Co-expression of fibulin-5 and VEGF165 increases long-term patency of synthetic vascular grafts seeded with autologous endothelial cells.

Small caliber synthetic vascular grafts are commonly used for bypass surgery and dialysis access sites but have high failure rates because of neointima formation and thrombosis. Seeding synthetic grafts with endothelial cells (ECs) provides a biocompatible surface that may prevent graft failure. However, EC detachment following exposure to blood flow still remains a major obstacle in the development of biosynthetic grafts. We tested the hypothesis that induced expression by the seeded EC, of vascular endothelial growth factor165 (VEGF165) and of fibulin-5, an extracellular matrix glycoprotein that has a crucial role in elastin fiber organization and increase EC adherence to surfaces, may improve long-term graft patency. Autologous ECs were isolated from venous segments, and were transduced with retroviral vectors expressing fibulin-5 and VEGF165. The modified cells were seeded on expanded polytetrafluoroethylene (ePTFE) grafts and implanted in a large animal model. Three months after transplantation, all grafts seeded with modified EC were patent on a selective angiography, whereas only a third of the control grafts were patent. Similar results were shown at 6 months. Thus, seeding ePTFE vascular grafts with genetically modified EC improved long-term small caliber graft patency. The biosynthetic grafts may provide a novel therapeutic modality for patients with peripheral vascular disease and patients requiring vascular access for hemodialysis.

Fibulin-5 regulates keloid-derived fibroblast-like cells through integrin beta-1.

OBJECTIVE: Keloid scar is pathological tissue that appears after skin injury, and that is more aggressive than hypertrophic scars. Keloid scars are characterized by increased proliferation of fibroblast-like cells (FLCs) and the accumulation of extracellular matrix, mainly collagen. Fibulin-5, a glycoprotein secreted by many cell types, is a component of the extracellular matrix. We investigated the effect of fibulin-5 on the adhesion and proliferation of FLCs derived from keloid scars and the role of integrin beta-1 in these activities. METHODS: Fibroblast-like cells were isolated from six keloid scars and cultured on plates coated with fibulin-5 or with gelatin. Cells were incubated for 72-96 h to examine proliferation rates and incubated for 240 min, with washings at 20, 40, 60, 90, 120, 180 min, to assess adhesion rates. To examine the role of integrin beta-1, the anti-human integrin beta-1 (CD29) antibody was added to the culture medium. RESULTS: Fibroblast-like cells from keloids cultured on a fibulin-5-coated surface showed a significantly reduced proliferation rate and a delayed adhesion rate, compared to cells cultured on gelatin-coated dishes. Adherence of these cells to fibulin-5 pre-coated wells was significantly reduced in the presence of anti-human integrin beta-1 (CD29) antibodies. Our current findings are similar to previously observed reduced proliferation in vascular smooth muscle cells overexpressing fibulin-5. We did not test the effects of fibulin-5 on normal fibroblasts. CONCLUSION: This study demonstrates the pivotal role of the extracellular protein, fibulin-5, on the adhesion and proliferation of human keloid-derived cells, through binding to integrin beta-1.

The formation of an anti-restenotic/anti-thrombotic surface by immobilization of nitric oxide synthase on a metallic carrier.

Coronary stenosis due to atherosclerosis, the primary cause of coronary artery disease, is generally treated by balloon dilatation and stent implantation, which can result in damage to the endothelial lining of blood vessels. This leads to the restenosis of the lumen as a consequence of migration and proliferation of smooth muscle cells (SMCs). Nitric oxide (NO), which is produced and secreted by vascular endothelial cells (ECs), is a central anti-inflammatory and anti-atherogenic player in the vasculature. The goal of the present study was to develop an enzymatically active surface capable of converting the prodrug l-arginine, to the active drug, NO, thus providing a targeted drug delivery interface. NO synthase (NOS) was chemically immobilized on the surface of a stainless steel carrier with preservation of its activity. The ability of this functionalized NO-producing surface to prevent or delay processes involved in restenosis and thrombus formation was tested. This surface was found to significantly promote EC adhesion and proliferation while inhibiting that of SMCs. Furthermore, platelet adherence to this surface was markedly inhibited. Beyond the application considered here, this approach can be implemented for the local conversion of any systemically administered prodrug to the active drug, using catalysts attached to the surface of the implant.

