"Affimer" synthetic protein scaffolds block oxidized LDL binding to the LOX-1 scavenger receptor and inhibit ERK1/2 activation.

In multicellular organisms, a variety of lipid-protein particles control the systemic flow of triacylglycerides, cholesterol, and fatty acids between cells in different tissues. The chemical modification by oxidation of these particles can trigger pathological responses, mediated by a group of membrane proteins termed scavenger receptors. The lectin-like oxidized low-density lipoprotein (LOX-1) scavenger receptor binds to oxidized low-density lipoprotein (oxLDL) and mediates both signaling and trafficking outcomes. Here, we identified five synthetic proteins termed Affimers from a phage display library, each capable of binding recombinant LOX-1 extracellular (oxLDL-binding) domain with high specificity. These Affimers, based on a phytocystatin scaffold with loop regions of variable sequence, were able to bind to the plasma membrane of HEK293T cells exclusively when human LOX-1 was expressed. Binding and uptake of fluorescently labeled oxLDL by the LOX-1-expressing cell model was inhibited with subnanomolar potency by all 5 Affimers. ERK1/2 activation, stimulated by oxLDL binding to LOX-1, was also significantly inhibited (p < 0.01) by preincubation with LOX-1-specific Affimers, but these Affimers had no direct agonistic effect. Molecular modeling indicated that the LOX-1-specific Affimers bound predominantly via their variable loop regions to the surface of the LOX-1 lectin-like domain that contains a distinctive arrangement of arginine residues previously implicated in oxLDL binding, involving interactions with both subunits of the native, stable scavenger receptor homodimer. These data provide a new class of synthetic tools to probe and potentially modulate the oxLDL/LOX-1 interaction that plays an important role in vascular disease.

The E2 ubiquitin-conjugating enzymes UBE2D1 and UBE2D2 regulate VEGFR2 dynamics and endothelial function.

Vascular endothelial growth factor receptor 2 (VEGFR2, encoded by KDR) regulates endothelial function and angiogenesis. VEGFR2 undergoes ubiquitination that programs this receptor for trafficking and proteolysis, but the ubiquitin-modifying enzymes involved are ill-defined. Herein, we used a reverse genetics screen for the human E2 family of ubiquitin-conjugating enzymes to identify gene products that regulate VEGFR2 ubiquitination and proteolysis. We found that depletion of either UBE2D1 or UBE2D2 in endothelial cells caused a rise in steady-state VEGFR2 levels. This rise in plasma membrane VEGFR2 levels impacted on VEGF-A-stimulated signalling, with increased activation of canonical MAPK, phospholipase Cγ1 and Akt pathways. Analysis of biosynthetic VEGFR2 is consistent with a role for UBE2D enzymes in influencing plasma membrane VEGFR2 levels. Cell-surface-specific biotinylation and recycling studies showed an increase in VEGFR2 recycling to the plasma membrane upon reduction in UBE2D levels. Depletion of either UBE2D1 or UBE2D2 stimulated endothelial tubulogenesis, which is consistent with increased VEGFR2 plasma membrane levels promoting the cellular response to exogenous VEGF-A. Our studies identify a key role for UBE2D1 and UBE2D2 in regulating VEGFR2 function in angiogenesis.

VEGFR endocytosis: Implications for angiogenesis.

The binding of vascular endothelial growth factor (VEGF) superfamily to VEGF receptor tyrosine kinases (VEGFRs) and co-receptors regulates vasculogenesis, angiogenesis and lymphangiogenesis. A recurring theme is that dysfunction in VEGF signaling promotes pathological angiogenesis, an important feature of cancer and pro-inflammatory disease states. Endocytosis of basal (resting) or activated VEGFRs facilitates signal attenuation and endothelial quiescence. However, increasing evidence suggest that activated VEGFRs can continue to signal from intracellular compartments such as endosomes. In this chapter, we focus on the evolving link between VEGFR endocytosis, signaling and turnover and the implications for angiogenesis. There is much interest in how such understanding of VEGFR dynamics can be harnessed therapeutically for a wide range of human disease states.

Monitoring VEGF-Stimulated Calcium Ion Flux in Endothelial Cells.

