Karen A. Vincent

Angiogenesis Is Induced in a Rabbit Model of Hindlimb Ischemia by Naked DNA Encoding an HIF-1α/VP16 Hybrid Transcription Factor

BackgroundHypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription factor that regulates expression of genes involved in O2 homeostasis, including vascular endothelial growth factor (VEGF), a potent stimulator of angiogenesis. We sought to exploit this native adaptive response to hypoxia as a treatment for chronic ischemia. Methods and ResultsA hybrid protein consisting of DNA-binding and dimerization domains from the HIF-1&agr; subunit and the transactivation domain from herpes simplex virus VP16 protein was constructed to create a strong, constitutive transcriptional activator. After transfection into HeLa, C6, and Hep3B cells, this chimeric transcription factor was shown to activate expression of the endogenous VEGF gene, as well as several other HIF-1 target genes in vitro. The bioactivity of HIF-1&agr;/VP16 hybrid gene transfer in vivo was examined in a rabbit model of hindlimb ischemia. Administration of HIF-1&agr;/VP16 was associated with significant improvements in calf blood pressure ratio, angiographic score, resting and maximal regional blood flow, and capillary density (all P <0.01). ConclusionsThe HIF-1&agr;/VP16 hybrid transcription factor is able to promote significant improvement in perfusion of an ischemic limb. These results confirm the feasibility of a novel approach for therapeutic angiogenesis in which neovascularization may be achieved indirectly by use of a transcriptional regulatory strategy.

Hypoxia-Inducible Factor-1 Mediates Activation of Cultured Vascular Endothelial Cells by Inducing Multiple Angiogenic Factors

Abstract— Hypoxia-inducible factor-1 (HIF-1) mediates transcriptional activation of vascular endothelial growth factor (VEGF) and other hypoxia-responsive genes. Transgenic expression of a constitutively stable HIF-1&agr; mutant increases the number of vascular vessels without vascular leakage, tissue edema, or inflammation. This study aimed to investigate the molecular basis by which HIF-1 mediates the angiogenic response to hypoxia. In primary human endothelial cells, hypoxia, desferrioxamine, or infection with Ad2/HIF-1&agr;/VP16, an adenoviral vector encoding a constitutively stable hybrid form of HIF-1&agr;, increased the mRNA and protein levels of VEGF, angiopoietin-2 (Ang-2), and angiopoietin-4 (Ang-4). Infection with Ad2/CMVEV (a control vector expressing no transgene) had no effect. Angiopoietin-1 (Ang-1) expression was not detected in human endothelial cells. Ang-4 was also induced by hypoxia or Ad2/HIF-1&agr;/VP16 in human cardiac cells, whereas Ang-1 expression remained unchanged. Recombinant Ang-4 protein protected endothelial cells against serum starvation-induced apoptosis and increased cultured endothelial cell migration and tube formation. Ad2/HIF-1&agr;/VP16 stimulated endothelial cell proliferation and tube formation. Hypoxia- or Ad2/HIF-1&agr;/VP16-induced tube formation was significantly reduced by a Tie-2 inhibitor. These results suggest that HIF-1 mediates the angiogenic response to hypoxia by upregulating the expression of multiple angiogenic factors. Ang-4 can function similarly as Ang-1 and substitute for Ang-1 to participate in hypoxia-induced angiogenesis. Activation of the angiopoietin/Tie-2 system may play a role in the ability of HIF-1 to induce hypervascularity without excessive permeability.

Scaleable chromatographic purification process for recombinant adeno-associated virus (rAAV)

Adeno‐associated virus (AAV) is a human parvovirus currently being developed as a vector for gene therapy applications. Traditionally AAV has been purified from cell lysates using CsCl gradients; this approach however is not likely to be useful in large‐scale manufacturing. Moreover gradient‐purified AAV vectors tend to be contaminated with significant levels of cellular and adenoviral proteins and nucleic acid. To address the issue of purification we have developed a process scale method for the rapid and efficient purification of recombinant AAV (rAAV) from crude cellular lysates.

