Gene Therapy with Endogenous Inhibitors of Angiogenesis for Neovascular Age-Related Macular Degeneration: Beyond Anti-VEGF Therapy

Age-related macular degeneration (AMD) is the leading cause of substantial and irreversible vision loss amongst elderly populations in industrialized countries. The advanced neovascular (or “wet”) form of the disease is responsible for severe and aggressive loss of central vision. Current treatments aim to seal off leaky blood vessels via laser therapy or to suppress vessel leakage and neovascular growth through intraocular injections of antibodies that target vascular endothelial growth factor (VEGF). However, the long-term success of anti-VEGF therapy can be hampered by limitations such as low or variable efficacy, high frequency of administration (usually monthly), potentially serious side effects, and, most importantly, loss of efficacy with prolonged treatment. Gene transfer of endogenous antiangiogenic proteins is an alternative approach that has the potential to provide long-term suppression of neovascularization and/or excessive vascular leakage in the eye. Preclinical studies of gene transfer in a large animal model have provided impressive preliminary results with a number of transgenes. In addition, a clinical trial in patients suffering from advanced neovascular AMD has provided proof-of-concept for successful gene transfer. In this mini review, we summarize current theories pertaining to the application of gene therapy for neovascular AMD and the potential benefits when used in conjunction with endogenous antiangiogenic proteins

Proopiomelanocortin gene delivery induces apoptosis in melanoma through NADPH oxidase 4-mediated ROS generation

AbstractHypoxia in the tumor microenvironment triggers differential signaling pathways for tumor survival. In this study, we characterize the involvement of hypoxia and reactive oxygen species (ROS) generation in the antineoplastic mechanism of proopiomelanocortin (POMC) gene delivery in a mouse B16-F10 melanoma model in vivo and in vitro. Histological analysis revealed increased TUNEL-positive cells and enhanced hypoxic activities in melanoma treated with adenovirus encoding POMC (Ad-POMC) but not control vector. Because the apoptotic cells were detected mainly in regions distant from blood vessels, it was hypothesized that POMC therapy might render melanoma cells vulnerable to hypoxic insult. Using a hypoxic chamber or cobalt chloride (CoCl2), we showed that POMC gene delivery elicited apoptosis and caspase-3 activation in cultured B16-F10 cells only under hypoxic conditions. The apoptosis induced by POMC gene delivery was associated with elevated ROS generation in vitro and in vivo. Blocking ROS generation using the antioxidant N-acetyl-l-cysteine abolished the apoptosis and caspase-3 activities induced by POMC gene delivery and hypoxia. We further showed that POMC-derived melanocortins, including α-MSH, β-MSH, and ACTH, but not γ-MSH, contributed to POMC-induced apoptosis and ROS generation during hypoxia. To elucidate the source of ROS generation, application of the NADPH oxidase inhibitor diphenyleneiodonium attenuated α-MSH-induced apoptosis and ROS generation, implicating the proapoptotic role of NADPH oxidase in POMC action. Of the NADPH oxidase isoforms, only Nox4 was expressed in B16-F10 cells, and Nox4 was also elevated in Ad-POMC-treated melanoma tissues. Silencing Nox4 gene expression with Nox4 siRNA suppressed the stimulatory effect of α-MSH-induced ROS generation and cell apoptosis during hypoxia. In summary, we demonstrate that POMC gene delivery suppressed melanoma growth by inducing apoptosis, which was at least partly dependent on Nox4 upregulation

NADPH oxidase-dependent redox signaling in TGF-β-mediated fibrotic responses

Uncontrolled fibrosis in organs like heart, kidney, liver and lung is detrimental and may lead to end-stage organ failure. Currently there is no effective treatment for fibrotic disorders. Transforming growth factor (TGF)-β has a fundamental role in orchestrating the process of fibrogenesis; however, interventions directly targeting TGF-β would have undesired systemic side effects due to the multiple physiological functions of TGF-β. Further characterization of the downstream signaling pathway(s) involved in TGF-β-mediated fibrosis may lead to discovery of novel treatment strategies for fibrotic disorders. Accumulating evidence suggests that Nox4 NADPH oxidase may be an important downstream effector in mediating TGF-β-induced fibrosis, while NADPH oxidase-dependent redox signaling may in turn regulate TGF-β/Smad signaling in a feed-forward manner. It is proposed that pharmacological inhibition of the Nox4 function may represent a novel approach in treatment of fibrotic disorders

Nox2 Knockout Delays Infarct Progression and Increases Vascular Recovery through Angiogenesis in Mice following Ischaemic Stroke with Reperfusion