The endothelial response to vascular endothelial growth factor A (VEGF-A) regulates many aspects of animal physiology in health and disease. Such VEGF-A-regulated phenomena include vasculogenesis, angiogenesis, tumor growth and progression. VEGF-A binding to receptor tyrosine kinases such as vascular endothelial growth factor receptor 2 (VEGFR2 ) activates multiple signal transduction pathways and changes in homeostasis, metabolism, gene expression, cell proliferation, migration, and survival. One such VEGF-A-regulated response is a rapid rise in cytosolic calcium ion levels which modulates different biochemical events and impacts on endothelial-specific responses. Here, we present a series of detailed and robust protocols for evaluating ligand-stimulated cytosolic calcium ion flux in endothelial cells. By monitoring an endogenous endothelial transcription factor (NFATc2 ) which displays calcium-sensitive redistribution, we can assess the relevance of cytosolic calcium to protein function. This protocol can be easily applied to both adherent and non-adherent cultured cells to evaluate calcium ion flux in response to exogenous stimuli such as VEGF-A.

A multicentre review comparing long term outcomes of endoscopic vein harvesting versus open vein harvesting for coronary artery bypass surgery.

Background: Utilisation of the Endoscopic Vein Harvesting (EVH) technique has been increasing for coronary artery bypass grafting (CABG) for the last two decades. Some surgeons remain concerned about the long-term patency of the long saphenous vein harvested endoscopically compared to traditional Open Vein Harvesting (OVH). The aim of this study was to perform a retrospective analysis of the outcomes between EVH and OVH from three UK centres with 10 years follow-up. Methods: 27,024 patients underwent CABG with long saphenous vein harvested by EVH (n=13,794) or OVH (n=13,230) in three UK centres between 2007 and 2019. Propensity modelling was used to calculate the Inverse Probability of Treatment Weights (IPTW). The primary endpoint was mortality from all causes and secondary endpoints were length of hospital stay, postoperative complications, and incidence of repeat coronary re-vascularisation for symptomatic patients. IPTW was used to balance the two intervention groups for baseline and preoperative co-morbidities. Results: Median follow-up time was 4.54 years for EVH and 6.00 years for OVH. Death from any cause occurred in 13.8% of the EVH group versus 20.8% in the OVH group over the follow-up period. The hazard ratio of death (EVH to OVH) was 0.823 (95% CI: 0.767, 0.884). Length of hospital stay was similar between the groups (p=0.86). Post-operative pulmonary complications were more common in EVH vs OVH (14.7% vs. 12.8%, p<0.001), but repeat coronary re-vascularisation was similar between the groups. Conclusions: This large retrospective multicentre analysis indicates that EVH has a lower risk of mortality compared with OVH during the follow-up period of the study. The observed benefits of EVH may outweigh the risks but should be considered on a case-by-case basis. We hope this review gives confidence to other cardiac centres that offering an EVH approach to conduit harvesting does not affect long term patient outcomes.

Use of keyhole vein removal technique has been increasing for coronary artery bypass surgery for last two decades. However, some surgeons remain worried about the quality and long-term effect of the vein tube removed using keyhole method compared to traditional Open Vein Harvesting (OVH). The aim of this study was to perform a retrospective analysis of the outcomes between keyhole and OVH from three UK centres with 10 years follow-up. In total, 27,024 patients underwent coronary artery bypass surgery with long saphenous vein harvested by either keyhole (n=13,794) or OVH (n=13,230) over a period of 2007­2019. The median follow-up time was 4.54 years for keyhole method and 6.00 years for OVH. Death from any cause occurred in 13.8% of the keyhole group versus 20.8% in the OVH group over the follow-up period. In conclusion, the keyhole surgery survival is not as bad as we hypothesised.

A post-preservation vascular flush removes significant populations of donor leukocytes prior to lung transplantation.

