Efficiency of intracoronary (IC) adenoviral vector transfection is impaired by the vascular endothelium. Ischemia and substances that increase vascular permeability (sodium nitroprusside, nitroglycerin) may augment adenoviral vector transfection efficiency (TE). We tested whether TE of adenoviral vector following IC infusion is improved by nitrates or by ischemia. Fluoroscopically guided angioplasty balloon catheters occluded the coronary artery in Yorkshire pigs and delivered adenoviral type 5 vector encoding the luciferase gene (Ad5Luc, 10(11) viral particles). TE (luciferase activity) was minimal and was not augmented by IC co-administration of 50 μg/min sodium nitroprusside to nonischemic myocardium. Two (but not one) 3-min episodes of occlusion tended to increase luciferase activity (p=0.06), and luciferase activity was further increased by IC co-administration of nitroglycerin (p<0.001). After 75 min of coronary artery occlusion, luciferase activity was greater than with shorter periods of ischemia, and was significantly greater in the ischemia-reperfused zone compared to the border zone 3 and 14 days after infusion; there was no transfection in nonischemic myocardium. IC delivery of Ad5Luc into post-ischemic myocardium caused no local inflammation or hemodynamic instability. We conclude that the uptake of IC Ad5 to ischemic reperfused myocardium validates use of IC Ad5 delivery protocols in future human gene therapy trials in patients following myocardial ischemia.

We tested the hypothesis that intracoronary delivery of an adenoviral vector (Ad5) encoding transgenes for either Insulin-like growth factor-I (IGF-1) or fibroblast growth factor 2 (FGF-2) would reduce myocardial I/R injury in a closed-chest porcine model. In Yorkshire pigs the LAD was occluded by a fluoroscopically guided angioplasty balloon for 75 min, and assigned to 4 groups based on an intracoronary infusion of 1) saline, 2) IGF-1, 3) FGF-2 or 4) Ad5 encoding luciferase (Ad5Luc to quantify transfection) at 15 min of reperfusion to the LAD and to LCx. Reperfusion was carried out for 3 or 14 days. There were no hemodynamic or rhythm disturbances associated with any infusion. After 3 days of R expression of luciferase was robust in samples from target myocardium, with sequentially less in the border zone and remote LV myocardium, confirming viral transfection. After 14 days of R, the viral transfection decreased significantly. Ejection fraction (EF) recovered toward baseline values in all groups (Figure A) without group differences. There were no group differences in systolic and diastolic performance (pressure-volume relations) among groups. Infarct size [% of LV or area at risk mass] was similar among the four groups (Figure B). In conclusion, Ad5 transfection with IGF-1 or FGF-2 was safe, but did not have overall treatment effect on LV performance indices or infarct size 14 days after I/R despite viral transfection.

ABSTRACTWe assessed the safety and efficacy of Formulated Collagen Gel (FCG) alone and with Ad5PDGF‐B (GAM501) compared with Standard of Care (SOC) in patients with 1.5–10.0 cm2 chronic diabetic neuropathic foot ulcers that healed <30% during Run‐in. Wound size was assessed by planimetry of acetate tracings and photographs in 124 patients. Comparison of data sets revealed that acetate tracings frequently overestimated areas at some sites. For per‐protocol analysis, 113 patients qualified using acetate tracings but only 82 qualified using photographs. Prior animal studies suggested that collagen alone would have little effect on healing and would serve as a negative control. Surprisingly trends for increased incidence of complete closure were observed for both GAM501 (41%) and FCG (45%) vs. Standard of Care (31%). By photographic data, Standard of Care had no significant effect on change in wound radius (mm/week) from during Run‐in to Week 1 (−0.06±0.32 to 0.78±1.53, p=ns) but both FCG (−0.08±0.61 to 1.97±1.77, p<0.002) and GAM501 (−0.02±0.58 to 1.46±1.37, p<0.002) significantly increased healing rates that gradually declined over subsequent weeks. Both GAM501 and FCG appeared to be safe and well tolerated, and alternate dosing schedules hold promise to improve overall complete wound closure in adequately powered trials.

