Brian H. Annex

The VIVA Trial

Background—Recombinant human vascular endothelial growth factor protein (rhVEGF) stimulates angiogenesis in animal models and was well tolerated in Phase I clinical trials. VIVA (Vascular endothelial growth factor in Ischemia for Vascular Angiogenesis) is a double-blind, placebo-controlled trial designed to evaluate the safety and efficacy of intracoronary and intravenous infusions of rhVEGF. Methods and Results—A total of 178 patients with stable exertional angina, unsuitable for standard revascularization, were randomized to receive placebo, low-dose rhVEGF (17 ng · kg−1 · min−1), or high-dose rhVEGF (50 ng · kg−1 · min−1) by intracoronary infusion on day 0, followed by intravenous infusions on days 3, 6, and 9. Exercise treadmill tests, angina class, and quality of life assessments were performed at baseline, day 60, and day 120. Myocardial perfusion imaging was performed at baseline and day 60. At day 60, the change in exercise treadmill test (ETT) time from baseline was not different between groups (placebo, +48 seconds; low dose, +30 seconds; high dose, +30 seconds). Angina class and quality of life were significantly improved within each group, with no difference between groups. By day 120, placebo-treated patients demonstrated reduced benefit in all three measures, with no significant difference compared with low-dose rhVEGF. In contrast, high-dose rhVEGF resulted in significant improvement in angina class (P =0.05) and nonsignificant trends in ETT time (P =0.15) and angina frequency (P =0.09) as compared with placebo. Conclusions—rhVEGF seems to be safe and well tolerated. rhVEGF offered no improvement beyond placebo in all measurements by day 60. By day 120, high-dose rhVEGF resulted in significant improvement in angina and favorable trends in ETT time and angina frequency.

Aging, Progenitor Cell Exhaustion, and Atherosclerosis

Background—Atherosclerosis is largely attributed to chronic vascular injury, as occurs with excess cholesterol; however, the effect of concomitant vascular aging remains unexplained. We hypothesize that the effect of time in atherosclerosis progression is related to obsolescence of endogenous progenitor cells that normally repair and rejuvenate the arteries. Methods and Results—Here we show that chronic treatment with bone marrow–derived progenitor cells from young nonatherosclerotic ApoE−/− mice prevents atherosclerosis progression in ApoE−/− recipients despite persistent hypercholesterolemia. In contrast, treatment with bone marrow cells from older ApoE−/− mice with atherosclerosis is much less effective. Cells with vascular progenitor potential are decreased in the bone marrow of aging ApoE−/− mice, but cells injected from donor mice engraft on recipient arteries in areas at risk for atherosclerotic injury. Conclusions—Our data indicate that progressive progenitor cell deficits may contribute to the development of atherosclerosis.

A novel protective effect of erythropoietin in the infarcted heart

Erythropoietin (EPO) has been shown to protect neurons from ischemic stroke, but can also increase thrombotic events and mortality rates in patients with ischemic heart disease. We reasoned that benefits of EPO might be offset by increases in hematocrit and evaluated the direct effects of EPO in the ischemic heart. We show that preconditioning with EPO protects H9c2 myoblasts in vitro and cardiomyocytes in vivo against ischemic injury. EPO treatment leads to significantly improved cardiac function following myocardial infarction. This protection is associated with mitigation of myocyte apoptosis, translating into more viable myocardium and less ventricular dysfunction. EPO-mediated myocyte survival appears to involve Akt activation. Importantly, cardioprotective effects of EPO were seen without an increase in hematocrit (eliminating oxygen delivery as an etiologic factor in myocyte survival and function), demonstrating that EPO can directly protect the ischemic and infarcted heart.

