Graft Endothelium Research Articles

Role of Procurement-Related Injury in Early Saphenous Vein Graft Failure after Coronary Artery Bypass Surgery

Saphenous vein graft thrombosis after coronary artery bypass graft surgery is a poorly understood problem that lessens the benefits of this procedure. Recent studies highlight the importance of injury sustained at the time of saphenous vein graft procurement in the pathogenesis of acute graft thrombosis. In particular, damage to the graft endothelium that occurs secondary to ex vivo pressure distention, a common practice during vein harvest, leads to a loss of antithrombotic factors and increased activity of subendothelial prothrombotic factors. The prothrombotic potential of damaged grafts is further exacerbated by an ischemic storage interval and subsequent exposure to arterial flow conditions after grafting. A clearer understanding of the mechanisms by which endothelial disruption leads to acute saphenous vein graft thrombosis may result in interventions for improving our procurement techniques, interrupting the downstream effects of the damaged saphenous vein graft and/or discriminating damage that is beyond an acceptable threshold of thrombotic risk.

STAT-1 and AP-1 decoy oligonucleotide therapy delays acute rejection and prolongs cardiac allograft survival

Acute myocardial rejection is a cell-mediated process characterized by increased leukocyte recruitment into the graft myocardial tissue. Transcription factors like STAT-1 and AP-1 are critically involved in this process by regulating vascular adhesion molecule expression. The aim of our study was to investigate the effect of decoy oligodeoxynucleotide (dODN) treatment targeting transcription factors AP-1 and STAT-1 on acute cardiac allograft rejection in a rat transplant model. Wistar-Furth (WF) cardiac allografts were transplanted into Lewis (LEW) rats after perfusion with STAT-1 or AP-1 dODN solution (5 micromol/l), buffer or the corresponding mutant control ODNs. Grafts were harvested and processed for histologic and immunohistochemical evaluation. As demonstrated by fluorescence dye-labelled dODN, exposure of the grafts to the dODNs during 45 min of warm ischemia resulted in a dominant uptake of naked DNA by the graft endothelium. Treatment with AP-1 and STAT-1 dODNs, but not with vehicle or the control dODNs, significantly prolonged cardiac allograft survival by approximately 40% from 5.6+/-0.5 days to 7.8+/-1.3 days and 7.4+/-0.5 days, respectively (mean+/-S.D., p<0.01, n=5 in each group). Immunohistochemical examination on days 1, 3 and 6 revealed a marked reduction of infiltrating leukocytes (AP-1 dODN: 85%, STAT-1 dODN: 50%), namely T-cells, in the dODN-perfused grafts at day 3 post transplantation. In addition, as demonstrated by immunohistochemical analysis, endothelial expression of ICAM-1 and VCAM-1 was found to be markedly reduced in dODN-treated grafts. Both AP-1 and STAT-1 dODN treatments suppress graft endothelial adhesion molecule expression, reduce graft infiltration and in turn significantly delay acute rejection. The utilization of dODNs in the cardioplegic solution might be a novel strategy to protect transplanted organs from early damage during transplantation, to preserve organ function and bridge the critical phase after transplantation when standard immunosuppression is not yet completely effective.

Tissue engineering of vascular conduits

Autologous conduits are not available in up to 40 per cent of patients with arteriopathy who require coronary or lower limb revascularization, and access sites for renal dialysis may eventually become exhausted. Synthetic prostheses achieve a poor patency rate in small-calibre anastomoses. This review examines how vascular tissue engineering may be used to address these issues. A Medline search was performed, using the keywords "vascular tissue engineering", "small diameter vascular conduit", "vascular cell biology", "biomechanics", "cell seeding" and "graft endothelialization". Key references were hand-searched for relevant papers. In vitro and in vivo approaches are currently being used for guided cell repopulation of both biological and synthetic scaffolds. The major clinical problem has been extended culture time (approximately 6 weeks), which precludes their use in the acute setting. However, recent advances have led not only to improved patency rates for prostheses, but also to a potential reduction in culture time. In addition, increased mobilization of endothelial progenitor cells in the presence of ischaemic tissue may increase the autologous cell yield for scaffold reseeding with further reduction in culture time.