High frequency of vitamin B12 deficiency in asymptomatic individuals homozygous to MTHFR C677T mutation is associated with endothelial dysfunction and homocysteinemia.

The aim of this study was to examine the association of homozygosity for the methylenetetrahydrofolate reductase (MTHFR) C677T mutation and vitamin B12 deficiency in 360 asymptomatic individuals and to investigate forearm endothelial function in C677T homozygotes. MTHFR C677T mutation and levels of vitamin B12, folic acid, and homocysteine were measured in study participants. Frequency of homozygosity for the C677T mutation was 67/360 (18.6%). Homocysteine levels were elevated in homozygous compared with heterozygous subjects or those without the mutation (20.6 +/- 18.8 vs. 9.4 +/- 3.2 mumol/l; P < 0.0001). The number of subjects with vitamin B12 deficiency (<150 pmol/l) was significantly higher among the homozygote than the heterozygote subjects or subjects without mutation [20/67 (29.8%) vs. 27/293 (9.2%); P < 0.0001]. Homozygote subjects had 4.2 times higher probability of having B12 deficiency (95% confidence interval = 2.1-8.3). Forearm endothelial function was assessed in 33 homozygote and 12 control subjects. Abnormal endothelial function was observed in homozygous subjects and was worse in homozygote subjects with vitamin B12 deficiency. Endothelial function was normalized after B12 and folic acid treatment. We found that homozygosity for the C677T mutation is strongly associated with B12 deficiency. Coexistence of homozygosity for the C677T mutation and B12 deficiency is associated with endothelial dysfunction and can be corrected with vitamin B12 and folic acid treatment.

Effects of fibulin-5 on attachment, adhesion, and proliferation of primary human endothelial cells.

BACKGROUND: Fibulin-5 is a novel extracellular protein that is thought to act as a bridging peptide between elastin fibers and cell surface integrins in blood vessel wall. Fibulin-5 binding to endothelial cell (EC) surface integrins may effect cell proliferation and cell attachment to extracellular matrix (ECM) or to artificial surfaces. In this paper, we describe the effects of fibulin-5 on attachment, adhesion, and proliferation of primary human EC. After demonstrating that fibulin-5 over-expression inhibited EC proliferation, we tested the hypothesis that co-expression of fibulin-5 and VEGF165 will lead to unique EC phenotype that will exhibit increased adherence properties and retain its proliferation capacity. METHODS AND RESULTS: Fibulin-5 and VEGF165 gene transfer to primary human saphenous vein endothelial cells was accomplished using retroviral vectors encoding the two genes. Transgene expression was verified using immunohistochemistry, Western blotting, and ELISA. Fibulin 5 over-expression tended to improve immediate EC attachment (30 min after seeding) and improved significantly adhesion (>40%) under shear stress tested 24h after EC seeding. The effects of fibulin-5 and VEGF165 on EC proliferation in the presence or absence of basic FGF were also tested. EC expressing fibulin-5 had reduced proliferation while VEGF165 co-expression ameliorated this effect. CONCLUSION: Fibulin-5 improved EC attachment to artificial surfaces. Dual transfer of fibulin-5 and VEGF165 resulted in EC phenotype with increased adhesion and improved proliferation. This unique EC phenotype can be useful for tissue engineering on endovascular prostheses.

Increased vascular endothelial growth factor 165 binding to kinase insert domain-containing receptor after infection of human endothelial cells by recombinant adenovirus encoding the Vegf(165) gene.

