Pathologic vs. protective roles of hypoxia-inducible factor 1 in RPE and photoreceptors in wet vs. dry age-related macular degeneration.

It has previously been reported that antioxidant vitamins can help reduce the risk of vision loss associated with progression to advanced age-related macular degeneration (AMD), a leading cause of visual impairment among the elderly. Nonetheless, how oxidative stress contributes to the development of choroidal neovascularization (CNV) in some AMD patients and geographic atrophy (GA) in others is poorly understood. Here, we provide evidence demonstrating that oxidative stress cooperates with hypoxia to synergistically stimulate the accumulation of hypoxia-inducible factor (HIF)-1α in the retinal pigment epithelium (RPE), resulting in increased expression of the HIF-1-dependent angiogenic mediators that promote CNV. HIF-1 inhibition blocked the expression of these angiogenic mediators and prevented CNV development in an animal model of ocular oxidative stress, demonstrating the pathological role of HIF-1 in response to oxidative stress stimulation in neovascular AMD. While human-induced pluripotent stem cell (hiPSC)-derived RPE monolayers exposed to chemical oxidants resulted in disorganization and disruption of their normal architecture, RPE cells proved remarkably resistant to oxidative stress. Conversely, equivalent doses of chemical oxidants resulted in apoptosis of hiPSC-derived retinal photoreceptors. Pharmacologic inhibition of HIF-1 in the mouse retina enhanced-while HIF-1 augmentation reduced-photoreceptor apoptosis in two mouse models for oxidative stress, consistent with a protective role for HIF-1 in photoreceptors in patients with advanced dry AMD. Collectively, these results suggest that in patients with AMD, increased expression of HIF-1α in RPE exposed to oxidative stress promotes the development of CNV, but inadequate HIF-1α expression in photoreceptors contributes to the development of GA.

Food and Drug Administration Public Workshop Summary-Development Considerations of Antifungal Drugs to Address Unmet Medical Need.

Pressing challenges in the treatment of invasive fungal infections (IFIs) include emerging and rare pathogens, resistant/refractory infections, and antifungal armamentarium limited by toxicity, drug-drug interactions, and lack of oral formulations. Development of new antifungal drugs is hampered by the limitations of the available diagnostics, clinical trial endpoints, prolonged trial duration, difficulties in patient recruitment, including subpopulations (eg, pediatrics), and heterogeneity of the IFIs. On 4 August 2020, the US Food and Drug Administration convened a workshop that included IFI experts from academia, industry, and other government agencies to discuss the IFI landscape, unmet need, and potential strategies to facilitate the development of antifungal drugs for treatment and prophylaxis. This article summarizes the key topics presented and discussed during the workshop, such as incentives and research support for drug developers, nonclinical development, clinical trial design challenges, lessons learned from industry, and potential collaborations to facilitate antifungal drug development.

Angiopoietin-like 4 is a potent angiogenic factor and a novel therapeutic target for patients with proliferative diabetic retinopathy.

Diabetic eye disease is the most common cause of severe vision loss in the working-age population in the developed world, and proliferative diabetic retinopathy (PDR) is its most vision-threatening sequela. In PDR, retinal ischemia leads to the up-regulation of angiogenic factors that promote neovascularization. Therapies targeting vascular endothelial growth factor (VEGF) delay the development of neovascularization in some, but not all, diabetic patients, implicating additional factor(s) in PDR pathogenesis. Here we demonstrate that the angiogenic potential of aqueous fluid from PDR patients is independent of VEGF concentration, providing an opportunity to evaluate the contribution of other angiogenic factor(s) to PDR development. We identify angiopoietin-like 4 (ANGPTL4) as a potent angiogenic factor whose expression is up-regulated in hypoxic retinal Müller cells in vitro and the ischemic retina in vivo. Expression of ANGPTL4 was increased in the aqueous and vitreous of PDR patients, independent of VEGF levels, correlated with the presence of diabetic eye disease, and localized to areas of retinal neovascularization. Inhibition of ANGPTL4 expression reduced the angiogenic potential of hypoxic Müller cells; this effect was additive with inhibition of VEGF expression. An ANGPTL4 neutralizing antibody inhibited the angiogenic effect of aqueous fluid from PDR patients, including samples from patients with low VEGF levels or receiving anti-VEGF therapy. Collectively, our results suggest that targeting both ANGPTL4 and VEGF may be necessary for effective treatment or prevention of PDR and provide the foundation for studies evaluating aqueous ANGPTL4 as a biomarker to help guide individualized therapy for diabetic eye disease.