Regional Angiogenesis With Vascular Endothelial Growth Factor in Peripheral Arterial Disease A Phase II Randomized, Double-Blind, Controlled Study of Adenoviral Delivery of Vascular Endothelial Growth Factor 121 in Patients With Disabling Intermittent Claudication

Background—“Therapeutic angiogenesis” seeks to improve perfusion by the growth of new blood vessels. The Regional Angiogenesis with Vascular Endothelial growth factor (RAVE) trial is the first major randomized study of adenoviral vascular endothelial growth factor (VEGF) gene transfer for the treatment of peripheral artery disease (PAD). Methods and Results—This phase 2, double-blind, placebo-controlled study was designed to test the efficacy and safety of intramuscular delivery of AdVEGF121, a replication-deficient adenovirus encoding the 121-amino-acid isoform of vascular endothelial growth factor, to the lower extremities of subjects with unilateral PAD. In all, 105 subjects with unilateral exercise-limiting intermittent claudication during 2 qualifying treadmill tests, with peak walking time (PWT) between 1 to 10 minutes, were stratified on the basis of diabetic status and randomized to low-dose (4×109 PU) AdVEGF121, high-dose (4×1010 PU) AdVEGF121, or placebo, administered as 20 intramuscular injections to the index leg in a single session. The primary efficacy end point, change in PWT (&Dgr;PWT) at 12 weeks, did not differ between the placebo (1.8±3.2 minutes), low-dose (1.6±1.9 minutes), and high-dose (1.5±3.1 minutes) groups. Secondary measures, including &Dgr;PWT, ankle-brachial index, claudication onset time, and quality-of-life measures (SF-36 and Walking Impairment Questionnaire), were also similar among groups at 12 and 26 weeks. AdVEGF121 administration was associated with increased peripheral edema. Conclusions—A single unilateral intramuscular administration of AdVEGF121 was not associated with improved exercise performance or quality of life in this study. This study does not support local delivery of single-dose VEGF121 as a treatment strategy in patients with unilateral PAD.

Therapeutic angiogenesis with recombinant fibroblast growth factor-2 for intermittent claudication (the TRAFFIC study): a randomised trial

BACKGROUND Recombinant fibroblast growth factor-2 (rFGF-2) improves perfusion in models of myocardial and hindlimb ischaemia. We investigated whether one or two doses of intra-arterial rFGF-2 improves exercise capacity in patients with moderate-to-severe intermittent claudication. METHODS 190 patients with intermittent claudication caused by infra-inguinal atherosclerosis were randomly assigned (1:1:1) bilateral intra-arterial infusions of placebo on days 1 and 30 (n=63); rFGF-2 (30 microg/kg) on day 1 and placebo on day 30 (single-dose, n=66); or rFGF-2 (30 microg/kg) on days 1 and 30 (double-dose, n=61). Primary outcome was 90-day change in peak walking time. Secondary outcomes included ankle-brachial pressure index and safety. The main analysis was per protocol. FINDINGS Before 90 days, six patients had undergone peripheral revascularisation and were excluded, and ten withdrew or had missing data. 174 were therefore assessed for primary outcome. Peak walking time at 90 days was increased by 0.60 min with placebo, by 1.77 min with single-dose, and by 1.54 min with double-dose. By ANOVA, the difference between groups was p=0.075. In a secondary intention-to-treat analysis, in which all 190 patients were included, the difference was p=0.034. Pairwise comparison showed a significant difference between placebo and single-dose (p=0.026) but placebo and double-dose did not differ by much (p=0.45). Serious adverse events were similar in all groups. INTERPRETATION Intra-arterial rFGF-2 resulted in a significant increase in peak walking time at 90 days; repeat infusion at 30 days was no better than one infusion. The findings of TRAFFIC provide evidence of clinical therapeutic angiogenesis by intra-arterial infusion of an angiogenic protein.

Rescue of Diabetes-Related Impairment of Angiogenesis by Intramuscular Gene Therapy with Adeno-VEGF