BACKGROUND: The angiogenic effect of vascular endothelial growth factor (VEGF(165)) is mediated mainly through the high-affinity tyrosine kinase receptor VEGF-R2 (KDR/flk-1). This study examined the effects of VEGF overexpression by primary human endothelial cells (ECs), which do not express VEGF under physiological conditions, on cell proliferation, VEGF binding to the kinase insert domain-containing receptor (KDR), and KDR expression. METHODS AND RESULTS: Human primary ECs and SMCs were infected by recombinant adenoviral vector encoding VEGF(165) (rAdVEGF). Proliferation rate, bromodeoxyuridine incorporation, (125)I-labeled VEGF(165) binding to the KDR receptor, and KDR expression were tested in the infected cells and in cells supplemented with VEGF protein. Enhanced proliferation and a significant increase in (125)I-VEGF(165) binding to the KDR receptor were induced by rAdVEGF infection of ECs (autocrine effect) as well as by addition of recombinant VEGF(165) to noninfected cells. Infection of ECs by rAdVEGF led to posttranscriptional upregulation of the KDR receptor, whereas KDR mRNA expression levels remained unchanged. Similar effects were observed with supplemented recombinant VEGF(165) to noninfected ECs; nevertheless, this phenomenon occurred only with high VEGF(165) concentrations (10 ng/mL). CONCLUSIONS: The effect of VEGF(165) on proliferation and upregulation of KDR receptor expression demonstrated an autocrine phenomenon of EC sensitization. The fact that high concentrations of VEGF may be achieved in vivo by local continuous overexpression of VEGF(165) by gene transfer emphasizes the potential advantage of gene transfer over protein supplementation for therapeutic angiogenesis.

Perceived benefit after participating in positive or negative/neutral heart failure trials: the patients' perspective.

BACKGROUND: Clinical trials, the gold standard for the evaluation of new therapeutic strategies, may prove a drug to be beneficial, harmful or neutral according to its effect on the end-point(s) under study. AIMS: To study the reaction and perspective of the patients participating in a clinical heart failure trial, particularly in relation to whether the trial subsequently proved to be positive, negative or neutral. METHODS: Anonymous self-completed questionnaire was sent to 78 and returned by 70 consecutive patients 1--6 months after participating in six clinical heart failure trials. The trial was neutral or negative regarding the primary end-point in four (47 patients) of the six studies (MACH-1 trial of mibefradil, REACH trial of bosentan, CASCO trial of calcium sensitizer, ecadotril trial of neutral endopeptidase inhibitor) and positive in two (23 patients) (ICARUS Israel carvedilol study, exercise study of candesartan cilexetil). RESULTS: Most patients reported subjective global clinical benefit (78% for positive, 74% for negative or neutral trial, NS) after participating in a clinical trial. After adjustment for age, sex, level of education, previous research, perceived comprehension, and treatment allocation (active drug/placebo) in a stepwise regression model, perceived global improvement was greater in older patients (P=0.02), after participation in a positive trial (P=0.05) and in females (P=0.07). The major reason given by the patient for perceived clinical improvement was better follow-up, some believed it was due to change in medication, particularly those who had participated in a positive trial. CONCLUSIONS: More than 70% of patients participating in clinical trials of new drugs for heart failure reported perceived global improvement. Clinical improvement was greater in, but not limited to, patients who participated in positive trials. These salutary findings support the continued recruitment of patients to clinical heart failure trials.

Similar late revascularization rates 10 to 12 years after angioplasty or bypass surgery for multivessel coronary artery disease: a report from the Lady Davis Carmel Medical Center (LDCMC) Registry.

We compared completed long-term outcome and late repeat revascularization rates in 272 consecutive patients with multivessel coronary disease who underwent revascularization (95 angioplasty cohort, 177 surgical cohort) between 1984 and 1986. Long-term survival was similar at 12 years in the angioplasty (70%) and surgical (74%) cohorts (p = NS), and repeat revascularization, although more frequent in the angioplasty patients during the first 5 years of follow-up, was performed equally in the 2 patient cohorts after 10 to 12 years of follow-up.

Therapeutic angiogenesis for ischemic syndromes.