HIF-1α accumulation in response to transient hypoglycemia may worsen diabetic eye disease.

Tight glycemic control (TGC), the cornerstone of diabetic management, reduces the incidence and progression of diabetic microvascular disease. However, TGC can also lead to transient episodes of hypoglycemia, which have been associated with adverse outcomes in patients with diabetes. Here, we demonstrate that low glucose levels result in hypoxia-inducible factor (HIF)-1-dependent expression of the glucose transporter, Glut1, in retinal cells. Enhanced nuclear accumulation of HIF-1α was independent of its canonical post-translational stabilization but instead dependent on stimulation of its translation and nuclear localization. In the presence of hypoxia, this physiologic response to low glucose resulted in a marked increase in the secretion of the HIF-dependent vasoactive mediators that promote diabetic retinopathy. Our results provide a molecular explanation for how early glucose control, as well as glycemic variability (i.e., oscillating serum glucose levels), contributes to diabetic eye disease. These observations have important implications for optimizing glucose management in patients with diabetes.

Targeting hypoxia-inducible factors with 32-134D safely and effectively treats diabetic eye disease in mice.

Many patients with diabetic eye disease respond inadequately to anti-VEGF therapies, implicating additional vasoactive mediators in its pathogenesis. We demonstrate that levels of angiogenic proteins regulated by HIF-1 and -2 remain elevated in the eyes of people with diabetes despite treatment with anti-VEGF therapy. Conversely, by inhibiting HIFs, we normalized the expression of multiple vasoactive mediators in mouse models of diabetic eye disease. Accumulation of HIFs and HIF-regulated vasoactive mediators in hyperglycemic animals was observed in the absence of tissue hypoxia, suggesting that targeting HIFs may be an effective early treatment for diabetic retinopathy. However, while the HIF inhibitor acriflavine prevented retinal vascular hyperpermeability in diabetic mice for several months following a single intraocular injection, accumulation of acriflavine in the retina resulted in retinal toxicity over time, raising concerns for its use in patients. Conversely, 32-134D, a recently developed HIF inhibitor structurally unrelated to acriflavine, was not toxic to the retina, yet effectively inhibited HIF accumulation and normalized HIF-regulated gene expression in mice and in human retinal organoids. Intraocular administration of 32-134D prevented retinal neovascularization and vascular hyperpermeability in mice. These results provide the foundation for clinical studies assessing 32-134D for the treatment of patients with diabetic eye disease.

PAI-1 is a vascular cell-specific HIF-2-dependent angiogenic factor that promotes retinal neovascularization in diabetic patients.

For patients with proliferative diabetic retinopathy (PDR) who do not respond adequately to pan-retinal laser photocoagulation (PRP) or anti-vascular endothelial growth factor (VEGF) therapies, we hypothesized that vascular cells within neovascular tissue secrete autocrine/paracrine angiogenic factors that promote disease progression. To identify these factors, we performed multiplex ELISA angiogenesis arrays on aqueous fluid from PDR patients who responded inadequately to anti-VEGF therapy and/or PRP and identified plasminogen activator inhibitor-1 (PAI-1). PAI-1 expression was increased in vitreous biopsies and neovascular tissue from PDR eyes, limited to retinal vascular cells, regulated by the transcription factor hypoxia-inducible factor (HIF)-2α, and necessary and sufficient to stimulate angiogenesis. Using a pharmacologic inhibitor of HIF-2α (PT-2385) or nanoparticle-mediated RNA interference targeting Pai1, we demonstrate that the HIF-2α/PAI-1 axis is necessary for the development of retinal neovascularization in mice. These results suggest that targeting HIF-2α/PAI-1 will be an effective adjunct therapy for the treatment of PDR patients.