BACKGROUND: Donor leukocytes are intrinsically involved in acute lung allograft rejection, via self-presentation of donor antigens to recipient leukocytes. Therapeutic modalities to remove donor leukocytes are currently unavailable. We evaluated if a vascular flush immediately following preservation can be used for this purpose. METHODS: A post-preservation flush was performed with STEEN solution in n = 6 porcine lungs following static cold storage. The first 500 ml effluent from the left atrium was collected and an inflammatory profile performed. RESULTS: A total of 1.17 billion (±2.8 × 108) viable leukocytes were identified within the effluent. T cells were the dominant cell population, representing 82% of the total mobilised leukocytes, of which <0.01% were regulatory T cells. IL-18 was the most abundant cytokine, with a mean concentration of 84,216 pg (±153,552 pg). In addition, there was a mean concentration of 8819 ng (±4415) cell-free mitochondrial DNA. CONCLUSION: There is an immediate transfer of donor leukocytes, cytokines and damage-associated molecular patterns following reperfusion. Such a pro-inflammatory donor load may enhance alloantigen presentation and drive recipient alloimmune responses. A post-preservation flush may therefore be an effective method for reducing the immune burden of the donor lung prior to transplantation.

Non-ischemic Heart Preservation via Hypothermic Cardioplegic Perfusion Induces Immunodepletion of Donor Hearts Resulting in Diminished Graft Infiltration Following Transplantation.

Introduction: Many donor organs contain significant leukocyte reservoirs which upon transplantation activate recipient leukocytes to initiate acute rejection. We aimed to assess whether non-ischemic heart preservation via ex vivo perfusion promotes immunodepletion and alters the inflammatory status of the donor organ prior to transplantation. Methods: Isolated porcine hearts underwent ex vivo hypothermic, cardioplegic perfusion for 8 h. Leukocyte populations were quantified in left ventricle samples by flow cytometry. Cell-free DNA, cytokines, and chemokines were quantified in the perfusate. Tissue integrity was profiled by targeted proteomics and a histological assessment was performed. Heterotopic transplants comparing ex vivo hypothermic preservation and static cold storage were utilized to assess graft infiltration as a solid clinical endpoint. Results:Ex vivo perfusion significantly immunodepleted myocardial tissue. The perfusate displayed a selective, pro-inflammatory cytokine/chemokine pattern dominated by IFN-γ. The tissue molecular profile was improved following perfusion by diminished expression of nine pro-apoptotic and six ischemia-associated proteins. Histologically, no evidence of tissue damage was observed and cardiac troponin I was low throughout perfusion. Cell-free DNA was detected, the source of which may be necrotic/apoptotic leukocytes. Post-transplant graft infiltration was markedly reduced in terms of both leucocyte distribution and intensity of foci. Conclusions: These findings demonstrate that ex vivo perfusion significantly reduced donor heart immunogenicity via loss of resident leukocytes. Despite the pro-inflammatory cytokine pattern observed, a pro-survival and reduced ischemia-related profile was observed, indicating an improvement in graft viability by perfusion. Diminished graft infiltration was observed in perfused hearts compared with those preserved by static cold storage following 48 h of transplantation.

Receptor Tyrosine Kinase Ubiquitination and De-Ubiquitination in Signal Transduction and Receptor Trafficking.

Receptor tyrosine kinases (RTKs) are membrane-based sensors that enable rapid communication between cells and their environment. Evidence is now emerging that interdependent regulatory mechanisms, such as membrane trafficking, ubiquitination, proteolysis and gene expression, have substantial effects on RTK signal transduction and cellular responses. Different RTKs exhibit both basal and ligand-stimulated ubiquitination, linked to trafficking through different intracellular compartments including the secretory pathway, plasma membrane, endosomes and lysosomes. The ubiquitin ligase superfamily comprising the E1, E2 and E3 enzymes are increasingly implicated in this post-translational modification by adding mono- and polyubiquitin tags to RTKs. Conversely, removal of these ubiquitin tags by proteases called de-ubiquitinases (DUBs) enables RTK recycling for another round of ligand sensing and signal transduction. The endocytosis of basal and activated RTKs from the plasma membrane is closely linked to controlled proteolysis after trafficking and delivery to late endosomes and lysosomes. Proteolytic RTK fragments can also have the capacity to move to compartments such as the nucleus and regulate gene expression. Such mechanistic diversity now provides new opportunities for modulating RTK-regulated cellular responses in health and disease states.

The effect of 1.5 T cardiac magnetic resonance on human circulating leucocytes.

