Georges von Degenfeld

Collagen Matrices Enhance Survival of Transplanted Cardiomyoblasts and Contribute to Functional Improvement of Ischemic Rat Hearts

Background— Cardiac cell transplantation is limited by poor graft viability. We aimed to enhance the survival of transplanted cardiomyoblasts using growth factor-supplemented collagen matrices. Methods and Results— H9c2 cardiomyoblasts were lentivirally transduced to express firefly luciferase and green fluorescent protein (GFP). Lewis rats underwent ligation of the left anterior descending artery (LAD) ligation to induce an anterior wall myocardial infarction. Hearts (n=9/group) were harvested and restored ex vivo with 1×106 genetically labeled H9c2 cells either in (1) saline-suspension, or seeded onto (2) collagen-matrix (Gelfoam [GF];), (3) GF/Matrigel (GF/MG), (4) GF/MG/VEGF (10 &mgr;g/mL), or (5) GF/MG/FGF (10 &mgr;g/mL). Hearts were then abdominally transplanted into syngeneic recipients (working heart model). Controls (n=6/group) underwent infarction followed by GF implantation or saline injection. Cell survival was evaluated using optical bioluminescence on days 1, 5, 8, 14, and 28 postoperatively. At 4 weeks, fractional shortening and ejection fraction were determined using echocardiography and magnetic resonance imaging, respectively. Graft characteristics were assessed by immunohistology. Bioluminescence signals on days 5, 8, and 14 were higher for GF-based grafts compared with plain H9c2 injections (P<0.03). Signals were higher for GF/MG grafts compared with GF alone (P<0.02). GFP-positive, spindle-shaped H9c2 cells were found integrated in the infarct border zones at day 28. Left ventricular (LV) function of hearts implanted with collagen-based grafts was better compared with controls (P<0.05). Vascular endothelial growth factor or fibroblast growth factor did not further improve graft survival or heart function. Conclusions— Collagen matrices enhance early survival of H9c2 cardiomyoblasts after transplantation into ischemic hearts and lead to improved LV function. Further optimization of the graft design should make restoration of large myocardial infarctions by tissue engineering approaches effective.

Overexpression of Dimethylarginine Dimethylaminohydrolase Reduces Tissue Asymmetric Dimethylarginine Levels and Enhances Angiogenesis

Background—This study was designed to determine whether overexpression of the enzyme dimethylarginine dimethylaminohydrolase (DDAH) could enhance angiogenesis by reducing levels of the endogenous nitric oxide synthase (NOS) inhibitor asymmetric dimethylarginine (ADMA). Methods and Results—In DDAH1 transgenic (TG) and wild-type mice (each n=42), the role of DDAH overexpression on angiogenesis was studied by use of the disk angiogenesis system and a murine model of hindlimb ischemia (each n=21). After surgery, animals were treated with either PBS or the NOS inhibitors ADMA or N&ohgr;-nitro-l-arginine methyl ester (L-NAME; each 250 &mgr;mol · kg−1 · d−1) by use of osmotic minipumps (each n=7). L-NAME was chosen to study an inhibitor that is not degraded by DDAH. Neovascularization in the disk angiogenesis system was impaired by both NOS inhibitors; however, TG animals were resistant to the effects of ADMA on neovascularization. Similarly, TG mice were more resistant to the inhibitory effect of ADMA on angioadaptation (angiogenesis and arteriogenesis) after hindlimb ischemia, as assessed by fluorescent microsphere studies and postmortem microangiograms. Enhanced neovascularization and limb perfusion in TG mice were associated with reduced plasma and tissue ADMA levels and enhanced tissue NOS enzyme activity. Conclusions—We describe a novel mechanism by which DDAH regulates postnatal neovascularization. Therapeutic manipulation of DDAH expression or activity may represent a novel approach to restore tissue perfusion.