Correction: Hypoxia-inducible factor 1 upregulation of both VEGF and ANGPTL4 is required to promote the angiogenic phenotype in uveal melanoma.

[This corrects the article DOI: 10.18632/oncotarget.6868.].

HIF-1α and HIF-2α redundantly promote retinal neovascularization in patients with ischemic retinal disease.

Therapies targeting VEGF have proven only modestly effective for the treatment of proliferative sickle cell retinopathy (PSR), the leading cause of blindness in patients with sickle cell disease. Here, we shift our attention upstream from the genes that promote retinal neovascularization (NV) to the transcription factors that regulate their expression. We demonstrated increased expression of HIF-1α and HIF-2α in the ischemic inner retina of PSR eyes. Although both HIFs participated in promoting VEGF expression by hypoxic retinal Müller cells, HIF-1 alone was sufficient to promote retinal NV in mice, suggesting that therapies targeting only HIF-2 would not be adequate to prevent PSR. Nonetheless, administration of a HIF-2-specific inhibitor currently in clinical trials (PT2385) inhibited NV in the oxygen-induced retinopathy (OIR) mouse model. To unravel these discordant observations, we examined the expression of HIFs in OIR mice and demonstrated rapid but transient accumulation of HIF-1α but delayed and sustained accumulation of HIF-2α; simultaneous expression of HIF-1α and HIF-2α was not observed. Staggered HIF expression was corroborated in hypoxic adult mouse retinal explants but not in human retinal organoids, suggesting that this phenomenon may be unique to mice. Using pharmacological inhibition or an in vivo nanoparticle-mediated RNAi approach, we demonstrated that inhibiting either HIF was effective for preventing NV in OIR mice. Collectively, these results explain why inhibition of either HIF-1α or HIF-2α is equally effective for preventing retinal NV in mice but suggest that therapies targeting both HIFs will be necessary to prevent NV in patients with PSR.

Identification of alternate transcripts of neuronal nitric oxide synthase in the mouse retina.

Nitric oxide (NO) is a major signaling molecule in the retina and CNS, with physiological roles in every cell type in the retina. Previous work shows that neuronal nitric oxide synthase (nNOS) is an important source of NO in the vertebrate retina. There are distinct, active alternative transcripts of nNOS observed in many tissues, including testes and brain, that may differ in both localization and enzyme kinetics. The present study characterized nNOS and the NO production from nNOS in the mouse retina in terms of its alternate transcripts, namely, nNOS alpha, nNOS beta, and nNOS gamma. We examined both basal and light-stimulated NO production as imaged using the NO-sensitive dye 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate-FM (DAF-FM), and we compared the NO production with the immunocytochemical localization of nNOS using antisera that recognize nNOS alpha/beta or nNOS alpha/beta/gamma. Western blots suggested the presence of NOS alpha/gamma protein in retina, but not nNOS beta, and we confirmed this at the message level by using a combination of RT-PCR and quantitative real-time PCR. Our findings indicated that the primary source of NO in the mammalian retina is nNOS alpha and that nNOS gamma may contribute to NO production as well.

Increased neuronal nitric oxide synthase activity in retinal neurons in early diabetic retinopathy.

PURPOSE: There are increased levels of nitric oxide (NO) in diabetic retinas. The purpose of this study was to determine the extent that neuronal nitric oxide synthase (nNOS) contributes to the increased levels of retinal NO in early diabetic retinopathy by examining the expression and activity of nNOS in retinal neurons after 5 weeks of diabetes. METHODS: Changes in NO levels were measured using NO imaging of retinal neurons in mice with streptozotocin-induced diabetes for five weeks. NO imaging was compared to nNOS localization using immunocytochemistry, and nNOS message and protein levels were measured using quantitative real-time PCR and western blots. RESULTS: There was a close anatomic correlation between the localization of the increased NO production and the nNOS immunoreactivity in the retinal plexiform layers of diabetic retinas. There was no change in nNOS message, but nNOS protein was decreased and its subcellular localization was altered. Treatment with insulin or aminoguanidine partially ameliorated the increase in NO in diabetic retinas. CONCLUSIONS: These results suggest that increased nNOS activity is responsible for the majority of increased NO in retinal neurons in early diabetic retinopathy. This supports a role for increased nNOS activity in the early neuronal dysfunction in the diabetic retina.