A Constitutively Active Hypoxia-Inducible Factor-1α/VP16 Hybrid Factor Activates Expression of the Human B-Type Natriuretic Peptide Gene

Hypoxia-inducible factor-1 (HIF-1) is a primary regulator of the physiological response to hypoxia. A recombinant adenovirus expressing a constitutively active hybrid form of the HIF-1α subunit (Ad2/HIF-1α/VP16) is being evaluated as a gene therapy for the treatment of peripheral vascular disease. Ad2/HIF-1α/VP16 up-regulates known HIF-1-responsive genes, including those involved in angiogenesis. Expression profile analysis revealed that the brain natriuretic peptide (BNP) gene was significantly up-regulated in response to HIF-1α/VP16 in human fetal cardiac cells. Real-time reverse transcription-polymerase chain reaction analyses confirmed transcriptional activation of the BNP gene by HIF-1α/VP16 in human but not rat cardiac cells. Because hypoxia itself did not increase human BNP gene expression in these analyses, the mechanism of the HIF-1α/VP16 effect was determined. Analyses of promoter deletion mutants suggested that the cis-acting sequence in the human BNP promoter mediating activation by HIF-1α/VP16 was a putative HIF-1 responsive element (HRE) located at -466. An SV40 basal promoter-luciferase plasmid containing a minimal BNP HRE was up-regulated by HIF-1α/VP16, whereas a similar construct carrying a mutation within the HIF-1 binding site was not. Mutation of an E-box motif within the BNP HRE reduced HIF-1α/VP16-mediated transcriptional activation by 50%. Gel-shift analyses showed that both the native HIF-1α and HIF-1α/VP16 are able to bind to a probe containing the HIF-1 binding site. These experiments demonstrate the existence of a functional HRE in the BNP promoter and further define the scope and mechanism of action of Ad2/HIF-1α/VP16.

Differential effects of membrane and soluble Fas ligand on cardiomyocytes: role in ischemia/reperfusion injury

Cardiomyocyte apoptosis by Fas ligand (FasL)/Fas signaling is associated with various pathophysiological conditions, such as ischemia/reperfusion injury and congestive heart failure. In this study, we tested the hypothesis that shedding of membrane FasL is a mechanism for downregulating FasL/Fas signaling and both membrane and soluble FasL are involved in cardiomyocyte hypoxia/reoxygenation (H/R) injury. We also examined the relative importance of mitochondrial damage and direct cleavage of the executioner caspases by activated initiator caspase 8 in the propagation of FasL/Fas signaling activated by either recombinant membrane FasL or H/R. We demonstrated that in neonatal rat cardiomyocytes maintained under normal culture conditions, recombinant human soluble FasL increased caspase 3 activation by twofold but did not reduce cell viability. In contrast, infection with a recombinant adenoviral vector expressing the non-cleavable human FasL (Ad2/nchFasL) resulted in cardiomyocyte death that was attenuated by soluble FasL. H/R increased the mRNA levels of both FasL and Fas and activated caspases 8, 9 and 3, indicating the activation of FasL/Fas signaling. Z-IETD.fmk and Z-LEHD.fmk, selective inhibitors for caspases 8 and 9, respectively, abolished caspase 3 activation induced by Ad2/nchFasL or H/R. Z-IETD.fmk also significantly reduced Ad2/nchFasL- or H/R-induced cardiomyocyte death. H/R potentiated membrane FasL-induced cell death. These results suggest that shedding of membrane FasL downregulates FasL/Fas signaling in cardiomyocytes and both membrane and soluble FasL contribute to H/R injury. Activation of FasL/Fas signaling by either recombinant membrane FasL under normal culture conditions or H/R causes cardiomyocyte death mainly through the mitochondrial damage/caspase 9 activation pathway.

A hypoxic inducible factor-1α hybrid enhances collateral development and reduces vascular leakage in diabetic rats

Diabetes mellitus is a common comorbidity of atherosclerosis. Hypoxia‐inducible factor‐1 (HIF‐1) is the master regulator of the angiogenic response to hypoxia.

Enhancement of Fas ligand-induced inhibition of neointimal formation in rabbit femoral and iliac arteries by coexpression of p35

Adenovirus-mediated gene transfer of Fas ligand (FasL) inhibits neointimal formation in balloon-injured rat carotid arteries. Vascular smooth muscle (VSM) cells coexpressing murine FasL and p35, a baculovirus gene that inhibits caspase activity, are not susceptible to FasL-mediated apoptosis in vitro but are capable of inducing apoptosis of VSM cells that do not express p35. We reasoned that coexpression of p35 in FasL-transduced VSM cells in vivo would promote their survival, enhance FasL-induced apoptosis of adjacent VSM cells, and thereby facilitate a greater inhibition of neointimal formation. In balloon-injured rabbit femoral arteries, either Ad2/FasL/p35 or Ad2/FasL was infused into the injured site and withdrawn 20 min later. Both vectors induced a dose-dependent reduction (p < 0.05) of the neointima-to-media ratio when assessed 14 days later. However, Ad2/FasL/p35 exhibited a significantly greater inhibition of neointimal formation than Ad2/FasL. In a more clinically relevant model of restenosis, rabbit iliac arteries were injured with an angioplasty catheter under fluoroscopic guidance. Adenoviral vectors were delivered locally to the injured site over a period of 2 min, using a porous infusion balloon catheter. Twenty-eight days after gene transfer angiographic and histologic assessments indicated a significant (p < 0.05) inhibition of iliac artery lumen stenosis and neointimal formation by Ad2/FasL/p35 (5 x 10(11) particles per artery). The extent of inhibition was comparable to that achieved with Ad2/TK, an adenoviral vector encoding thymidine kinase (5 x 10(11) particles per artery) and coadministration of ganciclovir for 7 days. These data suggest that coexpression of p35 in FasL-transduced VSM cells is more potent at inhibiting neointimal formation and as such represents an improved gene therapy approach for restenosis.