Microenvironmental VEGF distribution is critical for stable and functional vessel growth in ischemia

The critical role of vascular endothelial growth factor (VEGF) expression levels in developmental angiogenesis is well established. Nonetheless, the effects of different local (microenvironmental) VEGF concentrations in ischemia have not been studied in the adult organism, and VEGF delivery to patients has been disappointing. Here, we demonstrate the existence of both lower and upper threshold levels of microenvironmental VEGF concentrations for the induction of therapeutic vessel growth in ischemia. In the ischemic hind limb, implantation of myoblasts transduced to express VEGF164 at different levels per cell increased blood flow only moderately, and vascular leakage and aberrant preangiomatous vessels were always induced. When the same total dose was uniformly distributed by implanting a monoclonal population derived from a single VEGF‐expressing myoblast, blood flow was fully restored to nonischemic levels, collateral growth was induced, and ischemic damage was prevented. Hemangiomas were avoided and only normal, pericyte‐covered vessels were induced persisting over 15 mo. Surprisingly, clones uniformly expressing either lower or higher VEGF levels failed to provide any functional benefit. A biphasic effect of VEGF dose on vessel number and diameter was found. Blood flow was only improved if vessels were increased both in size and in number. Microenvironmental VEGF concentrations determine efficacy and safety in a therapeutic setting.—von Degenfeld, G., Banfi, A., Springer, M. L., Wagner, R. A., Jacobi, J., Ozawa, C. R., Merchant, M. J., Cooke, J. P., Blau, H. M. Microenvironmental VEGF distribution is critical for stable and functional vessel growth in ischemia. FASEB J. 20, E2277–E2287 (2006)

Luminescent imaging of beta-galactosidase activity in living subjects using sequential reporter-enzyme luminescence.

We generated a sequential reporter-enzyme luminescence (SRL) technology for in vivo detection of β-galactosidase (β-gal) activity. The substrate, a caged D-luciferin–galactoside conjugate, must first be cleaved by β-gal before it can be catalyzed by firefly luciferase (FLuc) to generate light. As a result, luminescence is dependent on β-gal activity. Using this technology, constitutive β-gal activity in engineered cells and inducible tissue-specific β-gal expression in transgenic mice can now be visualized noninvasively over time. A substantial advantage of β-gal as a bioluminescent probe is that the enzyme retains full activity outside of cells, unlike FLuc, which requires intracellular cofactors. As a result, antibodies conjugated to the recombinant β-gal enzyme can be used to detect endogenous cells and extracellular antigens in vivo. Thus, coupling the properties of FLuc to the advantages of β-gal permits bioluminescent imaging applications that previously were not possible.

Reevaluation of the Role of VEGF-B Suggests a Restricted Role in the Revascularization of the Ischemic Myocardium

Objective—The endogenous role of the VEGF family member vascular endothelial growth factor-B (VEGF-B) in pathological angiogenesis remains unclear. Methods and Results—We studied the role of VEGF-B in various models of pathological angiogenesis using mice lacking VEGF-B (VEGF-B−/−) or overexpressing VEGF-B167. After occlusion of the left coronary artery, VEGF-B deficiency impaired vessel growth in the ischemic myocardium whereas, in wild-type mice, VEGF-B167 overexpression enhanced revascularization of the infarct and ischemic border zone. By contrast, VEGF-B deficiency did not affect vessel growth in the wounded skin, hypoxic lung, ischemic retina, or ischemic limb. Moreover, VEGF-B167 overexpression failed to enhance vascular growth in the skin or ischemic limb. Conclusion—VEGF-B appears to have a relatively restricted angiogenic activity in the ischemic heart. These insights might offer novel therapeutic opportunities.