Disease caused by atherosclerosis are the most common causes of morbidity and mortality in western societies. The inadequacy of current therapeutic modalities is most pronounced in the significant proportion of patients with arterial obstructive disease, in whom anatomical and technical limitations rule out the possibility of angioplasty or surgery. Therefore, less invasive approaches are necessary to treat these patients. The development of collateral circulation improves blood flow to ischemic tissues and to alleviate ischemia-related symptoms. Our project concentrates on enhancement of the natural mechanism of angiogenesis by adenoviral based vector encoding vascular endothelial growth factor as an angiogenic factor. The aim of our study was to determine the efficacy of human vascular cell infection by adenoviral based vectors in vitro and in vivo. Human saphenous vein endothelial cells and smooth muscle cells were infected by adenoviral vectors encoding the lacZ and VEGF genes (rAdlacZ, rAdVEGF). VEGF expression by adenoviral vector-infected cells was detected by western analysis and its biological activity was examined by proliferation assay. The feasibility of adenoviral based gene transfer in vivo was evaluated after direct femoral artery injection of rAdlacZ in the rat. Vascular endothelial and smooth muscle cells expressed high levels of VEGF following rAdVEGF infection. The mitogenic effect of VEGF was validated by threefold increase in EC proliferation rate in comparison to the control groups. In vivo gene transfer was demonstrated using lacZ gene transfer to arterial wall cells in the superficial femoral artery. Efficient adenoviral based gene delivery was demonstrated both in vitro and in vivo. VEGF over-expression enhanced endothelial cell proliferation, which is the key step for induction of angiogenesis.

Late-onset heart failure as a mechanism for adverse long-term outcome in diabetic patients undergoing revascularization (a 13-year report from the Lady Davis Carmel Medical Center registry).

The adverse long-term prognosis following myocardial revascularization in diabetic patients has been ascribed to accelerated coronary disease, a higher incidence of late coronary restenosis after revascularization, and myocardial dysfunction. To examine the development of heart failure and its prognostic implications in diabetic patients, we analyzed the long-term (13-year) follow-up data of 363 patients-193 percutaneous transluminal coronary angioplasties and 170 coronary artery bypass operations-revascularized in a single cardiovascular center from 1984 to 1986. Baseline characteristics (age, previous infarction, baseline ventricular function) were similar in the 80 diabetic and 283 nondiabetic patients; multivessel disease and hypertension were marginally more common in diabetics (p = NS). Cumulative incidence of hospitalization for heart failure was high in the diabetic cohort (25% vs 11%, p = 0.001), with a rapidly increasing incidence after 5 years. Survival after first hospitalization for heart failure was markedly reduced in diabetics (11 of 20 [55%] vs 25 of 31 [81%] at 3 years; p = 0.04), as was survival free of further hospitalization for heart failure (5 of 20 [25%] vs 20 of 30 [63%]; p <0.005). Long-term 13-year survival (43% vs 78%, p <0.0001) and survival free of heart failure (33% vs 71%, p <0.0001) were decreased in diabetics, especially those with reduced ventricular function at baseline (17% vs 42%, p = 0.07). Multivariate analysis showed diabetes to be the strongest independent predictor of decreased survival (odds ratio 3. 6, 95% confidence interval 2.0 to 6.2; p <0.0001) and survival free of heart failure (odds ratio 4.0, 95% confidence interval 2.2 to 7. 1; p <0.0001) in patients undergoing revascularization. In summary, late-onset heart failure was frequent in diabetic patients after percutaneous transluminal coronary angioplasty or coronary artery bypass grafting, and once present heralded an unrelenting progressive downhill clinical course.

Same-day combined coronary angioplasty and minimally invasive coronary surgery.

Integrated myocardial revascularization combines the advantages of angioplasty, stenting, and minimally invasive surgery to revascularize patients with multivessel coronary artery disease without cardiopulmonary bypass. This pilot study showed that a new same-day management strategy, consisting of percutaneous coronary intervention followed immediately by minimally invasive surgery, was feasible and provided complete all-arterial revascularization with minimal surgical trauma, short hospital stay, and excellent early therapeutic result in 14 patients with multivessel coronary disease.

Long-term (10-year) outcome in patients with unstable angina pectoris treated by coronary balloon angioplasty.