Aims: Investigators have proposed that cardiovascular magnetic resonance (CMR) should have restrictions similar to those of ionizing imaging techniques. We aimed to investigate the acute effect of 1.5 T CMR on leucocyte DNA integrity, cell counts, and function in vitro, and in a large cohort of patients in vivo. Methods and results: In vitro study: peripheral blood mononuclear cells (PBMCs) were isolated from healthy volunteers, and histone H2AX phosphorylation (γ-H2AX) expression, leucocyte counts, and functional parameters were quantified using flow cytometry under the following conditions: (i) immediately following PBMC isolation, (ii) after standing on the benchside as a temperature and time control, (iii) after a standard CMR scan. In vivo study: blood samples were taken from 64 consecutive consenting patients immediately before and after a standard clinical scan. Samples were analysed for γ-H2AX expression and leucocyte counts. CMR was not associated with a significant change in γ-H2AX expression in vitro or in vivo, although there were significant inter-patient variations. In vitro cell integrity and function did not change with CMR. There was a significant reduction in circulating T cells in vivo following CMR. Conclusion: 1.5 T CMR was not associated with DNA damage in vitro or in vivo. Histone H2AX phosphorylation expression varied markedly between individuals; therefore, small studies using γ-H2AX as a marker of DNA damage should be interpreted with caution. Cardiovascular magnetic resonance was not associated with loss of leucocyte viability or function in vitro. Cardiovascular magnetic resonance was associated with a statistically significant reduction in viable leucocytes in vivo.

Ex-vivo flush of the limb allograft reduces inflammatory burden prior to transplantation.

BACKGROUND: Passenger leucocytes and inflammatory debris transferred from the donor limb to the recipient can induce allorecognition, which activates the host immune response. This is the first study to evaluate whether the transfer of this inflammatory burden can be reduced via post-preservation flush prior to revascularisation, and whether this is influenced by ischaemia. METHODS: Bilateral forelimbs from the same pig were procured and infused with preservation flush and stored on ice. Each limb from the same pig underwent a post-preservation intravascular flush with isotonic solution at either 2 or 6 h. Venous effluent underwent flow cytometry to phenotype leucocyte populations, with additional quantification of cytokines and cell-free DNA. RESULTS: We identified large populations of viable leucocytes in the flush effluent (8.65 × 108 ± 3.10 × 108 cells at 2 h and 1.02 × 109 ± 2.63 × 108 at 6 h). This comprised T cells, B cells, NK cells and monocytes. Post-preservation flush yielded significant concentrations of pro-inflammatory cytokines including IL-6, IL-18, GM-CSF, IL-1ß, IL1α and CXCL-8 and mitochondrial DNA. The regulatory cytokine, IL-10 was undetectable. CONCLUSIONS: This study supports the finding that a post-preservation flush removes leucocytes and inflammatory components that are responsible for direct presentation. This study also gives an indication of how ischaemia impacts on the inflammatory burden transferred to the recipient upon reperfusion.

Randomized Study Comparing the Effect of Carbon Dioxide Insufflation on Veins Using 2 Types of Endoscopic and Open Vein Harvesting.