Adenoviral Human BCL-2 Transgene Expression Attenuates Early Donor Cell Death After Cardiomyoblast Transplantation Into Ischemic Rat Hearts

Background— Cell transplantation for myocardial repair is limited by early cell death. Gene therapy with human Bcl-2 (hBcl-2) has been shown to attenuate apoptosis in the experimental setting. Therefore, we studied the potential benefit of hBcl-2 transgene expression on the survival of cardiomyoblast grafts in ischemic rat hearts. Methods and Results— H9c2 rat cardiomyoblasts were genetically modified to express both firefly luciferase and green fluorescent protein (mH9c2). The cells were then transduced with adenovirus carrying hBcl-2 (AdCMVhBcl-2/mH9c2). Lewis rats underwent ligation of the left anterior descending artery (LAD) to induce a sizable left ventricular (LV) infarct. Hearts were explanted and the infarcted region was restored using collagen matrix (CM) seeded with 1×106 mH9c2 cells (n=9) or AdCMVhBcl-2/mH9c2 cells (n=9). Control animals received CM alone (n=6) or no infarct (n=6). Restored hearts were transplanted into the abdomen of syngeneic recipients in a “working heart” model. Cell survival was evaluated using optical bioluminescence imaging on days 1, 5, 8, 14, and 28 after surgery. The left heart function was assessed 4 weeks postoperatively using echocardiography and magnetic resonance imaging. During 4 weeks after surgery, the optical imaging signal for the AdCMVhBCL2/mH9c2 group was significantly (P<0.05) higher than that of the mH9c2-control group. Both grafts led to better fractional shortening (AdCMVhBcl-2/mH9c2: 0.21±0.03; mH9c2: 0.21±0.04; control: 0.15±0.03; P=0.04) and ejection fraction (AdCMVhBcl-2/mH9c2: 47.0±6.2; mH9c2: 48.7±6.1; control: 34.3±6.0; P=0.02) compared with controls. Importantly, no malignant cells were found in postmortem histology. Conclusion— Transduction of mH9c2 cardiomyoblasts with AdCMVhBcl-2 increased graft survival in ischemic rat myocardium without causing malignancies. Both AdCMVhBcl-2/mH9c2 and mH9c2 grafts improved LV function.

Therapeutic angiogenesis due to balanced single-vector delivery of VEGF and PDGF-BB

Therapeutic angiogenesis by delivery of vascular growth factors is an attractive strategy for treating debilitating occlusive vascular diseases, yet clinical trials have thus far failed to show efficacy. As a result, limb amputation remains a common outcome for muscle ischemia due to severe atherosclerotic disease, with an overall incidence of 100 per million people in the United States per year. A challenge has been that the angiogenic master regulator vascular endothelial growth factor (VEGF) induces dysfunctional vessels, if expressed outside of a narrow dosage window. We tested the hypothesis that codelivery of platelet‐derived growth factor‐BB (PDGF‐BB), which recruits pericytes, could induce normal angiogenesis in skeletal muscle irrespective of VEGF levels. Coexpression of VEGF and PDGF‐BB encoded by separate vectors in different cells or in the same cells only partially corrected aberrant angiogenesis. In marked contrast, coexpression of both factors in every cell at a fixed relative level via a single bicistronic vector led to robust, uniformly normal angiogenesis, even when VEGF expression was high and heterogeneous. Notably, in an ischemic hindlimb model, single‐vector expression led to efficient growth of collateral arteries, revascularization, increased blood flow, and reduced tissue damage. Furthermore, these results were confirmed in a clinically applicable gene therapy approach by adenoviral‐mediated delivery of the bicistronic vector. We conclude that coordinated expression of VEGF and PDGF‐BB via a single vector constitutes a novel strategy for harnessing the potency of VEGF to induce safe and efficacious angiogenesis.—Banfi, A., von Degenfeld, G., Gianni‐Barrera, R., Reginato, S., Merchant, M. J., McDonald, D. M., Blau, H. M. Therapeutic angiogenesis due to balanced single‐vector delivery of VEGF and PDGF‐BB. FASEB J. 26, 2486‐2497 (2012). www.fasebj.org