Background: Inflammatory cells and fibroblasts orchestrate repair of hard and soft tissues by infiltrating the injured site and secreting growth factors and cytokines, stimulating tissue regeneration, progenitor cells, and laying down scar tissue. Although growth factor proteins have demonstrated efficacy in preclinical models of tissue repair, most have failed to produce significant improvements in the clinical setting.The Problem: The limited clinical success of growth factor proteins has been attributed to poor delivery methods and short half-lives. If the gene encoding a growth factor could be transfected into cells in the tissue defect, prolonged production of growth factor could improve efficacy over protein therapy. Combining gene therapy vectors with biocompatible matrices to hold the vector for transfection and provide a scaffolding for cell migration could improve tissue repair. This approach is called gene-activated matrix (GAM).Basic/Clinical Science Advances: Stimulation of tissue repair with GAMs has been demonstrated in preclinical models of cutaneous wound repair, nerve regeneration, ischemia, and bony defects. A phase 1/2 clinical study for the treatment of chronic diabetic neuropathic wounds utilizing the gene encoding human platelet-derived growth factor-B (Ad5PDGF-B in 2.6% collagen, GAM501) demonstrated safety and potential biological activity. A doubled-blinded, randomized, placebo-controlled phase 2b study to evaluate the safety and efficacy of a single and double application is currently underway.Clinical Care Relevance: New treatment options are needed to provide a better daily quality of life and avoid amputations in patients having diabetic foot ulcers. Advanced products such as GAM501 with a single or double administration could offer medical practitioners and patients an important new treatment option having a potentially higher therapeutic efficacy, as well as enhancing patient compliance.Conclusion: GAM501 is the first GAM product to progress to phase 2 development. GAMs offer promise in multiple areas of tissue regeneration and repair, including angiogenesis, bone fracture, nerve, cartilage, ligament, and surgical applications.

Background: The normal wound healing process is mediated by specific cells and cytokines, especially growth factors. In chronic wounds, there is often a deficiency of endogenous growth factors, which impairs healing.The Clinical Problem: Administration of exogenous growth factor proteins has had limited success because of inefficient delivery, inadequate persistence, and poor patient compliance with daily treatment regimens for prolonged periods of time.The Clinical Solution: If the gene encoding a growth factor could be transfected into cells in the tissue defect, prolonged production of growth factor could improve therapeutic efficacy. Formulating the DNA vector in a biocompatible matrix (Gene Activated Matrix) can further enhance the product potential. GAM501 (an adenovector encoding platelet-derived growth factor-B gene within 2.6% collagen matrix) has progressed to a phase 2b clinical trial.Market-History, Current, and Prospects: Only recombinant human platelet-derived growth factor-B homodimer (becaplermin) has been approved by the U.S. Food and Drug Administration for the treatment of diabetic ulcers. There remains a compelling medical need for a more practical and effective therapy for nonhealing ulcers, which is a large and expanding market.Business Challenges-Policies & Practices: An ideal wound healing product would promote faster healing, be more effective and easier to use, and be less expensive itself or require fewer associated medical resources than current products (i.e., faster, better, and cheaper). A combination product must generally demonstrate that both components are required for efficacy, or that the combination is superior to the individual components.Public-Private Partnership-SWOT (strength, weakness, opportunities, threat) Analysis: Developing a combination wound healing product requires collaboration with the U.S. Food and Drug Administration, and gene therapy trials must be reviewed by the Recombinant DNA Advisory Committee at National Institutes of Health.Conclusion: Collaboration with regulatory agencies during all phases of clinical development and periodic reanalysis of trial design strategies and commercial plans is essential in developing combination products for wound healing.

Background: Efficiency of intracoronary adenoviral vector (Ad) uptake in the heart may be impaired by permeability barrier function of the vascular endothelium. Substances that increase vascular pe...