<div><p>Evidence suggests the NADPH oxidases contribute to ischaemic stroke injury and Nox2 is the most widely studied subtype in the context of stroke. There is still conjecture however regarding the benefits of inhibiting Nox2 to improve stroke outcome. The current study aimed to examine the temporal effects of genetic Nox2 deletion on neuronal loss after ischaemic stroke using knockout (KO) mice with 6, 24 and 72 hour recovery. Transient cerebral ischaemia was induced via intraluminal filament occlusion and resulted in reduced infarct volumes in Nox2 KO mice at 24 h post-stroke compared to wild-type controls. No protection was evident at either 6 h or 72 h post-stroke, with both genotypes exhibiting similar volumes of damage. Reactive oxygen species were detected using dihydroethidium and were co-localised with neurons and microglia in both genotypes using immunofluorescent double-labelling. The effect of Nox2 deletion on vascular damage and recovery was also examined 24 h and 72 h post-stroke using an antibody against laminin. Blood vessel density was decreased in the ischaemic core of both genotypes 24 h post-stroke and returned to pre-stroke levels only in Nox2 KO mice by 72 h. Overall, these results are the first to show that genetic Nox2 deletion merely delays the progression of neuronal loss after stroke but does not prevent it. Additionally, we show for the first time that Nox2 deletion increases re-vascularisation of the damaged brain by 72 h, which may be important in promoting endogenous brain repair mechanisms that rely on re-vascularisation.</p></div

3',4'-Dihydroxyflavonol improves post-ischaemic coronary endothelial function following 7 days reperfusion in sheep

3′,4′-dihydroxyflavonol (DiOHF) is a potent antioxidant that reduces infarct size following myocardial ischaemia–reperfusion. Since oxidative stress induced by myocardial ischaemia–reperfusion impairs endothelium-dependent vasodilatation, we investigated whether DiOHF preserved coronary endothelial function following ischemia–reperfusion. One week after surgery conscious, instrumented sheep were subjected to 1 h of myocardial ischaemia followed by 7 days reperfusion. Immediately before reperfusion, sheep were injected with DiOHF (2 mg/kg iv, n = 4) or vehicle (dimethyl sulphoxide, n = 4). Coronary vascular responses to the endothelium-dependent vasodilator acetylcholine (ACh, 0.05–10.0 µg/kg/min iv), sodium nitroprusside and phenylephrine were determined. After ischaemia–reperfusion, dP/dtmax decreased from 1511 ± 93 to 1094 ± 53 mmHg/s, P < 0.05) at 24 h in the vehicle group, but by 7 days had returned towards baseline (1347 ± 91 mm Hg/s). DiOHF prevented the fall in dP/dtmax. Coronary conductance (CC) was increased (+ 34 ± 4%) by 10 µg/kg ACh given before ischaemia, but this vasodilatation was significantly reduced after 24 h and 7 days of reperfusion (+ 7 ± 2%, + 15 ± 2%, respectively, both P < 0.05). DiOHF partially preserved the coronary vasodilator response to ACh after 24 h reperfusion (basal 37 ± 7%, 24 h 18 ± 5%), and after 7 days reperfusion the response had recovered (31 ± 7%). DiOHF significantly decreased infarct size, expressed as a percentage of area-at-risk, by 40% after 7 days reperfusion (vehicle 80 ± 7%, DiOHF 46 ± 11%, P < 0.05). A single administration of DiOHF, during ischaemia and just prior to reperfusion, reduced infarct size, preserved ventricular contractility and caused a sustained protection against coronary endothelial dysfunction, with all these beneficial actions being preserved for 7 days reperfusion

NADPH-oxidase activity is elevated in penumbral and non-ischemic cerebral arteries following stroke

Reactive oxygen species play a role in neuronal damage following cerebral ischemia–reperfusion. We tested whether activity of the superoxide-generating enzyme, NADPH-oxidase, is enhanced in cerebral arteries within, adjacent and distant from the ischemic core. The right middle cerebral artery (MCA) of conscious rats was temporarily occluded by perivascular injection of endothelin-1 to induce stroke (ET-1; n = 19). Control rats were injected with saline (n = 9). At 24 h or 72 h post-administration of ET-1, the MCA and its branches within the ipsilateral penumbra and infarcted core, corresponding arteries in the contralateral hemisphere, and basilar artery were excised. Anatomically similar arteries were excised from saline-injected rats. At 24 h after stroke, NADPH-stimulated superoxide production by arteries from the infarcted core did not differ from levels generated by arteries from control rats, whereas levels were significantly lower 72 h after stroke. However, at both time points after stroke, superoxide production by arteries from the ischemic penumbra was 8-fold greater than levels generated by arteries from control rats. Surprisingly, even in the non-ischemic arteries from the contralateral hemisphere and in the basilar artery, superoxide production was increased ∼ 4- to 6-fold at 24 h, but had returned to normal 72 h after stroke. The NADPH-oxidase inhibitor, diphenyleneiodonium, virtually abolished superoxide production by all arteries. Thus, the activity of NADPH-oxidase is enhanced in cerebral arteries from the ischemic penumbra at 24 h and 72 h following cerebral ischemia. Additionally, NADPH-oxidase activity is temporarily enhanced after cerebral ischemia within arteries from non-ischemic parts of the brain