Modulating Expanded Polytetrafluoroethylene Vascular Graft Host Response via Citric Acid‐Based Biodegradable Elastomers

A biodegradable polyester elastomer, poly(1,8-octanediol citrate) (POC), is used to modify the lumen of expanded polytetrafluoroethylene (ePTFE) grafts via a spin-shearing method followed by interfacial in situ polycondensation. We demonstrate successful surface modification of ePTFE grafts without affecting graft compliance (see figure). The POC interface confers hydrophilicity, reduced thrombogenicity, facilitated graft endothelialization in vitro, and reduced macrophage infiltration on the ePTFE grafts.

Activation of Human Microvascular Endothelial Cells with TNF-Alpha and Hypoxia/Reoxygenation Enhances NK-cell Adhesion, but not NK-Cytotoxicity

Ischemia/reperfusion injury (I/R) and cellular rejection in solid organ transplantation are characterized by adhesion molecule up-regulation on the graft endothelium, a prerequisite for leukocyte recruitment. The contribution of NK cells to I/R and allograft rejection is not well understood. The aim of the present study was to investigate allogeneic interactions between human NK cells and microvascular endothelial cells (MVEC) with special regard to the differential impact of TNF-alpha and hypoxia/reoxygenation in an in vitro model of I/R. MVEC were stimulated in vitro for 8 h with TNF-alpha, exposed to hypoxia (1% O2), hypoxia/reoxygenation, and combinations thereof in a hypoxia chamber. Cell surface expression of adhesion molecules on MVEC was analyzed by flow cytometry, and adhesion molecule shedding by ELISA. NK cell adhesion on MVEC was determined under shear stress, and NK cytotoxicity using Cr-release assays. Surface expression of ICAM-1, VCAM-1, and E-/P-selectin on MVEC was up-regulated by TNF-alpha but unaffected by hypoxia/reoxygenation in the absence of TNF-alpha. ICAM-1 expression was further increased by a combination of TNF-alpha and hypoxia/reoxygenation, whereas TNF-alpha-induced E-/P-selectin expression was strongly reversed by hypoxia/reoxygenation. NK cell adhesion increased after exposing MVEC to TNF-alpha and hypoxia/reoxygenation. Susceptibility of MVEC to NK cytotoxicity was enhanced by TNF-alpha and slighty reduced by hypoxia/reoxygenation. Endothelial activation with TNF-alpha, but not hypoxia/reoxygenation, induced NK cytotoxicity whereas the combination thereof induced the strongest NK cell adhesion. Our findings suggesting a role for NK cells in allograft responses support the development of anti-inflammatory treatment strategies to prevent I/R.

Neutrophil Adherence to Activated Saphenous Vein and Mammary Endothelium After Graft Preparation

Interaction of circulating leukocytes and vascular endothelium plays an important role in vasoconstriction, endothelial dysfunction, and vascular injury. Dilation procedures of grafts before coronary artery bypass graft surgery might lead to vascular injury and subsequent bypass graft disease. We analyzed in vitro the adherence of fluorescence-labeled polymorphonuclear neutrophils (PMNs) to endothelium of human saphenous vein grafts or internal mammary artery grafts after stimulation with thrombin (0.5 to 2 U/mL) or dilating procedures. Furthermore, we investigated endothelial function of prepared grafts. Thrombin stimulation resulted in a dose-dependent increase of PMN adherence to the endothelium of saphenous vein and internal mammary artery, which was attenuated by the selectin-blocking carbohydrate fucoidin or anti-P-selectin monoclonal antibody. Mechanical dilation of saphenous vein or internal mammary artery led to a marked increase in PMN adherence (65 +/- 5 versus 5 +/- 3 PMN/mm2; p < 0.01), which was significantly attenuated by fucoidin or anti-P-selectin monoclonal antibodies. Treatment of internal mammary artery with the vasodilator papaverine led to a marked increase of PMN adherence (59 +/- 8 versus 12 +/- 4 PMN/mm2; p < 0.01) when papaverine was administered directly into the vessel. However, external treatment with papaverine did not affect PMN adhesion. Endothelial dysfunction was observed in dilated venous grafts and in arterial grafts internally treated with papaverine; in contrast, external treatment did not affect endothelial function. This study showed that mechanical or pharmacologic dilation of venous or arterial coronary grafts, usually performed before anastomosis of aortocoronary bypass grafts, led to increased selectin-mediated PMN adhesion on vascular endothelium and subsequent endothelial dysfunction.