Angiopoietin-like 4 binds neuropilins and cooperates with VEGF to induce diabetic macular edema.

The majority of patients with diabetic macular edema (DME), the most common cause of vision loss in working-age Americans, do not respond adequately to current therapies targeting VEGFA. Here, we show that expression of angiopoietin-like 4 (ANGPTL4), a HIF-1-regulated gene product, is increased in the eyes of diabetic mice and patients with DME. We observed that ANGPTL4 and VEGF act synergistically to destabilize the retinal vascular barrier. Interestingly, while ANGPTL4 modestly enhanced tyrosine phosphorylation of VEGF receptor 2, promotion of vascular permeability by ANGPTL4 was independent of this receptor. Instead, we found that ANGPTL4 binds directly to neuropilin 1 (NRP1) and NRP2 on endothelial cells (ECs), leading to rapid activation of the RhoA/ROCK signaling pathway and breakdown of EC-EC junctions. Treatment with a soluble fragment of NRP1 (sNRP1) prevented ANGPTL4 from binding to NRP1 and blocked ANGPTL4-induced activation of RhoA as well as EC permeability in vitro and retinal vascular leakage in diabetic animals in vivo. In addition, sNRP1 reduced the stimulation of EC permeability by aqueous fluid from patients with DME. Collectively, these data identify the ANGPTL4/NRP/RhoA pathway as a therapeutic target for the treatment of DME.

Hypoxia-Inducible Factor-Dependent Expression of Angiopoietin-Like 4 by Conjunctival Epithelial Cells Promotes the Angiogenic Phenotype of Pterygia.

Purpose: Disappointing results from clinical studies assessing the efficacy of therapies targeting vascular endothelial growth factor (VEGF) for the treatment of pterygia suggest that other angiogenic mediators may also play a role in its development. We therefore explore the relative contribution of VEGF, hypoxia-inducible factor (HIF)-1α (the transcription factor that regulates VEGF expression in ocular neovascular disease), and a second HIF-regulated mediator, angiopoietin-like 4 (ANGPTL4), to the angiogenic phenotype of pterygia. Methods: Expression of HIF-1α, VEGF, and ANGPTL4 were examined in surgically excised pterygia, and in immortalized human (ih) and primary rabbit (pr) conjunctival epithelial cells (CjECs). Endothelial cell (EC) tubule formation assays using media conditioned by ihCjECs in the presence or absence of inducers/inhibitors of HIF-1 or RNA interference (RNAi) targeting VEGF, ANGPTL4, or both were used to assess their relative contribution to the angiogenic potential of these cells. Results: HIF-1α and VEGF expression were detected in 6/6 surgically excised pterygia and localized to CjECs. Accumulation of HIF-1α in was confirmed in ihCjECs and prCjECs, including stratified prCjECs grown on collagen vitrigel, and resulted in expression of VEGF and the promotion of EC tubule formation; the latter effect was partially blocked using RNAi targeting VEGF mRNA expression. We demonstrate expression of a second HIF-regulated angiogenic mediator, ANGPTL4, in CjECs in culture and in surgically excised pterygia. RNAi targeting ANGPTL4 inhibited EC tubule formation and was additive to RNAi targeting VEGF. Conclusions: Our results support the development of therapies targeting both ANGPTL4 and VEGF for the treatment of patients with pterygia.

Expression Pattern of HIF-1α and VEGF Supports Circumferential Application of Scatter Laser for Proliferative Sickle Retinopathy.