Discordant effects of a soluble VEGF receptor on wound healing and angiogenesis

Soluble receptors to vascular endothelial growth factor (VEGF) can inhibit its angiogenic effect. Since angiogenesis is involved in wound repair, we hypothesized that adenovirus-mediated gene transfer of a soluble form of VEGF receptor 2 (Flk-1) would attenuate wound healing in mice. C57Bl/6J and genetically diabetic (db/db) mice (each n=20) received intravenous (i.v.) injections of recombinant adenoviruses (109 PFU) encoding the ligand-binding ectodomain of VEGF receptor 2 (Flk-1) or cDNA encoding the murine IgG2α Fc fragment (each n=10). At 4 days after gene transfer, two full-thickness skin wounds (0.8 cm) were created on the dorsum of each animal. Wound closure was measured over 9–14 days after which wounds were resected for histological analysis. Prior to killing, fluorescent microspheres were systemically injected for quantitation of wound vascularity. Single i.v. injections of adenoviruses encoding soluble Flk-1 significantly decreased wound angiogenesis in both wild-type and diabetic mice. Fluorescence microscopy revealed a 2.0-fold (wild type) and 2.9-fold (diabetic) reduction in wound vascularity in Flk-1-treated animals (p<0.05). Impairment of angiogenesis was confirmed by CD31 immunohistochemistry. Interestingly, despite significant reductions in wound vascularity, wound closure was not grossly delayed. Our data indicates that while VEGF function is essential for optimal wound angiogenesis, it is not required for wound closure.

Myoblast-mediated gene transfer for therapeutic angiogenesis and arteriogenesis

Therapeutic angiogenesis aims at generating new blood vessels by delivering growth factors such as VEGF and FGF. Clinical trials are underway in patients with peripheral vascular and coronary heart disease. However, increasing evidence indicates that the new vasculature needs to be stabilized to avoid deleterious effects such as edema and hemangioma formation. Moreover, a major challenge is to induce new vessels that persist following cessation of the angiogenic stimulus. Mature vessels may be generated by modulating timing and dosage of growth factor expression, or by combination of ‘growth’ factors with ‘maturation’ factors like PDGF‐BB, angiopoietin‐1 or TGF‐β. Myoblast‐mediated gene transfer has unique characteristics that make it a useful tool for studying promising novel approaches to therapeutic angiogenesis. It affords robust and long‐lasting expression, and can be considered as a relatively rapid form of ‘adult transgenesis’ in muscle. The combined insertion of different gene constructs into single myoblasts and their progeny allows the simultaneous expression of different ‘growth’ and ‘maturation’ factors within the same cell in vivo. The additional insertion of a reporter gene makes it possible to analyze the phenotype of the vessels surrounding the transgenic muscle fibers into which the myoblasts have fused. The effects of timing and duration of gene expression can be studied by using tetracycline‐inducible constructs, and dosage effects by selecting subpopulations consistently expressing distinct levels of growth factors. Finally, the autologous cell‐based approach using transduced myoblasts could be an alternative gene delivery system for therapeutic angiogenesis in patients, avoiding the toxicities seen with some viral vectors.

A universal technology for monitoring G-protein-coupled receptor activation in vitro and noninvasively in live animals

G‐protein coupled receptors (GPCRs) are a versatile and ubiquitous family of membrane receptors that transmit extracellular signals to mammalian cells and constitute the most important class of drug targets. Yet, sensitive and specific methods are lacking that would allow quantitative comparisons of pharmacologic properties of these receptors in physiological or pathological settings in live animals. We sought to overcome these limitations by employing low affinity, reversible β‐galactosidase complementation to quantify GPCR activation via interaction with β‐arres‐tin. A panel of cell lines was engineered expressing different GPCRs together with the reporter system. In vitro evaluation revealed highly sensitive, dynamic, and specific assessment of GPCR agonists and antagonists. Following implantation of the cells into mice, it was possible for the first time to monitor pharmacological GPCR activation and inhibition in their physiological context by noninvasive bioluminescence imaging in living animals. This technology has unique advantages that enable novel applications in the functional investigation of GPCR modulation in live animals in biological research and drug discovery.— von Degenfeld G., Wehrman T. S., Hammer, M. M., Blau H. M. A universal technology for monitoring G‐protein‐coupled receptor activation in vitro and noninvasively in live animals. FASEB J. 21, 3819–3826 (2007)