Biomimetic materials for injectable tissue engineering: studies of acute, lasting and unexpected angiogenesis response

Challenges for therapeutic manipulation of adult angiogenesis with exogenous growth factors are to counteract (1) aberrant vessel growth (2) vessel leakage (3) vessel regression. We describe tissue engineering solutions, based on fibrin or poly ethyleneglycol-peptide implant matrices, for localized and prolonged delivery of VEGF that help ensure localized growth of structurally normal and lasting vessels and minimize adverse side effects. These matrices bind VEGF such that decisions on dosage, location, and duration of released factor are coupled to cellular proteolytic activity at the treatment site, i.e. biological feedback regulation of release. We describe acute angiogenesis response to such schemes of cell-demanded release using video microscopy, morphometry, casting techniques and scanning electron microscopy. Further, we have used biophotonic measurements in transgenic Vegfr2-luc (Xenogen) to correlate transcriptional activation of angiogenesis with immunohistochemical analysis of lasting vessel growth at and around VEGF- releasing implants. We have used VEGF-fibrin matrices applied to the flow surface of synthetic ePTFE grafts in pig trials of spontaneous graft endothelialization. We report an unexpected, untoward effect of VEGF for increasing intimal thickening, indicative of an effect for smooth muscle cell activation. Sponsored by Gebert Rüf Foundation.

Nanocomposite Containing Bioactive Peptides Promote Endothelialisation by Circulating Progenitor Cells: An In vitro Evaluation

Objective The formation of an endothelial cell layer on the luminal surface of cardiovascular devices, especially bypass grafts, is an important attribute in order to improve their patency. Endothelial progenitor cells (EPCs) have a potential role in the endothelialisation of bypass grafts. We hypothesised that a novel approach to improve endothelialisation of bypass grafts by EPCs would be the creation on the graft lumen of a microenvironment that supports EPC adhesion and differentiation. Methods A new generation of nanocomposite based on silsesquioxane in the form of polyhedral oligomeric silsesquioxane (POSS) nanocages which incorporate bioactive peptides (RGD) was made into sheets. Peripheral blood mononuclear cells (PBMCs) containing EPCs isolated from six consenting young, healthy, adult volunteers were then plated both on (1) sheets of the nanocomposite with the bioactive peptide, (2) sheets of the nanocomposite without the bioactive peptide, (3) culture dishes as control and then cultured in presence of vascular endothelial growth factor (VEGF). Confirmation of endothelial and EPCs markers was carried out using fluorescence-activated cell sorter (FACS) analysis, reverse transcription polymerase chain reaction (RT-PCR) and immunostaining. Results One to two percent of PBMCs expressed CD34 as determined by FACS analysis. Cells were demonstrated to express mRNA for the EPC markers CD34, platelet-endothelial cell adhesion molecule-1 (CD31), CD133 and vascular endothelial growth factor receptor-2(FlK-1/KDR). Endothelial cell-colony forming units were formed between day 5 and day 7 after plating. Colonies were confirmed to be endothelial like cells by immunostaining. There were significantly greater numbers of EPC colonies on the bioactive nanocomposites as compared to the nanocomposite alone and the uncoated dishes. Conclusion We report a new nanocomposite based biomaterial that has been demonstrated, in vitro, to promote endothelialisation from PBMCs containing EPCs.

Letter Regarding Article by Rotmans et al, “In Vivo Cell Seeding With Anti-CD34 Antibodies Successfully Accelerates Endothelialization but Stimulates Intimal Hyperplasia in Porcine Arteriovenous Expanded Polytetrafluoroethylene Grafts”

To the Editor: Rapid (re)endothelialization of autologous and synthetic vascular grafts is regarded as a key element in reducing the risk of acute thrombosis and, by limiting adverse vessel wall remodeling, which includes intimal hyperplasia, preserving long-term patency. Rotmans et al1 (and the accompanying editorial2) challenge this tenet of vascular biology in their report of human anti-CD34 antibody–coated polytetrafluoroethylene (PTFE) grafts in a porcine AV fistula model. Rapid “endothelialization” of grafts is described, which, rather than improving functional outcome, was associated with significantly increased intimal hyperplasia at the venous anastomosis. The authors conclude that future studies will need to address a putative deficiency in the functional capability of captured endothelial progenitor cells (EPCs), highlighting approaches based on the use of regulatory proteins or alternative cell combinations. In our opinion, the data do not support these conclusions. The graft endoluminal cells were inadequately characterized with lectin (a surface antigen shared by several blood-borne …