Purpose: Retinal vascular occlusions in sickle cell anemia patients cause tissue ischemia and the release of angiogenic mediators that promote the development of retinal neovascularization, initiating proliferative sickle retinopathy (PSR). Laser photocoagulation (LPC) has emerged as the most common treatment for PSR. Nonetheless, only two randomized controlled clinical trials have evaluated the use of LPC for PSR, and both failed to definitively demonstrate efficacy of this approach. This may be due to a lack of knowledge regarding the appropriate location for placement of laser coagulations in PSR eyes. To help address this question, we examined the expression of hypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF) in PSR eyes. Methods: The expression pattern of HIF-1α and VEGF in PSR (n = 5) and control (n = 3) eyes was examined by immunohistochemistry in different retinal regions defined by the presence or absence of retinal vessels. Results: Hypoxia-inducible factor 1α and VEGF were expressed in the inner retina of 5/5 untreated PSR eyes adjacent to retinal neovascularization; expression of HIF-1α was not detected (and VEGF only lightly detected) in normal retinal and choroidal vasculature of 3/3 control eyes. Hypoxia-inducible factor 1α and VEGF were strongly expressed in retinal cells within avascular (nonperfused) retina, anterior to the boundary between perfused and nonperfused retina, as well as in posterior ischemic retina in the presence or absence of neovascular sea fans. Conclusions: If the goal of LPC in PSR is to quench the expression of HIF-1-driven angiogenic mediators, our results support broad application of peripheral laser for its treatment.

Hypoxia-inducible factor 1 upregulation of both VEGF and ANGPTL4 is required to promote the angiogenic phenotype in uveal melanoma.

PURPOSE: Expression of the hypoxia-inducible factor (HIF)-1-regulated gene product, vascular endothelial growth factor (VEGF), correlates with tumor vascularity in patients with uveal melanoma (UM). While the relationship between HIF-1 and VEGF in cancer is well-studied, their relative contribution to the angiogenic phenotype in UM has not previously been interrogated. Here we evaluate the contribution of HIF-1, VEGF, and a second HIF-1-regulated gene product, angiopoietin-like 4 (ANGPTL4), to angiogenesis in UM. EXPERIMENTAL DESIGN: UM cells were examined for expression of HIF-1α, VEGF, and ANGPTL4. Their contribution to the angiogenic potential of UM cells was assessed using the endothelial cell tubule formation and directed in vivo angiogenesis assays. These results were corroborated in tissue from UM animal models and in tissue from patients with UM. RESULTS: Inhibition of VEGF partially reduced tubule formation promoted by conditioned medium from UM cells. Inhibition of ANGPTL4, which was highly expressed in hypoxic UM cells, a UM orthotopic transplant model, a UM tumor array, and vitreous samples from UM patients, inhibited the angiogenic potential of UM cells in vitro and in vivo; this effect was additive to VEGF inhibition. CONCLUSIONS: Targeting both ANGPTL4 and VEGF may be required for the effective inhibition of angiogenesis in UM.

Altered prostanoid signaling contributes to increased skin tumorigenesis in Tpl2 knockout mice.

Squamous cell carcinoma is the second most common form of skin cancer with the incidence expected to double over the next 20 years. Inflammation is believed to be a critical component in skin cancer progression. Therefore, understanding genes involved in the regulation of inflammatory pathways is vital to the design of targeted therapies. Numerous studies show cyclooxygenases (COXs) play an essential role in inflammation-associated cancers. Tpl2 (MAP3K8) is a protein kinase in the MAP Kinase signal transduction cascade. Previous research using a two-stage skin carcinogenesis model revealed that Tpl2(-/-) mice have significantly higher tumor incidence and inflammatory response than wild-type (WT) controls. The current study investigates whether cyclooxygenase-2 (COX-2) and COX-2- regulated prostaglandins and prostaglandin receptors drive the highly tumorigenic state of Tpl2(-/-) mice by investigating the relationship between Tpl2 and COX-2. Keratinocytes from newborn WT or Tpl2(-/-) mice were treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) for various times over 24 hours. Western analysis revealed significant differences in COX-2 and COX-2 dependent prostanoids and prostanoid receptors. Additionally, in vivo experiments confirmed that COX-2 and COX-2 downstream factors were elevated in TPA-treated Tpl2(-/-) skin, as well as in papillomas from Tpl2(-/-) mice. Use of the selective COX-2 inhibitor Celecoxib showed the increased tumorigenesis in the Tpl2(-/-) mice to primarily be mediated through COX-2. These experiments illustrate COX-2 induction in the absence of Tpl2 may be responsible for the increased tumorigenesis found in Tpl2(-/-) mice. Defining the relationship between Tpl2 and COX-2 may lead to new ways to downregulate COX-2 through the modulation of Tpl2.