OBJECTIVE: The aim of the study was to assess whether the use of carbon dioxide insufflation has any impact on integrity of long saphenous vein comparing 2 types of endoscopic vein harvesting and traditional open vein harvesting. METHODS: A total of 301 patients were prospectively randomized into 3 groups. Group 1 control arm of open vein harvesting (n = 101), group 2 closed tunnel (carbon dioxide) endoscopic vein harvesting (n = 100) and Group 3 open tunnel (carbon dioxide) endoscopic vein harvesting (open tunnel endoscopic vein harvesting) (n = 100). Each group was assessed to determine the systemic level of partial arterial carbon dioxide, end-tidal carbon dioxide, and pH. Three blood samples were obtained at baseline, 10 minutes after start of endoscopic vein harvesting, and 10 minutes after the vein was retrieved. Vein samples were taken immediately after vein harvesting without further surgical handling to measure the histological level of endothelial damage. A modified validated endothelial scoring system was used to compare the extent of endothelial stretching and detachment. RESULTS: The level of end-tidal carbon dioxide was maintained in the open tunnel endoscopic vein harvesting and open vein harvesting groups but increased significantly in the closed tunnel endoscopic vein harvesting group (P = 0.451, P = 0.385, and P < 0.001). Interestingly, partial arterial carbon dioxide also did not differ over time in the open tunnel endoscopic vein harvesting group (P = 0.241), whereas partial arterial carbon dioxide reduced significantly over time in the open vein harvesting group (P = 0.001). A profound increase in partial arterial carbon dioxide was observed in the closed tunnel endoscopic vein harvesting group (P < 0.001). Consistent with these patterns, only the closed tunnel endoscopic vein harvesting group demonstrated a sudden drop in pH over time (P < 0.001), whereas pH remained stable for both open tunnel endoscopic vein harvesting and open vein harvesting groups (P = 0.105 and P = 0.869, respectively). Endothelial integrity was better preserved in the open vein harvesting group compared with open tunnel endoscopic vein harvesting or closed tunnel endoscopic vein harvesting groups (P = 0.012) and was not affected by changes in carbon dioxide or low pH. Significantly greater stretching of the endothelium was observed in the open tunnel endoscopic open tunnel endoscopic vein harvesting group compared with the other groups (P = 0.003). CONCLUSIONS: This study demonstrated that the different vein harvesting techniques impact on endothelial integrity; however, this does not seem to be related to the increase in systemic absorption of carbon dioxide or to the pressurized endoscopic tunnel. The open tunnel endoscopic harvesting technique vein had more endothelial stretching compared with the closed tunnel endoscopic technique; this may be due to manual dissection of the vein. Further research is required to evaluate the long-term clinical outcome of these vein grafts.

Study Comparing Vein Integrity and Clinical Outcomes in Open Vein Harvesting and 2 Types of Endoscopic Vein Harvesting for Coronary Artery Bypass Grafting: The VICO Randomized Clinical Trial (Vein Integrity and Clinical Outcomes).

BACKGROUND: Current consensus statements maintain that endoscopic vein harvesting (EVH) should be standard care in coronary artery bypass graft surgery, but vein quality and clinical outcomes have been questioned. The VICO trial (Vein Integrity and Clinical Outcomes) was designed to assess the impact of different vein harvesting methods on vessel damage and whether this contributes to clinical outcomes after coronary artery bypass grafting. METHODS: In this single-center, randomized clinical trial, patients undergoing coronary artery bypass grafting with an internal mammary artery and with 1 to 4 vein grafts were recruited. All veins were harvested by a single experienced practitioner. We randomly allocated 300 patients into closed tunnel CO2 EVH (n=100), open tunnel CO2 EVH (n=100), and traditional open vein harvesting (n=100) groups. The primary end point was endothelial integrity and muscular damage of the harvested vein. Secondary end points included clinical outcomes (major adverse cardiac events), use of healthcare resources, and impact on health status (quality-adjusted life-years). RESULTS: The open vein harvesting group demonstrated marginally better endothelial integrity in random samples (85% versus 88% versus 93% for closed tunnel EVH, open tunnel EVH, and open vein harvesting; P<0.001). Closed tunnel EVH displayed the lowest longitudinal hypertrophy (1% versus 13.5% versus 3%; P=0.001). However, no differences in endothelial stretching were observed between groups (37% versus 37% versus 31%; P=0.62). Secondary clinical outcomes demonstrated no significant differences in composite major adverse cardiac event scores at each time point up to 48 months. The quality-adjusted life-year gain per patient was 0.11 (P<0.001) for closed tunnel EVH and 0.07 (P=0.003) for open tunnel EVH compared with open vein harvesting. The likelihood of being cost-effective, at a predefined threshold of £20 000 per quality-adjusted life-year gained, was 75% for closed tunnel EVH, 19% for open tunnel EVH, and 6% for open vein harvesting. CONCLUSIONS: Our study demonstrates that harvesting techniques affect the integrity of different vein layers, albeit only slightly. Secondary outcomes suggest that histological findings do not directly contribute to major adverse cardiac event outcomes. Gains in health status were observed, and cost-effectiveness was better with closed tunnel EVH. High-level experience with endoscopic harvesting performed by a dedicated specialist practitioner gives optimal results comparable to those of open vein harvesting. CLINICAL TRIAL REGISTRATION: URL: https://www.isrctn.com. International Standard Randomised Controlled Trial Registry Number: 91485426.