Enhancement of in vivo endothelialization of tissue‐engineered vascular grafts by granulocyte colony‐stimulating factor

Successful reconstruction of large-diameter blood vessel in humans has been demonstrated using the tissue engineering technique, but improvement in patency of small-diameter bioartificial vascular graft remains a great challenge. This study reports that granulocyte colony-stimulating factor (G-CSF) can enhance in vivo endothelialization of tissue-engineered vascular grafts, which could be used to improve patency of small-diameter vascular graft. Vascular grafts were tissue engineered with decellularized canine abdominal aortas and canine autologous bone marrow-derived cells. Prior to cell seeding onto decellularized graft matrices, bone marrow-derived cells were induced to differentiate into endothelial cells and smooth muscle cells. The cell-seeded vascular grafts were implanted into the abdominal aortas of bone marrow donor dogs. Before and after graft implantation, G-CSF was administered subcutaneously to the dogs (n = 3). The grafts implanted into the dogs not receiving G-CSF were used as controls (n = 3). Eight weeks after implantation, grafts in both groups showed regeneration of vascular tissues including endothelium and smooth muscle. Importantly, endothelium formation was more extensive in the G-CSF-treated grafts than in the control grafts, as assessed with reverse transcription polymerase chain reaction, western blot, and immunohistochemistry. In addition, intimal hyperplasia was significantly reduced in the G-CSF-treated grafts compared to the control grafts. This study suggests that G-CSF administration could be applied to improve patency of small-diameter tissue-engineered vascular grafts.

Effects of Resveratrol in Storage Solution on Adhesion Molecule Expression and Nitric Oxide Synthesis in Vein Grafts

Endothelial injury in human saphenous vein grafts may occur during harvesting and storage, which may have an adverse effect on coronary artery bypass grafting outcome. In this study, we sought to determine whether resveratrol, a natural antioxidant enriched in grape, can limit endothelial activation and reduce endothelial injury in human saphenous vein grafts. Human saphenous vein grafts, obtained from 8 patients and divided into two equal groups of control and study specimens, were stored in either heparinized blood (group A) or heparinized blood containing 50 microg/mL resveratrol (group B) for 1 hour at room temperature. Specimens were analyzed by Western blotting to quantify intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and inducible nitric oxide synthase-2 expression, as well as tissue cyclic guanylate monophosphate levels. Myeloperoxidase activity, a marker of neutrophil sequestration in human saphenous vein grafts, was also measured in each group. Intercellular adhesion molecule-1 expression (1,674 +/- 332 versus 559 +/- 282; p = 0.003), vascular cell adhesion molecule-1 expression (753 +/- 183 versus 472 +/- 151; p = 0.025), and myeloperoxidase activity (7.00 +/- 1.05 versus 1.33 +/- 0.45 nm/min; p = 0.004) were significantly lower in group B. In contrast, inducible nitric oxide synthase-2 expression (548 +/- 237 versus 2,234 +/- 726; p = 0.004) and tissue cyclic guanylate monophosphate levels (2.02 +/- 0.53 versus 5.61 +/- 0.89 pmol/mL; p = 0.001) were significantly higher in group B. Resveratrol reduced upregulation of leukocyte-endothelial cell adhesion molecule expression in human saphenous vein graft endothelium and decreased neutrophil adhesion to saphenous vein graft endothelium. Resveratrol also augmented inducible nitric oxide synthase-2 expression and increased cyclic guanylate monophosphate levels. These results suggest that resveratrol might improve vascular homeostasis and reduce endothelial injury during the hypoxic storage period of human saphenous vein grafts for coronary artery bypass grafting.

Catastrophic microangiopathy induced by high‐titre factor VIII inhibitors after liver transplantation for haemophilia A with cirrhosis

Liver transplantation may induce immune tolerance to factor VIII inhibitors but de novo development of inhibitors after transplantation may cause intractable haemorrhage. We report a patient with mild haemophilia A and high-titre FVIII inhibitors who received an orthotopic liver transplantation for complications of hepatitis C virus cirrhosis. Recombinant activated FVII was used in addition to routine haemostatic agents. Conventional immunosuppression was supplemented with antithymocyte globulin and cyclophosphamide. FVIII inhibitors disappeared from the circulation with liver transplantation but they were found to have bound to the graft endothelium, which became activated and induced catastrophic microangiopathy. A subsequent anamnestic response resulted in FVIII inhibitor titres of 1000 Bethesda Units. Uncontrollable haemorrhage persisted until the recipient's death. In patients with high-titre FVIII inhibitors resilient desensitization is required before liver transplantation.