Diabetes is a major risk factor for coronary and peripheral artery diseases. Although diabetic patients often present with advanced forms of these diseases, it is not known whether the compensatory mechanisms to vascular ischemia are affected in this condition. Accordingly, we sought to determine whether diabetes could: 1) impair the development of new collateral vessel formation in response to tissue ischemia and 2) inhibit cytokine-induced therapeutic neovascularization. Hindlimb ischemia was created by femoral artery ligation in nonobese diabetic mice (NOD mice, n = 20) and in control C57 mice (n = 20). Hindlimb perfusion was evaluated by serial laser Doppler studies after the surgery. In NOD mice, measurement of the Doppler flow ratio between the ischemic and the normal limb indicated that restoration of perfusion in the ischemic hindlimb was significantly impaired. At day 14 after surgery, Doppler flow ratio in the NOD mice was 0.49+/-0.04 versus 0.73+/-0.06 for the C57 mice (P< or =0.005). This impairment in blood flow recovery persisted throughout the duration of the study with Doppler flow ratio values at day 35 of 0.50+/-0.05 versus 0.90+/-0.07 in the NOD and C57 mice, respectively (P< or =0.001). CD31 immunostaining confirmed the laser Doppler data by showing a significant reduction in capillary density in the NOD mice at 35 days after surgery (302+/-4 capillaries/mm2 versus 782+/-78 in C57 mice (P< or =0.005). The reduction in neovascularization in the NOD mice was the result of a lower level of vascular endothelial growth factor (VEGF) in the ischemic tissues, as assessed by Northern blot, Western blot and immunohistochemistry. The central role of VEGF was confirmed by showing that normal levels of neovascularization (compared with C57) could be achieved in NOD mice that had been supplemented for this growth factor via intramuscular injection of an adenoviral vector encoding for VEGF. We conclude that 1) diabetes impairs endogenous neovascularization of ischemic tissues; 2) the impairment in new blood vessel formation results from reduced expression of VEGF; and 3) cytokine supplementation achieved by intramuscular adeno-VEGF gene transfer restores neovascularization in a mouse model of diabetes.

Atherosclerotic Vascular Disease Conference Writing Group III: Pathophysiology

The existence of atherosclerosis has been recognized for >500 years; as a pathological condition, it has been recognized for >150 years. Understanding of atherosclerotic vascular disease (AVD) has evolved most dramatically over the past 25 years with the growth of the field of vascular biology.1 Numerous studies have described this disease as a diffuse and progressive process with a variable distribution and clinical presentation that is dependent on the regional circulation involved. Factors that may influence these differences include the size and structure of the affected artery, local and regional flow, changes in microcirculatory alterations, and end-organ damage. This report discusses the general concepts of atherosclerosis, pathophysiology, microcirculatory disturbances, regional responses to atherosclerosis and ischemia, and recommendations for future research, programs, and advocacy. Atherosclerosis involves several highly interrelated processes, including lipid disturbances, platelet activation, thrombosis, endothelial dysfunction, inflammation, oxidative stress, vascular smooth cell activation, altered matrix metabolism, remodeling, and genetic factors.2 This sequence is shown schematically in Figure 1 and described in detail below. Figure 1. The 7 stages of development of an atherosclerotic plaque. First LDL moves into the subendothelium and is oxidized by macrophage and SMCs (1 and 2). Release of growth factors and cytokines attracts additional monocytes (3 and 4). Foam cell accumulation and SMC proliferation result in growth of the plaque (6, 7, and 8). Risk factors play an important role in initiating and accelerating the complex process of atherosclerosis. Risk factors for atherosclerosis are also the primary method of risk assessment and the target for therapeutic intervention in the prevention of premature vascular disease. Interestingly, the impact of these risk factors on disease development and progression in the peripheral vasculature are not the same as those in the coronary vessels and may represent an avenue of investigation to explain variations in clinical presentation …

Results of a Double-Blind, Placebo-Controlled Study to Assess the Safety of Intramuscular Injection of Hepatocyte Growth Factor Plasmid to Improve Limb Perfusion in Patients With Critical Limb Ischemia