Adenovirus-Mediated Expression of β-Adrenergic Receptor Kinase C-Terminus Reduces Intimal Hyperplasia and Luminal Stenosis of Arteriovenous Polytetrafluoroethylene Grafts in Pigs

Background—Hemodialysis vascular access dysfunction is the single most important cause of morbidity in kidney hemodialysis patients. Failure of an arteriovenous polytetrafluoroethylene (PTFE) graft, the most common form of hemodialysis access, is primarily due to intimal hyperplasia and thrombosis at the venous anastomosis. Methods and Results—This study was aimed at evaluating the efficacy and safety of an adenoviral vector (Ad2/&bgr;ARKct) encoding the carboxyl terminus of &bgr;-adrenergic receptor kinase (&bgr;ARKct) in a pig model of arteriovenous PTFE graft failure. Transduction of the external jugular vein with Ad2/&bgr;ARKct (5E9, 5E10, or 5E11 particles per vein) did not result in systemic toxicity, as measured by clinical and pathological assessments. Ad2/&bgr;ARKct significantly reduced neointimal hyperplasia in the graft/vein anastomosis. It also improved the graft patency rate and angiographic score, as measured histologically and angiographically, compared with vehicle or empty viral vector controls. Conclusions—Our results suggest that local administration of adenoviral vectors encoding &bgr;ARKct into the jugular vein represents a viable strategy to treat AV graft hemodialysis vascular access failure.

Catheter-mediated delivery of adenoviral vectors expressing β-adrenergic receptor kinase C-terminus inhibits intimal hyperplasia and luminal stenosis in rabbit iliac arteries

Previous studies have shown that incubation of balloon‐injured rat carotid arteries with adenoviral vectors encoding the carboxyl terminus of the β‐adrenergic receptor kinase (Ad2/βARKct) for 30 min reduces neointima formation. However, it is unclear whether this beneficial effect of βARKct could be achieved using a catheter‐based vector delivery system and whether the observed inhibition of neointima formation translated into a reduction of vessel stenosis.

92. Evaluation of Producer Cell Line Platform for Production of Oversized AAV-FVIII Vectors

Recombinant adeno-associated virus (rAAV) vectors are being evaluated as gene delivery vehicles in several clinical trials. The 4.7 kb wild-type (WT) size genome of AAV presents a challenge for incorporating larger transgenes with incomplete vector genome (vg) packaging being a frequent outcome. To test the feasibility of producing oversized rAAV production using the producer cell line (PCL) method, we generated slightly oversized rAAV vectors (harboring 5.1 or 5.4 kb sized vgs) containing a liver-restricted promoter (mTTR) and a codon-optimized cDNA encoding human B-domain deleted FVIII (FVIIIco). Genomes were packaged into the AAVrh8R serotype vector using the PCL process and compared to matched vectors generated via the triple transfection (TXN) method. Vectors were then characterized for production yields, integrity of packaged genomes and homogeneity. The data showed that the PCL platform was able to produce oversized AAV vectors at levels that were 10- to 100-fold higher than the TXN process with yields greater than 100,000 vg/cell. The PCLs were stable with consistent production maintained up to 60 passages. Southern and dot blot analyses of the packaged genomes demonstrated encapsidation of genomes larger than 4.7 kb in the PCL generated vector while the majority of genomes packaged via the TXN method were 4.7 kb in size. Furthermore, the PCL process generated more vector DNA-containing particles and less packaging of non-vector DNA. Testing the PCL generated vectors in the hemophilia A knock-out (KO) mouse model showed a 2-fold higher plasma FVIII activity (Coatest) and vg copies in the livers than obtained with the vectors made by the TXN process. In summary, the PCL production process generated higher yields of oversized rAAV/FVIIIco vectors as well as higher quality vectors than the TXN method. Hence, the PCL platform may be used for producing greater quality oversized rAAV vectors at levels that can meet the needs for clinical studies.