Characterizing the early inflammatory contribution of the donor kidney following reperfusion.

BACKGROUND: Donor kidneys contain a large reservoir of passenger leucocytes that contribute to acute rejection via direct alloantigen presentation and pro-inflammatory cytokine secretion. However, the early contribution of these cells following revascularization has not previously been described. We performed a secondary, high-volume preservation flush following cold storage to characterize the inflammatory contribution of the donor kidney upon reperfusion. METHODS: Porcine kidneys were retrieved using a protocol analogous to current UK clinical practice. Following 2 h of cold static preservation, kidneys underwent a secondary flush with Ringer's solution. The venous effluent was collected and leucocytes phenotyped via flow cytometry. Inflammatory mediators, including cytokines and cell-free DNA, were then assessed to determine the inflammatory contribution of the donor kidney. RESULTS: Upon reperfusion, a significant population of donor-derived CD45 + leucocytes mobilized from the renal vasculature via the renal vein [mean 4.738 × 10 8 (SD 1.348 × 10 8 )]. Within this population, T cells were dominant, representing >60% of the leucocyte repertoire. Granulocytes, monocytes and natural killer cells were also identified, but in comparatively lower numbers. Significant concentrations of cytokines and cell-free DNA were also eluted upon reperfusion. CONCLUSIONS: The donor kidney contains a significant immune load that rapidly mobilizes following reperfusion. Performing a secondary preservation flush prior to implantation may reduce this inflammatory burden via diversion of donor leucocytes and inflammatory mediators from entry into the recipient circulation. This may modulate direct presentation and reduce the inflammatory contribution of the donor kidney following transplantation.

A comprehensive review on learning curve associated problems in endoscopic vein harvesting and the requirement for a standardised training programme.

Endoscopic vein harvesting is becoming one of the most favourable vein harvesting techniques in multiple bypass coronary surgery, due to its short term post-operative benefits with high patient satisfaction. However, long-term graft patency has been both supported and questioned in the literature. Graft failure can be affected by harvesting methods and operator's experience. Endoscopic vein harvesting is associated with a learning curve period, during which the incidence of vein trauma is high due to unfamiliarity with the surgical technique. There is a paucity of structured learning tools for novice practitioners, meaning that training differs significantly between hospital centres. Inconsistent training methods can lead to poor surgical technique, which can have a significant impact on vein quality and stress level of the practitioner. In turn, this can lead to increased postoperative complications and longer surgical duration. The main aim of this literature review is to understand the impact of the learning curve on the vein conduit and whether there is a requirement for a standardised training programme for the novice practitioners.

Ex Vivo Normothermic Perfusion Induces Donor-Derived Leukocyte Mobilization and Removal Prior to Renal Transplantation.

INTRODUCTION: Ex vivo normothermic perfusion offers an alternative method of organ preservation, allowing donor kidneys to be reanimated and evaluated prior to transplantation. Beyond preservation, it can be used to characterize the immunological contribution of the donor kidney in isolation. Furthermore, it has the potential to be used as an immunomodulatory strategy to manipulate donor kidneys prior to transplantation. METHODS: Explanted porcine kidneys underwent 6 hours of perfusion. Sequential perfusate samples were collected and leukocytes characterized via flow cytometry. An inflammatory profile was generated via cytokine quantification. Cell-free DNA was also determined as markers of cell death. RESULTS: All kidneys functioned within normal parameters and met the criteria for transplantation at the end of perfusion. Throughout perfusion there were continuous increases in pro-inflammatory cytokines, including large concentrations of interferon-γ, suggesting that perfusion drives a significant inflammatory response. Increasing concentrations in cell-free DNA were also observed, suggesting cell death. During perfusion there was a marked cellular diapedesis of T cells, B cells, natural killer (NK) cells, and monocytes from the kidney into the circuit. Minor populations of granulocytes and macrophages were also detected. DISCUSSION: We demonstrate that ex vivo normothermic perfusion initiates an inflammatory cytokine storm and release of mitochondrial and genomic DNA. This is likely to be responsible for immune cell activation and mobilization into the circuit prior to transplantation. Interestingly this did not have an impact on renal function. These data therefore suggest that normothermic perfusion can be used to immunodeplete and to saturate the pro-inflammatory capacity of donor kidneys prior to transplantation.

