Vascular Regeneration in Ischemic Hindlimb by Adeno-Associated Virus Expressing Conditionally Silenced Vascular Endothelial Growth Factor.

BACKGROUND: Critical limb ischemia (CLI) is the extreme manifestation of peripheral artery disease, a major unmet clinical need for which lower limb amputation is the only option for many patients. After 2 decades in development, therapeutic angiogenesis has been tested clinically via intramuscular delivery of proangiogenic proteins, genes, and stem cells. Efficacy has been modest to absent, and the largest phase 3 trial of gene therapy for CLI reported a worsening trend of plasmid fibroblast growth factor. In all clinical trials to date, gene therapy has used unregulated vectors with limited duration of expression. Only unregulated extended expression vectors such as adeno-associated virus (AAV) and lentivirus have been tested in preclinical models. METHODS AND RESULTS: We present preclinical results of ischemia (hypoxia)-regulated conditionally silenced (CS) AAV-human vascular endothelial growth factor (hVEGF) gene delivery that shows efficacy and safety in a setting where other strategies fail. In a BALB/c mouse model of CLI, we show that gene therapy with AAV-CS-hVEGF, but not unregulated AAV or plasmid, vectors conferred limb salvage, protection from necrosis, and vascular regeneration when delivered via intramuscular or intra-arterial routes. All vector treatments conferred increased capillary density, but organized longitudinal arteries were selectively generated by AAV-CS-hVEGF. AAV-CS-hVEGF therapy reversibly activated angiogenic and vasculogenic genes, including Notch, SDF1, Angiopoietin, and Ephrin-B2. Reoxygenation extinguished VEGF expression and inactivated the program with no apparent adverse side effects. CONCLUSIONS: Restriction of angiogenic growth factor expression to regions of ischemia supports the safe and stable reperfusion of hindlimbs in a clinically relevant murine model of CLI.

Prevention of nocturnal elevation of intraocular pressure by gene transfer of dominant-negative RhoA in rats.

IMPORTANCE: We developed a gene transfer tool for the control of nocturnal elevated intraocular pressure (IOP). OBJECTIVE: To demonstrate that inhibiting the trabecular meshwork RhoA pathway by delivering a mutated, dominant-negative RhoA gene (dnRhoA) carried inside a long-expressing recombinant virus would reduce nocturnal elevated IOP in a living animal. DESIGN AND SETTING: We generated an optimized recombinant viral molecule by inserting a mutated RhoA complementary DNA with a translation enhancer-promoter into a specially designed plasmid containing mutated viral terminal repeats. We then generated the virus particle, self-complementary adeno-associated virus serotype 2 carrying the mutated gene (scAAV2.dnRhoA) and assessed its function in vitro by infecting primary human trabecular meshwork cells and in vivo by injecting living rats intracamerally with therapeutic and control viruses. Three different models of 12-hour light and dark cycles were used. Viruses were injected when animals showed the circadian dark IOP elevation. The IOP measurements were conducted with a tonometer at 2 to 4 hours after onset of the nocturnal and diurnal cycles. Values at preinjection time were used as baselines. Animals were euthanized at 4 to 8 weeks after injection. EXPOSURES: Intraocular injection of rodent eyes with the recombinant viral vector scAAV2.dnRhoA. MAIN OUTCOMES AND MEASURES: Nocturnal elevation of IOP blocked for prolonged periods by transferred RhoA gene. RESULTS: By visual inspection, human trabecular meshwork cells infected with scAAV2.dnRhoA showed diminished stress fiber formation. Living rats exhibited a circadian IOP cycle that could be reset by adjusting light conditions to facilitate light and dark nocturnal IOP studies. A single-dose injection of scAAV2.dnRhoA into the rat eyes prevented elevation of IOP during the nocturnal cycle for at least 4 weeks (mean [SE], 9.2 [0.2] mm Hg light IOP and 9.6 [0.4] mm Hg dark IOP), while control eyes showed a significantly higher IOP over baseline (9.5 [0.4] mm Hg light IOP and 13.5 [0.3] mm Hg dark IOP). CONCLUSIONS AND RELEVANCE: To our knowledge, this is the first example of a gene transfer strategy that prevents nocturnal IOP elevation in living animals for prolonged periods. Inhibiting the RhoA pathway upstream of Rho kinase with a safe gene drug could provide a new enhanced treatment for long-term management of elevated nocturnal IOP.

Bone marrow transplantation transfers age-related susceptibility to neovascular remodeling in murine laser-induced choroidal neovascularization.