Monitoring of the Intestinal Mucosal Perfusion Using Laser Doppler Flowmetry After Multivisceral Transplantation

Background Graft endothelium constitutes a prime target during acute rejection. Infiltration of T cells, monocytes, and enhanced endothelial-leukocyte interactions result in microvascular impairment and altered perfusion. Materials and methods We measured mucosal blood flow using a laser Doppler flowmeter in three patients undergoing multivisceral transplantation. Thirty-seven measurements were performed through the ileostomy over the first 4 weeks posttransplantation. Most measurements were performed within a 24-hour interval from endoscopy and biopsy. Results Mucosal perfusion increased throughout the first postoperative week and eventually stabilized around levels specific for each patient. Mucosal perfusion remained stable during graft pancreatitis, but decreased 35% to 55% from baseline (the average value of the previous measurements) during acute rejection and sepsis. During the first week posttransplantation there was a gradual increase in mucosal perfusion, which might reflect regeneration after reperfusion injury. Increased mucosal perfusion did not seem to correlate with rejection or other adverse clinical events. A sudden decrease in mucosal perfusion of 30% or more compared to the previous measurements was associated with septic episodes and/or rejection.

Disruption of Graft Endothelium Correlates With Early Failure After Off-Pump Coronary Artery Bypass Surgery

Saphenous vein graft failure after coronary artery bypass surgery may be as high as 5% to 10% in the first postoperative week. We hypothesized that identifying damage sustained by saphenous vein endothelium before grafting predicts early graft attrition after off-pump coronary artery bypass graft surgery. Intraoperative graft flow, platelet function, and endothelial integrity were analyzed in 125 patients undergoing off-pump coronary artery bypass graft surgery. Endothelial integrity was assessed in an excess vein segment from each graft using immunohistochemistry (CD31 staining). Platelet function was monitored just before and immediately after revascularization and on postoperative days 1 and 3 using whole blood aggregometry, thrombelastography, and platelet activated clotting time. Platelet activation was monitored using flow cytometry. Intraoperative conduit blood flow, measured by transit time ultrasonography, was used to detect and rectify anastomotic problems. Early graft patency was determined on postoperative day 5 using gated multichannel computed tomography angiography. In 106 patients undergoing postoperative computed tomography evaluation, 10 vein grafts in 10 patients were discovered to have developed early thrombosis, representing 4% (10 of 217) of all vein grafts. Endothelial integrity was 10.75% +/- 17.56% in 10 grafts that failed early compared with 51.45% +/- 36.29% in patent grafts (p = 0.04). Perioperative platelet function and graft flow did not differ significantly between the two groups. Although endothelial disruption predicts early failure of bypass grafts, the importance of a hypercoaguable state and low graft flow as a cause of early graft thrombosis after off-pump coronary artery bypass graft surgery was not supported by our preliminary results. A means to assess, prevent, and treat intraoperative vein graft damage will likely improve early graft patency.

Cardiovascular tissue engineering: state of the art

In patients requiring coronary or peripheral vascular bypass procedures, autogenous arterial or vein grafts remain as the conduit of choice even in the case of redo patients. It is in this class of redo patients that often natural tissue of suitable quality becomes unavailable; so that prosthetic material is then used. Prosthetic grafts are liable to fail due to graft occlusion caused by surface thrombogenicity and lack of elasticity. To prevent this, seeding of the graft lumen with endothelial cells has been undertaken and recent clinical studies have evidenced patency rates approaching reasonable vein grafts. Recent advances have also looked at developing a completely artificial biological graft engineered from the patient’s cells with surface and viscoelastic properties similar to autogenous vessels. This review encompasses both endothelialisation of grafts and the construction of biological cardiovascular conduits.