A randomized study comparing traditional monofilament knotted sutures with barbed knotless sutures for donor leg wound closure in coronary artery bypass surgery.

OBJECTIVES: Surgical knots on the suture line provide an anchoring function, but also represent a potential source of infection and irritation on the donor leg after coronary artery bypass surgery. Knotless barbed sutures were designed to prevent knot-related complications. This study compared knot-related wound complication rates between patients receiving traditional monofilament sutures and those receiving barbed knotless sutures for closure of the donor leg. METHODS: One hundred and forty-two patients were randomized into two groups. Group 1 (n = 70) received traditional monofilament sutures and Group 2 (n = 72) received barbed knotless sutures. All wounds were assessed on postoperative days 3 and 5 and weeks 2, 4 and 6 using a validated wound scoring system. Antibiotics usage and general practitioner and district nurse visits were recorded. RESULTS: No demographic differences were observed between groups. Leg wound skin closure times were significantly shorter in Group 2 compared with Group 1 (P < 0.001). Group 1 demonstrated a greater incidence of excessive scarring (P < 0.001), itching (P < 0.001), irritation (P < 0.001) and adverse skin tissue reactions (P < 0.001) than Group 2. Fewer general practitioner visits were recorded in Group 1 compared with Group 2 (P = 0.051). CONCLUSION: Knotless barbed suture usage significantly reduces the incidence of knot-related leg wound complications compared with traditional monofilament knotted sutures. This may be related to differences in the rate of absorption of the suture material or an associated decrease in the incidence of adverse skin tissue reactions that may delay postoperative wound healing.

Histological and immunohistochemical evaluation of human saphenous vein harvested by endoscopic and open conventional methods.

OBJECTIVES: The introduction of endoscopic saphenous vein harvesting (ESVH) has been reported to decrease saphenectomy-associated wound pain and infection, compared with the traditional open conventional saphenous vein harvesting (OCSVH) technique. Despite all these benefits, the rate of adoption among surgeons has been variable. Criticism of this technique centres on the risk of injury at the time of vein harvest with its potential detrimental effect on structural viability and long-term patency. The aim of our study is to investigate the endothelial preservation of saphenous vein grafts harvested by various extraction methods. METHODS: A prospective, observational study of 30 human saphenous vein grafts was performed to evaluate endothelial preservation by haematoxylin-eosin and CD 31 staining methods. The saphenous vein was harvested endoscopically either by an open tunnel (OT-ESVH), closed tunnel (CT-ESVH) or an OCSVH harvesting technique. Research samples were collected without distension to avoid intraluminal dilatation and endothelial disruption. Both haematoxylin-eosin and immunohistochemistry slides were imaged by a high-resolution slide-scanning system. RESULTS: Haematoxylin-eosin staining of the CT-ESVH group showed mostly preserved endothelium (P = 0.398) with some endothelial stretching (P = 1.0) and no endothelial detachment (P = 0.197). The OT-ESVH group showed marked endothelial stretching (P = 0.053). However, the OCSVH group showed significantly more endothelial detachment than the endoscopic groups (P = 0.01). The mean grading score of immunohistochemistry using the CD 31 antibody was much lower in the OT-ESVH group (1.6 ± 0.84, P = 0.009), showing more poorly preserved endothelial cells than the CT-ESVH and OCSVH groups. CONCLUSIONS: We observed more endothelial stretching in the OT-ESVH group, which in our opinion, was due to lack of subcutaneous tissue separation, poor visualization and traction stresses across the wall of the saphenous vein. However, the OCSVH method revealed poor endothelial protection with areas of endothelial detachment, not observed with both endoscopic techniques. Interestingly, most preserved endothelium was found in the CT-ESVH group, which was previously known to be associated with worse graft patency.

Does the introduction of a comprehensive structured training programme for endoscopic vein harvesting improve conduit quality? A multicentre pilot study.