OBJECTIVES: We sought to examine completed 10-year survival and event-free survival in patients with stable and unstable angina pectoris treated by coronary balloon angioplasty. BACKGROUND: Patients with unstable angina are at increased risk for recurrent acute coronary events. METHODS: The study included 208 consecutive patients (133 with stable and 75 with unstable angina pectoris) undergoing angioplasty from 1984 to 1986. The balloon crossed the lesion in 185 patients (121 with stable and 64 with unstable angina pectoris). Angioplasty was performed in patients with unstable angina pectoris 12+/-15 days (median 8) after symptom onset. Patients with unstable angina pectoris were classified retrospectively into Braunwald class I (n=3), class II (n=20), class III (n=28), class B (n=52) and class C (n=12). Follow-up data were obtained from hospital charts, telephone interview and official death certificates where applicable. The study had >80% power to detect a clinically significant 20% difference in survival and a 20% difference in event-free survival between the stable and unstable patient groups. RESULTS: Despite similar baseline characteristics, early (40-day) mortality was slightly higher in patients with unstable angina (4.7% [3 of 64 patients] vs. 0.8% [1 of 121 patients], p=NS). Long-term outcome was not different, because survival curves were parallel thereafter (10-year survival was 83% for those with stable and 77% for those with unstable angina, p=NS). Survival free of myocardial infarction or coronary artery bypass graft surgery at 10 years was 53% in patients with stable and 47% in patients with unstable angina (p=NS), and survival free of infarction, bypass surgery or repeat angioplasty was 32% for both groups at 10 years. In patients with Braunwald class III unstable angina, 10-year survival was 80%, as compared with 85% in other patients with unstable angina, due to the early hazard (p=NS). Survival and event-free survival were similar in patients who had had a recent myocardial infarction (Braunwald class C) and in patients with acute electrocardiographic changes. Repeat hospital admissions were not more frequent in patients with unstable angina (3.1+/-3.5 vs. 3.0+/-2.6, p=NS). CONCLUSIONS: Ten-year survival and event-free survival were similar in patients with stable and unstable angina pectoris treated by coronary balloon angioplasty, with no evidence of an increased rate of recurrent cardiovascular events in the unstable group.

Importance of diabetes mellitus and systemic hypertension rather than completeness of revascularization in determining long-term outcome after coronary balloon angioplasty (the LDCMC registry). Lady Davis Carmel Medical Center.

The study examined the 10-year outcome in a cohort of 227 unselected, consecutive patients (age 58+/-10 years) undergoing coronary balloon angioplasty between 1984 and 1986 and followed in a single cardiac center (Lady Davis Carmel Medical Center registry). In particular, we sought to identify the relative importance of the systemic risk factors diabetes and hypertension and the extent of coronary disease as opposed to procedure-related technical variables, the immediate success of the procedure, or completeness of revascularization. By life-table analysis (99% follow-up), 94% of the patients were alive at 5 years, and 77% at 10 years after angioplasty. Ten-year survival was reduced in patients with diabetes mellitus (59% vs 83%, p = 0.0008), in patients with previous myocardial infarction (68% vs 85%, p = 0.01), in patients with ejection fraction <50% (55% vs 82%, p = 0.005), and in patients with 3-vessel disease (58% vs 84% and 86% for 1- and 2-vessel disease, respectively, p = 0.04). Diabetes mellitus was the major independent predictor of poor survival (adjusted odds ratio 3.1, 95% confidence interval 1.55 to 6.19, p = 0.001). Survival at 10 years was identical in 199 patients in whom angioplasty was complete and in 25 in whom the balloon catheter did not cross the lesion, although bypass surgery was more frequent in the latter group (45% vs 21%, p = 0.001). Incomplete revascularization did not predict poor survival (72% vs 79% with complete angioplasty, p = NS). Event-free survival at 10 years for the whole group was 29%, and 49% of patients survived with no event other than a single repeat angioplasty procedure. Multivessel disease, hypertension, and diabetes mellitus were independent predictors of decreased event-free survival, but incomplete revascularization was not. Thus, long-term outcome after coronary balloon angioplasty was related to diabetes mellitus, systemic hypertension, and extent of coronary disease, but not to the immediate success of the procedure or completeness of revascularization.

Ventricular remodeling: from bedside to molecule.

The multiple mechanisms that bring about the decompensation of the hypertrophic remodeled myocardium are synergistic and not fully understood. Our current hypothesis is that the increased stress on the ventricle is initially offset by compensatory myocardial hypertrophy. In many instances, however, progressive ventricular dilatation and heart failure occur as a result of maladaptive hypertrophy (abnormal myosin-actin production), programmed cell death (apoptosis) and/or changes in the interstitial vasculature and collagen composition. The molecular and genetic background to these processes includes changes in myocardial gene expression, activation of the local tissue renin-angiotensin and other neurohormonal systems, increased matrix metalloproteinase activity (including collagenase), and expression of certain components of the immune system, such as TNF-alpha. Future research will hopefully provide better methods for limiting the remodeling-ventricular dilatation process by novel pharmacotherapies, gene therapy and, possibly, surgical therapy, and determine the impact of such interventions on survival.