Comparison of the Rheologic Parameters in Left Internal Thoracic Artery Grafts With Those in Saphenous Vein Grafts

Left internal thoracic artery (LITA) grafts have superior patency to saphenous vein grafts (SVG). Because shear stress augments the release of nitric oxide throughout the LITA endothelium, shear stress and shear rate in coronary artery bypass grafts (CABG) may play an important role in the higher patency, so the aim of the present study was to evaluate and compare the rheologic parameters in CABG using LITA and SVG. Rheologic examinations were done in 197 patients using a vacuum-suction glass tube viscometer after CABG surgery was completed. Shear stress and shear rate were calculated from the geometry of the graft, blood flow in the graft and blood viscosity. Of 197 patients, 177 underwent LITA grafting to the left anterior descending artery (LAD) and 160 had SVG anastomosis to coronary arteries. Mean wall shear stress in the LITA grafts to the LAD (13.8+/-1.0 dyne/cm2) was nearly 4-6-fold larger than that in the SVG grafts. Mean shear rate (559.1+/-57.0 s(-1)) of LITA-LAD grafts was approximately 2-3-fold higher than that of SVG. These results suggest that high wall shear stress and shear rate play an important role in the higher patency rate of LITA grafts.

Circulating endothelial (progenitor) cells reflect the state of the endothelium: vascular injury, repair and neovascularization

An increase in the number of circulating endothelial cells (CEC) and of bone marrow-derived endothelial progenitor cells (EPC) in the peripheral blood is associated with vascular injury, repair and neovascularization. The phenotype and number of CEC may serve as diagnostic or prognostic parameters of vascular injury and tumour growth. An increase in the number of EPC may reflect repair of ischaemic vascular injury, a finding which has resulted in the initiation of clinical cardiovascular pilot trials using cell therapy. However, there is no consensus on the exact phenotype of the EPC and haematopoietic stem cells (HSC) and therefore the best candidate cell for transplant has not been established. Although the use of peripheral blood stem cells following mobilization, or of ex vivo-expanded cells, may improve EPC-mediated vascular graft endothelialization or tissue vascularization, sustained EPC-induced neovascularization still needs to be proven. Flow cytometric characterization, in combination with functional assays, will further elucidate the phenotype of the CEC and EPC, thereby providing reliable detection to appreciate their role in vascular diseases and cancer and to evaluate and, if possible, improve their therapeutic potential.

Influence of ischemic injury on vein graft remodeling: Role of cyclic adenosine monophosphate second messenger pathway in enhanced vein graft preservation

Objective Endothelial injury during the harvest of saphenous vein grafts might play an important role in the development of vein graft disease after coronary artery bypass grafting. Using a murine autologous arterialized vein patch model, we tested whether the initial ischemic insult of vein grafts was linked to the later development of graft neointimal hyperplasia and whether the restoration of the cyclic adenosine monophosphate second messenger pathway would attenuate the development of neointimal hyperplasia. Methods A segment of the external jugular vein of a mouse was grafted onto its abdominal aorta. Three weeks after the operation, the degree of neointimal hyperplasia of the implanted graft was compared among (1) grafts without preservation, (2) grafts with 2 hours of preservation (25°C) in heparinized saline, and (3) grafts with 2 hours of preservation in heparinized saline in the presence of a cyclic adenosine monophosphate analog. In addition, cyclic adenosine monophosphate contents of vein grafts and leukocyte adherence to the graft endothelium were assessed. Results Cyclic adenosine monophosphate contents were significantly decreased after 2 hours of preservation (212 ± 8 vs 156 ± 5 pmol/L, P < .01). The grafts preserved for 2 hours showed greater neointimal hyperplasia compared with the grafts without preservation (neointimal expansion, 68.7% ± 9.6% vs 46.1% ± 4.8%; P < .01). The addition of a cyclic adenosine monophosphate analog to the preservation solution significantly suppressed neointimal hyperplasia of grafts preserved for 2 hours (44.3% ± 5.0%). Inhibiting the cyclic adenosine monophosphate–dependent protein kinase by adding Rp-cAMPS abrogated the beneficial effects. Furthermore, grafts preserved for 2 hours had significantly more leukocytes adhering to the graft endothelium 24 hours after the operation compared with nonpreserved grafts, which was significantly reduced by the cyclic adenosine monophosphate treatment. Conclusions Ischemic insult during vein graft harvest and preservation is a key factor in the development of vein graft neointimal hyperplasia at least in part caused by the depletion of cyclic adenosine monophosphate. We conclude that stimulation of the cyclic adenosine monophosphate second messenger pathway might be a potential strategy for the prevention of vein graft disease.