OBJECTIVES:The goal of this study was to explore the effects of angiogenic gene therapy.BACKGROUND:Preclinical studies with intracoronary administration of Ad5FGF-4 (alferminogene tadenovec, Generx, Berlex Biosciences, Richmond, California) suggested it could induce angiogenesis and provide a new clinical approach to the treatment of chronic angina pectoris. Two preliminary clinical trials provided evidence that it could improve exercise treadmill test (ETT) time and myocardial perfusion. The AGENT (Angiogenic GENe Therapy)-3 and -4 trials of a low and high dose of Ad5FGF-4 for chronic angina were initiated in the U.S. and other countries and enrolled 532 patients in a randomized, double-blind, placebo-controlled fashion. Both studies were halted when an interim analysis of the AGENT-3 trial indicated that the primary end point change from baseline in total ETT time at 12 weeks would not reach significance.METHODS:We performed a pooled data analysis from the 2 nearly identical trials to investigate possible treatment effects on primary and secondary end points in prespecified subgroups.RESULTS:The effect of placebo was large and not different than active treatment in men, but the placebo effect in women was negligible and the treatment effect was significantly greater than placebo. We found a significant, gender-specific beneficial effect of Ad5FGF-4 on total ETT time, time to 1 mm ST-segment depression, time to angina, and Canadian Cardiovascular Society class in women. This is the first clinical report of a gender difference in response to cardiac angiogenic therapy.CONCLUSIONS:The potential importance of the observed gender-specific angiogenic response on the clinical treatment of refractory angina is substantial and deserves further investigation. (Efficacy and Safety of Intracoronary Ad5FGF-4 in Patients With Stable Angina; http://www.clinicaltrials.gov/ct/show/NCT00346437; NCT00346437) (Safety and Efficacy of Intracoronary Ad5FGF-4 in Patients With Stable Angina [AGENT-4]; http://www.clinicaltrials.gov/ct/show/NCT00185263; NCT00185263) (AWARE; http://www.clinicaltrials.gov/ct/show/NCT000438867; NCT000438867).

Conclusions The potential importance of the observed gender-specific angiogenic response on the clinical treatment of refractory angina is substantial and deserves further investigation. (Efficacy and Safety of Intracoronary Ad5FGF-4 in Patients With Stable Angina; http://www.clinicaltrials.gov/ct/show/NCT00346437 ; NCT00346437 ) (Safety and Efficacy of Intracoronary Ad5FGF-4 in Patients With Stable Angina [AGENT-4]; http://www.clinicaltrials.gov/ct/show/NCT00185263 ; NCT00185263 ) (AWARE; http://www.clinicaltrials.gov/ct/show/NCT00438867 ; NCT00438867 )

In this issue of Circulation,' Hori and colleagues report the results of using three antioxidant agents in an attempt to reduce ischemic injury in a coronary microembolization model. Repeated injections of 15-,um microspheres resulted in progressive microvascular obstruction, decreased function, and edema until the extent of embolization decreased flow to nearly zero (less than 0.08 ml/min/ g). In this model, adenosine release from ischemic regions induces hyperemia in nonobstructed vessels. With successive injections, progressively smaller border zones surround areas of increasing microvascular obstruction until the extent of embolization occludes