Angiogenesis by gene therapy: a new horizon for myocardial revascularization?

The concept of therapeutic angiogenesis is based on the premise that the potential for vascular growth inherent in vascular tissue can be utilized to promote the development of new blood vessels under the influence of the appropriate growth factors. Direct application of growth factors of the fibroblast (acidic, basic fibroblast growth factor, FGF-5), endothelial (vascular endothelial growth factor) and other series has been effective in preliminary studies. Angiogenesis by gene transfer provides an attractive alternative, with the advantage that the protein may continue to be secreted for a longer period of time and that the gene may be targeted to specific tissues to enhance efficacy and reduce systemic side effects. Angiogenesis by gene transfer is currently under investigation using a variety of growth factors and a wide array of potential delivery systems. These include application of the gene as naked DNA or by viral vector in the proximal vessel by direct intravascular injection, interventional cardiologic techniques (hydrogel coating on balloon, double balloon system, stent implantation) or by direct application to adventitia, pericardium or ischemic tissue distal to the site of arterial obstruction. As our understanding of the molecular and genetic processes underlying angiogenesis increases, and as we examine the results of preliminary animal and human protocols, we hope to develop the potential of angiogenesis by gene transfer for therapeutic use.

Importance of immediate and very early postprocedural angiographic and thallium-201 single photon emission computed tomographic perfusion measurements in predicting late results after coronary intervention.

We examined prospectively the hypothesis that the adequacy of initial dilatation may be a major determinant of the late result of coronary angioplasty and that a better assessment of initial dilatation can be made from a combined angiographic and perfusion study than from angiography alone. Angiographic and perfusion (thallium-201 single-photon-emission computed tomography) measurements were made very early (18 to 24 hours) after coronary angioplasty in 59 patients (67 lesions) and also immediately (37 +/- 16 minutes) after the procedures in 19 of them (23 lesions). The early measurements, singly, in combination, and as a restenosis index (restenosis index = thallium-201 ischemic score (units) - minimal luminal area (squared millimeters) were examined as predictors of the late angiographic result. At late angiography (5.5 +/- 2.2 months after angioplasty), residual stenosis was related to the immediate and very early postangioplasty minimal luminal dimension, thallium-201 ischemic score, and restenosis index, and also to day-1 loss and lesion length. The combination of a normal result in the immediate or early thallium-201 perfusion study with a large ( > or = 2 mm) angiographic luminal dimension stratified a group of patients with better long-term results after angioplasty and a lower incidence of late restenosis (p = 0.03). The findings emphasize the importance of the initial procedure as a determinant of the late result of angioplasty.

Inhibition of intravascular thrombosis and vascular smooth muscle cell proliferation by gene therapy.

Intravascular thrombosis, smooth muscle cell proliferation and matrix production lead to arterial luminal narrowing and reduction of blood flow to various organs. Alteration of gene expression of the arterial wall cells to inhibit thrombosis and smooth muscle activation is emerging as a new and exciting therapeutic modality for cardiovascular pathology. We have used genetically modified endothelial cells to seed endovascular prostheses and tested cell adhesion to the prostheses both in vitro and in vivo. We also used two catheter-based systems to deliver genes directly to the arterial wall cells in vivo employing retroviral and adenoviral vectors. With efficient vectors for gene transfer and high level expression of proteins by the transduced cells, gene therapy will serve as a major therapy for post-angioplasty restenosis, unstable angina pectoris and vascular grafts stenosis.

Vascular gene therapy.

Gene therapy is emerging as a new and exciting therapeutic modality for cardiovascular pathology. The work reported here was carried out in the National Heart, Lung and Blood Institute (NHLBI) in Bethesda, MD, USA, where genetically engineered endothelial cells were used to seed endovascular prostheses and cell adhesion to the prostheses was tested both in vitro and in vivo. Two catheter based systems were used to deliver genes to the arterial wall cells in vivo, employing retroviral and adenoviral vectors. Efficient gene transfer to vascular cells in vivo was achieved with adenoviral vectors.