Reduction of ICAM-1 and LFA-1-positive leukocytes in the perivascular space of arteries under mycophenolate mofetil therapy reduces rat heart transplant vasculopathy

Background The interaction of lymphocyte function-associated antigen-1 (LFA-1)–positive host leukocytes with intercellular adhesion molecule-1 (ICAM-1) on graft endothelium may play a key role in allograft recognition, triggering the development of transplant vasculopathy (TVP). We investigated the correlation between TVP and ICAM-1 expression and accumulation of LFA-1–positive leukocytes in the perivascular space (PVS) of arteries under different immunosuppressive drugs. Methods After cardiac transplantation (Lewis to Fisher) animals were randomized 4 groups: cyclosporine (CsA), 3 mg/kg/day ( n = 74); mycophenolate mofetil (MMF), 40 mg/kg/day ( n = 96); FK 506, 0.3 mg/kg/day ( n = 96); and control, no therapy ( n = 74). Three or 4 animals from each group were harvested at intervals of 1 to 4 days within the study period of 60 days. Using immunohistochemistry, LFA-1–positive leukocytes were analyzed in intra- and epicardial arteries. ICAM-1 expression was scored histologically. TVP was assessed by digitizing morphometry and expressed as mean vascular occlusion. Results Accumulation of LFA-1–positive leukocytes in the PVS of arteries and the myocardium correlated with expression of ICAM-1 on graft endothelium. The severity of TVP in arteries correlated with the accumulation of LFA-1–positive leukocytes in PVS. All immunosuppressive drugs significantly reduced ICAM-1 expression, LFA-1 accumulation and extent of TVP, compared with controls. In MMF-treated animals, we also found a significant reduction of ICAM-1 expression, LFA-1 accumulation and extent of TVP compared with the groups treated with CsA and FK 506 ( p < 0.005). Conclusion These data support an essential role of LFA-1/ICAM-1 interaction in the genesis of TVP that may be abrogated, especially by the use of MMF.

Non-viral in vivo thrombomodulin gene transfer prevents early loss of thromboresistance of grafted veins

Immediate loss of thrombomodulin activity in the endothelium of vein grafts has been demonstrated during 90 min exposure to arterial circulation; this loss of activity is ascribed as an important cause of early thrombosis. Conventional ex vivo gene transfection after vein harvest cannot cover this acute period immediately after implantation. We have established a highly efficient non-viral gene therapy protocol utilizing modified transferrin receptor-facilitated gene transfer. Using this technique, we examined whether in vivo thrombomodulin gene therapy, directed to the endothelium of rat veins 2 days prior to grafting, may prevent thromboresistance impairment of vein grafts under simulated arterial circulation. Abdomen of SD rat was opened and cationic liposome:transferrin:thrombomodulin gene complexes or the vector without DNAs were applied to the inferior vena cava of rats while blood flow was reduced by proximal and distal clamping. After 2 days, the transfected veins were harvested and thrombomodulin expression and thromboresistance properties determined before and after exposure to an artificial circuit. The trial of gene transfection using variable doses of DNAs confirmed that 7.5 microg of total DNAs was the most efficient quantity for thrombomodulin gene transfection to IVCs, although accompanying an increase of gene expression in other downstream organs. By transfection of the thrombomodulin gene in IVCs, the generation capacity of activated protein C in venous endothelium increased three-fold compared with veins treated with vector alone (P<0.01). Under simulated arterial circulation, perfusion of veins treated with vector alone decreased thrombomodulin activity to 36% of preperfused levels (P<0.01), whereas transfected grafts preserved the activity at normal vein endothelium levels even after perfusion. Consequently, the increase in endothelial thrombin activity induced by simulated arterial circulation was markedly attenuated in transfected veins (P<0.01), while immunohistochemistry confirmed the preservation of endothelial lining. Transferrin receptor-facilitated in vivo gene transfer to the inferior vena cava resulted in sufficient thrombomodulin gene expression immediately after graft implantation and subsequent maintenance of thromboresistance even after exposure to arterial pressure. Although further studies are needed, the present results suggest the possibility of gene therapy targeting acute phases of vein graft disease.