared to arterial grafts. NT SV grafts showed higher activity for e-NOS compared to CT SV grafts. Paper III is a RCT comparing patency rates between NT SV and RA grafts, three years after surgery, showing a significantly higher patency rate for NT SV grafts. Paper IV is a RCT comparing patency rates for ITA graft harvested with and without surrounding tissue and did not show any difference between graft preparations. In conclusion, the NT for SV graft harvesting preserves an intact vasa vasorum and associated NO production. NT SV grafts show a higher patency rate than RA grafts. Harvesting of ITA with or without surrounding tissue does not affect patency rate.

Introdução: A termoablação vem substituindo a cirurgia convencional no tratamento cirúrgico do refluxo da veia safena magna em pacientes portadores de varizes dos membros inferiores. Contudo, a termoablação apresenta elevados custos. A Eletrocoagulação endovenosa pode, seletivamente e de forma segura, causar necrose da parede da veia safena magna, mas seus resultados clínicos nunca foram estudados previamente. O objetivo deste estudo é comparar a Eletrocoagulação e a Radiofrequência no tratamento da insuficiência da veia safena magna, considerando eficácia, complicações e impacto na qualidade de vida. Métodos: Trata-se de um ensaio clinico prospectivo, randomizado e duplo-cego. Os pacientes portadores de varizes de membros inferiores e refluxo de veia safena magna ao Eco Doppler colorido foram randomizados em dois grupos de tratamento: Eletrocoagulação ou Radiofrequência. O seguimento dos pacientes ocorreu após uma semana, três meses e seis meses do procedimento. O desfecho primário foi considerado como oclusão da veia safena magna ao Eco Doppler colorido e o desfecho secundário, como a taxa de complicações e a melhora na qualidade de vida, mediante pontuação do Escore de Gravidade Clínica Venosa e Questionário Aberdeen para Veias Varicosas. Resultados: Foram incluídos no estudo 57 pacientes, totalizando 85 veias safenas magnas tratadas, sendo que 43 foram submetidas à Radiofrequência e 42, à Eletrocoagulação. Não houve diferença estatisticamente significante entre os grupos, no pré-operatório, em relação à idade (P = 0,264), gênero (P = 0,612), Escore de Gravidade Clínica Venosa (P = 0,125), Questionário Aberdeen para Veias Varicosas (P = 0,054), diâmetro (P = 0,880) e profundidade (P = 0,763) da veia safena magna tratada. No intraoperatório, imediatamente após a realização da termoablação, todas as veias safenas magnas submetidas à eletrocoagulação apresentaram ausência de fluxo no segmento tratado e incompressibilidade, enquanto 12 membros ainda exibiam fluxo em sua veia safena magna tratada (P < 0,001) e 9 veias apresentavam-se compressíveis (P < 0,001), quando submetidas à Radiofrequência. A principal complicação pós-operatória encontrada foi a parestesia, não havendo significância estatística quanto à sua presença entre os grupos (P = 0,320). O tempo de retorno às atividades rotineiras foi menor no grupo da Eletrocoagulação em relação ao grupo da Radiofrequência (P = 0,026). Não houve diferença entre os grupos em relação à taxa de oclusão da veia safena magna no seguimento de 3 meses (P = 0,157) e 6 meses (P = 0,157), bem como na melhora da pontuação do Questionário Aberdeen para veias varicosas após 3 meses (P = 0,786) e 6 meses (P = 0,401) e na melhora da pontuação do Escore de Gravidade Clínica Venosa após 3 meses (P = 0,324) e 6 meses (P = 0,367). Conclusões: A Eletrocoagulação revelou-se um método eficaz para ablação da veia safena magna, com taxa de oclusão venosa, ocorrência de complicações e impacto na qualidade de vida semelhantes àqueles encontrados na Radiofrequência. / Background: Thermoablation has been replacing conventional surgery in the surgical treatment of great saphenous vein reflux in patients with lower limb varicose veins; however, thermoablation is expensive. Intravenous Electrocoagulation may, selectively and safely, cause necrosis of the great saphenous vein wall, but the clinical results have never been studied. The objective of this study was to compare Electrocoagulation and Radiofrequency in the treatment of great saphenous vein insufficiency, considering efficacy, complications and effect on quality of life. Methods: This is a prospective, double-blind, randomized clinical trial. Patients with lower limb varicose veins and great saphenous vein reflux confirmed by duplex ultrasonography were randomized into two treatment groups: Electrocoagulation or Radiofrequency. Patients were followed-up 1 week, 3 months and 6 months after the procedure. Occlusion of the great saphenous vein confirmed by duplex ultrasonography was considered the primary outcome and the rate of complications and improvement in quality of life, using the Aberdeen Varicose Veins Questionnaire and Venous Clinical Severity Score, were the secondary outcome. Results: Fifty-seven patients were included, with a total of 85 treated great saphenous veins; 43 were treated with Radiofrequency and 42 with Electrocoagulation. There was no statistically significant difference between the groups regarding age (P = 0,264), sex (P = 0,612), Venous Clinical Severity Score (P = 0,125), Aberdeen Varicose Veins Questionnaire score (P = 0,054), diameter (P = 0,880) and depth (P = 0,763) of the treated great saphenous vein. In the intraoperative period, immediately after thermoablation, all great saphenous veins treated with Electrocoagulation presented no flow in the treated segment and incompressibility, while 12 limbs still had flow in treated great saphenous vein (P < 0,001), and 9 veins showed compressibility (P < 0,001) when treated with Radiofrequency. The main postoperative complication was paresthesia; however there was no statistical significance between the groups (P = 0,320) regarding its presence. Time to return to routine activities was lower in the Electrocoagulation group than in the Radiofrequency group (P = 0,026). There was no difference between the groups at the 3-month (P = 0,157) and 6-month (P = 0,157) follow-ups regarding occlusion of the great saphenous vein and in improvement of Aberdeen Varicose Veins Questionnaire score after 3 months (P = 0,786) and 6 months (P = 0,401) and Venous Clinical Severity Score after 3 months (P = 0,324) and 6 months (P = 0,367). Conclusions: Electrocoagulation has been shown to be an effective method for ablation of the great saphenous vein, with venous occlusion rate, occurrence of complications, and effect on the quality of life similar to that with Radiofrequency.