Lipopolysaccharide (LPS) from gram-negative bacteria circulates in acute, subacute, and chronic conditions. It was hypothesized that LPS directly induces cardiac apoptosis. In adult rat ventricular myocytes (isolated with depyrogenated digestive enzymes to minimize tolerance), LPS (10 ng/ml) decreased the ratio of Bcl-2 to Bax at 12 h; increased caspase-3 activity at 16 h; and increased annexin V, propidium iodide, and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining at 24 h. Apoptosis was blocked by the caspase inhibitor benzyloxycarbonyl-valine-alanine-aspartate fluoromethylketone (Z-VAD-fmk), captopril, and angiotensin II type 1 receptor (AT(1)) inhibitor (losartan), but not by inhibitors of AT(2) receptors (PD-123319), tumor necrosis factor-alpha (TNFRII:Fc), or nitric oxide (N(G)-monomethyl-L-arginine). Angiotensin II (100 nmol/l) induced apoptosis similar to LPS without additive effects. LPS in vivo (1 mg/kg iv) increased apoptosis in left ventricular myocytes for 1-3 days, which dissipated after 1-2 wk. Losartan (23 mg. kg(-1). day(-1) in drinking water for 3 days) blocked LPS-induced in vivo apoptosis. In conclusion, low levels of LPS induce cardiac apoptosis in vitro and in vivo by activating AT(1) receptors in myocytes.

Background—The angiogenic response to myocardial ischemia can be augmented in animal models by gene transfer with the use of a replication defective adenovirus (Ad) containing a human fibroblast growth factor (FGF) gene. Methods and Results—The objectives of the Angiogenic GENe Therapy (AGENT) trial were to evaluate the safety and anti-ischemic effects of 5 ascending doses of Ad5-FGF4 in patients with angina and to select potentially safe and effective doses for subsequent study. Seventy-nine patients with chronic stable angina Canadian Cardiovascular Society class 2 or 3 underwent double-blind randomization (1:3) to placebo (n=19) or Ad5-FGF4 (n=60). Safety evaluations were performed at each visit and exercise treadmill testing (ETT) at baseline and at 4 and 12 weeks. Single intracoronary administration of Ad5-FGF4 seemed to be safe and well tolerated with no immediate adverse events. Fever of <1-day duration occurred in 3 patients in the highest-dose group. Transient, asymptomatic elevations in liver enzymes occurred in 2 patients in lower-dose groups. Serious adverse events during follow-up (mean, 311 days) were not different between placebo and Ad5-FGF4. Overall, patients who received Ad5-FGF4 tended to have greater improvements in exercise time at 4 weeks (1.3 versus 0.7 minutes, P =NS, n=79). A protocol-specified, subgroup analysis showed the greatest improvement in patients with baseline ETT ≤10 minutes (1.6 versus 0.6 minutes, P =0.01, n=50). Conclusions—Results show evidence of favorable anti-ischemic effects with Ad5-FGF4 compared with placebo, and it appears to be safe. Angiogenic gene transfer with Ad5-FGF4 shows promise as a new therapeutic approach to the treatment of angina pectoris.

(21) and induce apoptosis in cardiac myocytes (24), it has been assumed that secondary mediators are responsible for the cardiotoxic effects of LPS. However, cardiac myocytes are sensitive to the direct effects of low levels of LPS, independently of mediators released from noncardiac myocytes. We found that clinically relevant levels of LPS (low ng/ml) activate cardiac myocytes within hours to depress myofilament responsiveness to calcium (41) and impair cell volume regulation (30). Cardiac myocytes may be directly sensitive to LPS due to the expression of Toll-like receptor 4 (11), the transmembrane component of the LPS receptor (3). Apoptosis, an energy-dependent process of programmed cell death, is increased in heart failure from multiple causes (18), including sepsis (31). In sepsis, it is unknown whether cardiac apoptosis is caused by LPS itself or is a consequence of secondary effects activated by LPS. If low levels of LPS induce cardiac apoptosis, this has important implications because LPS circulates in several subacute and chronic conditions with less prominent activation of secondary mediators than in sepsis. Decompensated heart failure (32), pancreatitis, liver disease, chronic infections, smoking, and exercise (15), for example, are associated with plasma LPS levels in the picograms per milliliter to the nanograms per milliliter range. It was hypothesized that low levels of LPS induce apoptosis in cardiac myocytes. The rationales for this hypothesis were the following points. First, cardiac apoptosis may occur in subacute or chronic conditions associated with circulating LPS. Second, blockade of secondary mediators may not prevent LPS-induced cardiac apoptosis. This is analogous to the failure for inhibitors of TNF-, IL-1, platelet-activating factor, bradykinin, prostaglandins (43), and NO (14) to reduce mortality in sepsis and septic shock in several large,