PURPOSE: Neovascular remodeling (NVR), the progression of small capillaries into large-caliber arterioles with perivascular fibrosis, represents a major therapeutic challenge in neovascular age-related macular degeneration (AMD). Neovascular remodeling occurs after laser-induced choroidal neovascularization (CNV) in aged but not young mice. Additionally, bone marrow-derived cells, including macrophages, endothelial precursor cells, and mesenchymal precursor cells, contribute to CNV severity. In this study, we investigated the impact of aged bone marrow transplantation (BMT) on the degree of fibrosis, size, and vascular morphology of CNV lesions in a mouse model of laser-induced CNV. METHODS: Young (2 months) and old (16 months) mice were transplanted with green fluorescent protein (GFP)-labeled bone marrow isolated from either young or old donors. Laser CNV was induced 1 month following transplant, and eyes were analyzed via choroidal flat mounts and immunohistochemistry 1 month postlaser. The identity of cells infiltrating CNV lesions was determined using specific markers for the labeled transplanted cells (GFP+), macrophages (F4/80+), perivascular mesenchymal-derived cells (smooth muscle actin, SMA+), and endothelial cells (CD31+). RESULTS: Bone marrow transplantation from aged mice transferred susceptibility to NVR into young recipients. Inversely, transplantation of young marrow into old mice prevented NVR, preserving small size and minimal fibrosis. Mice with NVR demonstrated a greater relative contribution of marrow-derived SMA+ perivascular mesenchymal cells as compared to other cells. CONCLUSIONS: Our findings indicate that the status of bone marrow is an important determining factor of neovascular severity. Furthermore, we find that perivascular mesenchymal cells, rather than endothelial cells, derived from aged bone marrow may contribute to increased CNV severity in this murine model of experimental neovascularization.

Treatment of sheep steroid-induced ocular hypertension with a glucocorticoid-inducible MMP1 gene therapy virus.

PURPOSE: To investigate whether intracameral injection of the adenovirus vector AdhGRE.MMP1 would reduce or prevent elevated intraocular pressure (IOP) induced by corticosteroids in living animals. METHODS: Glucocorticoid-inducible adenovirus vectors carrying wild-type or mutant forms of human metalloproteinase 1 (MMP1 and mutMMP1) cDNAs were generated. An adenovirus carrying no gene (Ad5.CMV.Null) was used as an additional control. Sheep were injected intracamerally with 30 microL of each vector, either previously or after the induction of increased IOP with topical prednisolone or sub-Tenon triamcinolone under various protocols. IOP was measured with a Perkins tonometer. Inflammation was monitored by visual inspection. RESULTS: In eyes in which IOP was already elevated to 24 to 30 mm Hg, injection of AdhGRE.MMP1 reduced IOP by 70% in 24 hours and to 10 to 13 mm Hg in 48 hours. In eyes with normal IOP (9-11 mm Hg), preinjection of the virus protected against the increase in IOP normally produced by the corticosteroid. IOP remained at a level of approximately 12 mm Hg for 5 days despite the continuous application of the corticosteroid. Injections of the control viruses had no hypotensive effects. There were no signs of ocular inflammation or discomfort to the animals. CONCLUSIONS: A single dose of a gene therapy vector carrying an inducible metalloproteinase human gene can both protect against the IOP increase produced by corticosteroid instillation in the sheep model and quickly reverse the IOP increase previously elicited by the corticosteroid. These results are a first step toward a treatment of steroid-glaucoma with inducible overexpression of extracellular matrix modulator genes.

Development of a gene therapy virus with a glucocorticoid-inducible MMP1 for the treatment of steroid glaucoma.