Background— The Study to Assess the Safety of Intramuscular Injection of Hepatocyte Growth Factor Plasmid to Improve Limb Perfusion in Patients With Critical Limb Ischemia (HGF-STAT trial) determined the effect of hepatocyte growth factor (HGF) plasmid on safety and limb tissue perfusion as measured by transcutaneous oxygen tension (TcPo2) in patients with critical limb ischemia (CLI). Methods and Results— Randomized patients with rest pain or ischemic ulcers and TcPo2 <40 mm Hg and/or toe pressure <50 mm Hg received placebo or HGF-plasmid intramuscular injection as follows: 0.4 mg at days 0, 14, and 28 (low dose); 4.0 mg at days 0 and 28 (middle dose); or 4.0 mg at days 0, 14, and 28 (high dose). Patients were evaluated for safety, changes in TcPo2 and ankle and toe pressure, amputation, and wound healing. Ninety-three of 104 treated patients were evaluated for safety (mean age 70 years, 63% male, 53% diabetic, 64% with tissue loss, mean ankle-brachial index 0.41, and mean toe pressure 26 mm Hg). Adverse events occurred in 86% of the patients, most of which were related to CLI or comorbid conditions and were not different between groups. TcPo2 (mean±SE) increased at 6 months in the high-dose group (24.0±4.2 mm Hg, 95% CI 15.5 to 32.4 mm Hg) compared with the placebo (9.4±4.2 mm Hg, 95% CI 0.9 to 17.8), low-dose (11.1±3.7 mm Hg, CI 3.7 to 18.7 mm Hg), and middle-dose (7.3±4.8 mm Hg, CI −2.2 to 17.0 mm Hg) groups (ANCOVA P=0.0015). There was no difference between groups in secondary end points, including ankle-brachial index, toe-brachial index, pain relief, wound healing, or major amputation. Conclusions— Intramuscular injection of HGF plasmid was safe and well tolerated. Larger studies to determine whether HGF plasmid can improve wound healing and limb salvage in patients with CLI are warranted.

A target-mediated model to describe the pharmacokinetics and hemodynamic effects of recombinant human vascular endothelial growth factor in humans

The Vascular Endothelial Growth Factor (VEGF) in Ischemiafor Vascular Angiogenesis (VIVA) trial was a double‐blind, placebo‐controlled, phase II clinical trial designed to evaluate the safety, efficacy, and pharmacokinetics of combinedintracoronary and intravenous infusions of recombinant human vascular endothelial growth factor (rhVEGF165) for therapeutic angiogenesis. This study describes the use of a mechanism‐based model to characterize the nonlinear kinetics observed after intravenous administration of rhVEGF165. The model predicts that rhVEGF165 distribution occurs through both saturable binding to high‐affinity receptors and reversible interactions with low‐affinity binding sites.

Capillary density of skeletal muscle: A contributing mechanism for exercise intolerance in class II-III chronic heart failure independent of other peripheral alterations

OBJECTIVES The study was conducted to determine if the capillary density of skeletal muscle is a potential contributor to exercise intolerance in class II-III chronic heart failure (CHF). BACKGROUND Previous studies suggest that abnormalities in skeletal muscle histology, contractile protein content and enzymology contribute to exercise intolerance in CHF. METHODS The present study examined skeletal muscle biopsies from 22 male patients with CHF compared with 10 age-matched normal male control patients. Aerobic capacities, myosin heavy chain (MHC) isoforms, enzymes, and capillary density were measured. RESULTS The patients with CHF demonstrated a reduced peak oxygen consumption when compared to controls (15.0+/-2.5 vs. 19.8+/-5.0 ml x kg(-1) x min(-1), p <0.05). Using cell-specific antibodies to directly assess vascular density, there was a reduction in capillary density in CHF measured as the number of endothelial cells/fiber (1.42+/-0.28 vs. 1.74+/-0.35, p = 0.02). In CHF, capillary density was inversely related to maximal oxygen consumption (r = 0.479, p = 0.02). The MHC IIx isoform was found to be higher in patients with CHF versus normal subjects (28.5+/-13.6 vs. 19.5+/-9.4, p <0.05). CONCLUSIONS There was a significant reduction in microvascular density in patients with CHF compared with the control group, without major differences in other usual histologic and biochemical aerobic markers. The inverse relationship with peak oxygen consumption seen in the CHF group suggests that a reduction in microvascular density of skeletal muscle may precede other skeletal muscle alterations and play a critical role in the exercise intolerance characteristic of patients with CHF.