Protection of ischemic myocardium is an important unmet need in reperfusion therapy of acute myocardial infarction. Myocardial ischemia and reperfusion induce necrosis and apoptosis in cardiomyocytes. Caspase processing and activation are critical steps in most receptor and nonreceptor pathways of apoptosis. Caspase inhibitors have been shown to reduce ischemia reperfusion injury in cardiac muscle. Information about dose response and time of administration are needed to optimize the design of preclinical studies. We used isolated adult rabbit cardiomyocytes subjected to metabolic inhibition (MI) and recovery to examine the role of caspases and caspase inhibitors, the dose response, and the timing of administration. In vitro inhibitory concentrations (Ki) were determined for purified caspases. Cardiomyocytes subjected to MI were treated with peptidomimetic fluoromethyl ketone inhibitors of caspases before or during MI, or at recovery. Caspase inhibitors were most effective when added before MI and included throughout recovery, but were partially protective if added after MI. The optimal concentration of the inhibitors tested was approximately 10 microM. Protection was sustained when cells were allowed to recover for 4 or 24 h. These results suggest that caspase activation is an important component of myocyte injury mediated by MI and recovery. Low doses of caspase inhibitors were identified that reduce injury in this model system, and further investigations using in vivo models are warranted.

The signal transduction pathways by which ischemia-reperfusion leads to apoptosis may involve the JNK pathway, ceramide generation, and inhibition of protective PKC pathways, The biochemical events associated with apoptosis include mitochondrial inactivation, cytochrome c dislocation, caspase activation, and cytoplasmic acidification. Through the concerted efforts of multiple classes of enzymes, apoptosis is accomplished, resulting In the death of a cell in which potentially transforming oncogenes have been degraded and inflammatory contents are contained within the plasma membrane until the fragments can be ingested by phagocytes, This non-inflammatory mode of cell death permits tissue remodeling with minimal scar formation, and so is preferable to necrotic cell death, The distinction between apoptosis and necrosis, which implies different mechanisms of cell death, is blurred in the case of a pathologic insult such as ischemia-reperfusion. It is suggested that it is more useful to view cell death in the context of whether or not it can be prevented.

Recently, we found that vacuolar proton ATPase (VPATPase) operates in cardiomyocytes as a complementary proton-extruding mechanism, Its activity was increased by preconditioning with resultant attenuation of intracellular acidification during ischemia. In this study, we examined whether VPATPase-mediated proton efflux during metabolic inhibition/recovery may spare Na+ overload via Na+-H+ exchange, attenuate Na+-Ca2+ exchange, and decrease apoptosis. Neonatal rat cardiomyocytes were subjected to 2- to 3-hour metabolic inhibition with cyanide and 2-deoxyglucose and 24-hour recovery. The effect of VPATPase inhibition by 50 nmol/L bafilomycin A(1) on apoptosis, pH(i), and [Ca2+](i) was studied by flow cytometry with propidium iodide, seminaphthorhodafluor (SNARF)-1-AM, and indo-1-AM staining, respectively. VPATPase inhibition increased the amount of apoptosis measured after 24 hours of recovery and abrogated the protective effect of inhibition of Na+-H+ exchange by (5-N-ethyl-N-isopropyl)amiloride (EIPA). Dual blockade of VPATPase and Na+-H+ exchange was additive in effect with EIPA on pH(i) during metabolic inhibition/recovery and recovery from the acid challenge with sodium propionate. VPATPase blockade increased the rate of accumulation of intracellular Ca2+ at the beginning of metabolic inhibition and abrogated the delaying effect of EIPA on intracellular Ca2+ accumulation. These results indicate that VPATPase plays an important accessory role in cardiomyocyte protection by reducing acidosis and Na+-H+ exchange-induced Ca2+ overload.