PURPOSE: To design a glucocorticoid-inducible virus vector overexpressing recombinant matrix metalloproteinase 1 (MMP1) and counteract extracellular matrix deposition in the trabecular meshwork only when steroid is present. METHODS: Endogenous MMP1 expression was measured in primary human trabecular meshwork cells (HTM) treated with dexamethasone (DEX), triamcinolone acetate, and prednisolone acetate by TaqMan PCR. Wild-type and mutant MMP1 cDNAs were cloned downstream of a glucocorticoid response element (GRE) and P(TAL) promoter. Adenoviruses AdhGRE.MMP1 and AdhGRE.mutMMP1 were generated by homologous recombination. HTM cells and perfused human anterior segments were infected with the viruses, with and without DEX. MMP1 mRNA and protein were analyzed by TaqMan PCR, Western blot analysis, and ELISA. Activity of secreted MMP1 was evaluated by FRET and rat tail collagen type I assays. Immunohistochemistry was performed by double-labeling with anti-human MMP1 and collagen type I antibodies. RESULTS: Endogenous MMP1 expression was greatly downregulated by the steroids. DEX-treated cells and perfused organ cultures infected with AdhGRE.MMP1 secreted high levels of MMP1. Induction of MMP1 cycled on and off with the addition or removal of DEX. Secreted wild-type MMP1 degraded collagen type I after activation, whereas secreted mutMMP1 did not. Immunohistochemistry showed faint staining of collagen type I in areas of trabecular meshwork with high MMP1 transgene expression. CONCLUSIONS: The authors have developed a novel glucocorticoid-inducible adenovirus vector that overproduces MMP1 only in the presence of DEX. The availability of this vector sets up the foundation for the development of gene therapy drugs for the potential treatment of ocular hypertension in steroid-responsive patients.

The effect of the controlled release of basic fibroblast growth factor from ionic gelatin-based hydrogels on angiogenesis in a murine critical limb ischemic model.

The localized delivery of exogenous, angiogenic growth factors has become a promising alternative treatment of peripheral artery disease (PAD) and critical limb ischemia. In the present study, we describe the development of a novel controlled release vehicle to promote angiogenesis in a murine critical limb ischemic model. Ionic, gelatin-based hydrogels were prepared by the carbodiimide-mediated amidation reaction between the carboxyl groups of gelatin or poly-L-glutamic acid molecules and the amine groups of poly-L-lysine or gelatin molecules, respectively. The degree of swelling of the synthesized hydrogels was assessed as a function of EDC/NHS ratios and the pH of the equilibrating medium, while the release kinetic profile of basic fibroblast growth factor (FGF-2) was evaluated in human fibroblast cultures. The degree of swelling (DS) decreased from 26.5+/-1.7 to 18.5+/-2.4 as the EDC concentration varied from 0.75 to 2.5 mg/ml. Eighty percent of the FGF-2 was released at controlled rates from gelatin-polylysine (gelatin-PLL) and gelatin-polyglutamic acid (gelatin-PLG) hydrogel scaffolds over a period of 28 days. Cell adhesion studies revealed that the negatively charged surface of the gelatin-PLG hydrogels exhibited superior adhesion capabilities in comparison to gelatin-PLL and control gelatin surfaces. Laser Doppler perfusion imaging as well as CD31(+) capillary immunostaining demonstrated that the controlled release of FGF-2 from ionic gelatin-based hydrogels is superior in promoting angiogenesis in comparison to the bolus administration of the growth factor. Over 4 weeks, FGF-2 releasing gelatin-PLG hydrogels exhibited marked reperfusion with a Doppler ratio of 0.889 (+/-0.04) which was 69.3% higher than in the control groups.

The CENP-B homolog, Abp1, interacts with the initiation protein Cdc23 (MCM10) and is required for efficient DNA replication in fission yeast.

Abp1, and the closely related Cbh1 and Cbh2 are homologous to the human centromere-binding protein CENP-B that has been implicated in the assembly of centromeric heterochromatin. Fission yeast cells lacking Abp1 show an increase in mini-chromosome instability suggesting that Abp1 is important for chromosome segregation and/or DNA synthesis. Here we show that Abp1 interacts with the DNA replication protein Cdc23 (MCM10) in a two-hybrid assay, and that the Deltaabp1 mutant displays a synthetic phenotype with a cdc23 temperature-sensitive mutant. Moreover, genetic interactions were also observed between abp1+ and four additional DNA replication initiation genes cdc18+, cdc21+, orc1+, and orc2+. Interestingly, we find that S phase is delayed in cells deleted for abp1+ when released from a G1 block. However, no delay is observed when cells are released from an early S phase arrest induced by hydroxyurea suggesting that Abp1 functions prior to, or coincident with, the initiation of DNA replication.

Redox stress and the contributions of BH3-only proteins to infarction.