OBJECTIVES: Endoscopic vein harvesting is one of the most popular minimally invasive vein-harvesting techniques for coronary artery bypass graft surgery. It is associated with improved cosmetic outcome and fewer wound-related problems compared with the conventional open technique. However, its efficacy with regard to conduit damage and long-term patency has recently been questioned. Learning curve-associated trauma to the vein has a major impact on vein quality and the incidence of graft failure post-surgery. In an attempt to address this problem, we have devised and tested a learning tool termed Manchester Endoscopic Learning Tool (MELT). In this study, we compare vein quality following MELT training with standard recommended training. METHODS: Fourteen practitioners across seven UK centres were enrolled into the study. Practitioners were categorized into two groups receiving MELT or standard training. Data were collected prospectively from the first eight vein retrievals per operator following training. A total of n = 112 vein-harvesting procedures were included in the study. RESULTS: Veins harvested by MELT practitioners had fewer small avulsions (P <0.001), required fewer repairs (P <0.001) and experienced a lower incidence of bruising (P <0.001) than veins obtained by practitioners receiving standard training. The incidence of very short side branches requiring repair was also significantly reduced (P <0.001) in the MELT group compared with standard training. CONCLUSIONS: Our formalized training programme consistently minimizes vein trauma resulting in better-quality conduits when compared with the current standard training. Exposure of surgical practitioners to the structured curriculum during their endoscopic vein harvesting training will enhance their learning and lead to better-quality conduits. This is likely to impart clinical benefit post-surgery.

Heart rate after cardiac transplantation-lessons from the tortoise and the shrew.

There is a striking consistency in the total number of heart beats accrued over a lifetime across a range of animal species despite vast differences in size. Moreover, an inverse relationship is observed between heart rate and lifespan, leading to speculation that elevated heart rate could significantly affect longevity. It is the aim of this review to analyze heart rate as a contributing factor in defining the functional lifespan of the transplanted human heart, which may unavoidably determine the longevity of the recipient. Sinus tachycardia occurs as a result of sympathetic/parasympathetic denervation, an unavoidable consequence of transplantation. The effect of elevated heart rate in this cohort has been scarcely reported. We highlight herein multitudinous mechanisms whereby elevated heart rate accelerates the deterioration in cardiac function and arterial elasticity due to injury and stress accumulation. Additionally, we propose a significant role for heart rate in confounding the alloimmune response. Tachycardia exacerbates injurious episodes of myocardial ischemia and significantly increases the production of reactive oxygen species via increased metabolism. These factors promote immune infiltration and activation, contributing to acute and chronic rejection. Further research is required to assess the potential therapeutic benefits of heart rate reduction.

Characterisation of the T cell and dendritic cell repertoire in a murine model of mucopolysaccharidosis I (MPS I).

BACKGROUND: Mucopolysaccharidosis I (MPS I) is a metabolic disorder caused by α-L-Iduronidase (IDUA) deficiency, resulting in lysosomal accumulation of heparan (HS) and dermatan sulphate (DS). This has been reported in microglia, yet currently the effect of IDUA deficiency on T cells and dendritic cells (DC) and their functionality in disease pathogenesis remains unclear. METHODS: Peripheral blood was collected from 3 month old C57BL/6 MPS I (n = 11) and wildtype (WT) (n = 6) mice. T cell and DC phenotype and functional characteristics were identified by flow cytometry. RESULTS: MPS I mice exhibited a reduction in DC (p = <0.001) along with CD8+ cytotoxic (p = 0.01) and CD4+ T helper (p = 0.032) cells, compared to WT controls. MPS I DC displayed a significant decrease in cell surface CD123 (p = 0.02) and CD86 (p = 0.006) expression. Furthermore, CD45RB expression was significantly reduced on T helper cells in the MPS I population (p = 0.019). CONCLUSION: We report a reduction in circulating DC and T cells in the MPS I mouse; indicative of adaptive immune dysfunction. DC reduction may occur in response to down-regulation of the IL-3 receptor (CD123), necessary for DC survival. We also report down-regulation of cell surface CD86, a molecule required for T cell co-stimulation. T helper cell down-regulation of CD45RB is redolent of an anti-inflammatory phenotype with poor proliferative capacity. The definitive causes of our findings and the consequences and role that these findings play in the pathogenesis of MPS are unclear, but may be in response to lysosomal storage of unmetabolized HS and DS.