Ischemia followed by reperfusion is the primary cause of tissue injury and infarction during heart attack and stroke. The initiating stimulus is believed to involve reactive oxygen species that are produced during reperfusion when electron transport resumes in the mitochondria after suppression by ischemia. Programmed death has been shown to be a significant component of infarction, and evidence indicates that multiple pathways are initiated during both ischemia and reperfusion phases. Major infarction is preceded by severe ischemia that includes hypoxia, intracellular acidosis, glucose depletion, loss of ATP, and elevation of cytoplasmic calcium. The superimposition of a reactive oxygen surge on the latter condition provides the impetus for maximal damage. Compelling evidence implicates mitochondria not only as the source of initiating ROS but also as the focal sensors that translate the redox stress signal into a cellular-death response. Pivotal to this response are the BH3-only proteins that are activated by death signals and regulate mitochondrial communication with executioner proteins in the cytoplasm. The BH3-only proteins do this by controlling the activity of pores and channels in the outer mitochondrial membrane. To date at least six BH3-only proteins have been shown to contribute to ischemia-reperfusion death pathways in heart and/or brain; these include Bnip3, PUMA, Bid, Bad, HGTD-P, and Noxa. Here we review the evidence for these cell-death pathways and discuss their relevance to ischemic disease and infarction.

Identification and cloning of two putative subunits of DNA polymerase epsilon in fission yeast.

DNA polymerase epsilon (Pol epsilon) is a multi-subunit enzyme required for the initiation of chromosomal DNA replication. Here, we report the cloning of two fission yeast genes, called dpb3+ and dpb4+ that encode proteins homologous to the two smallest subunits of Pol epsilon. Although Dpb4 is not required for cell viability, Deltadpb4 mutants are synthetically lethal with mutations in four genes required for DNA replication initiation, cdc20+ (encoding DNA Pol epsilon), cut5+ (homologous to DPB11/TopBP1), sna41+ (homologous to CDC45) and cdc21+ (encoding Mcm4, a component of the pre-replicative complex). In contrast to Dpb4, Dpb3 is essential for cell cycle progression. A glutathione S-transferase pull-down assay indicates that Dpb3 physically interacts with both Dpb2 and Dpb4, suggesting that Dpb3 associates with other members of the Pol epsilon complex. Depletion of Dpb3 leads to an accumulation of cells in S phase consistent with Dpb3 having a role in DNA replication. In addition, many of the cells have a bi-nucleate or multinucleate phenotype, indicating that cell separation is also inhibited. Finally, we have examined in vivo localization of green fluorescent protein (GFP)-tagged Dpb3 and Dpb4 and found that both proteins are localized to the nucleus consistent with their proposed role in DNA replication. However, in the absence of Dpb3, GFP-Dpb4 appears to be more dispersed throughout the cell, suggesting that Dpb3 may be important in establishing or maintaining normal localization of Dpb4.

A unique pathway of cardiac myocyte death caused by hypoxia-acidosis.

Chronic hypoxia in the presence of high glucose leads to progressive acidosis of cardiac myocytes in culture. The condition parallels myocardial ischemia in vivo, where ischemic tissue becomes rapidly hypoxic and acidotic. Cardiac myocytes are resistant to chronic hypoxia at neutral pH but undergo extensive death when the extracellular pH (pH[o]) drops below 6.5. A microarray analysis of 20 000 genes (cDNAs and expressed sequence tags) screened with cDNAs from aerobic and hypoxic cardiac myocytes identified >100 genes that were induced by >2-fold and approximately 20 genes that were induced by >5-fold. One of the most strongly induced transcripts was identified as the gene encoding the pro-apoptotic Bcl-2 family member BNIP3. Northern and western blot analyses confirmed that BNIP3 was induced by 12-fold (mRNA) and 6-fold (protein) during 24 h of hypoxia. BNIP3 protein, but not the mRNA, accumulated 3.5-fold more rapidly under hypoxia-acidosis. Cell fractionation experiments indicated that BNIP3 was loosely bound to mitochondria under conditions of neutral hypoxia but was translocated into the membrane when the myocytes were acidotic. Translocation of BNIP3 coincided with opening of the mitochondrial permeability pore (MPTP). Paradoxically, mitochondrial pore opening did not promote caspase activation, and broad-range caspase inhibitors do not block this cell death pathway. The pathway was blocked by antisense BNIP3 oligonucleotides and MPTP inhibitors. Therefore, cardiac myocyte death during hypoxia-acidosis involves two distinct steps: (1) hypoxia activates transcription of the death-promoting BNIP3 gene through a hypoxia-inducible factor-1 (HIF-1) site in the promoter and (2) acidosis activates BNIP3 by promoting membrane translocation. This is an atypical programmed death pathway involving a combination of the features of apoptosis and necrosis. In this article, we will review the evidence for this unique pathway of cell death and discuss its relevance to ischemic heart disease. The article also contains new evidence that chronic hypoxia at neutral pH does not promote apoptosis or activate caspases in neonatal cardiac myocytes.