Characterization of nitric oxide signaling pathways in the mouse retina.

Nitric oxide (NO) is a gaseous neuromodulator with physiological functions in every retinal cell type. NO is synthesized by several nitric oxide synthases (NOS) and often functions through its second messenger, cyclic guanosine monophosphate (cGMP), and protein kinase G (PKG). This study combined NO imaging, immunocytochemistry, biochemistry, and molecular biology to localize NO and its downstream signaling pathways in the mouse retina. Neuronal NOS (nNOS) was localized primarily in puncta in the inner plexiform layer, in amacrine cells, and in somata in the ganglion cell layer. Endothelial NOS was in blood vessels. Light-stimulated NO production imaged with diaminofluorescein was present in somata in the inner nuclear layer and in synaptic boutons in the inner plexiform layer. The downstream target of NO, soluble guanylate cyclase (sGC), was in somata in the inner and outer nuclear layers and in both plexiform layers. Cyclic GMP immunocytochemistry was used functionally to localize sGC that was activated by an NO donor in amacrine, bipolar, and ganglion cells. Cyclic GMP-dependent protein kinase (PKG) Iα was found in bipolar cells, ganglion cells, and both plexiform layers, whereas PKG II was found in the outer plexiform layer, amacrine cells, and somata in the ganglion cell layer. This study shows that the NO/cGMP/PKG signaling pathway is functional and widely distributed in specific cell types in the outer and inner mouse retina. A better understanding of these signaling pathways in normal retina will provide a firm basis for targeting their roles in retinal pathology.

Inhibition of tumor cell surface ATP synthesis by pigment epithelium-derived factor: implications for antitumor activity.

Recently, we have shown that the antiangiogenic pigment epithelium-derived factor (PEDF) can bind the catalytic ß-subunit of F1-ATP synthase and inhibit endothelial cell surface ATP synthase activity. This factor can additionally restrict tumor growth, invasion and metastasis, and can directly induce death on several tumor cell types. Active cell surface ATP synthase is also present in certain tumor cells and its ATP product is considered a stimulus for tumor growth. The present study aimed to elucidate the biological implications of the interactions between the extracellular PEDF and tumor cell surface ATP synthase. Incubation of T24 human urinary bladder carcinoma cells in media containing human recombinant PEDF protein for 48-96 h dramatically decreased cell viability in a concentration-dependent fashion as monitored by real-time cell impedance with a microelectronic system, microscopic imaging and biomarkers of live cells. Intact tumor cells exhibited cell surface ATP synthesis activity, which was inhibited by piceatannol, a specific inhibitor of F1/F0-ATP synthase. Immunoblotting revealed that the ß subunit of F1-ATP synthase was present in plasma membrane fractions of these cells. Interestingly, pre-incubation of tumor cells with PEDF inhibited the activity of cell surface ATP synthase in a concentration-dependent fashion. The PEDF-derived peptide 34-mer decreased tumor cell viability and inhibited extracellular ATP synthesis to the same extent as full-length PEDF. Moreover, ATP additions attenuated both the PEDF-mediated decrease in tumor cell viability and the inhibition of endothelial cell tube formation. The results lead to conclude that PEDF is a novel inhibitor of tumor cell surface ATP synthase activity that exhibits a cytotoxic effect on tumor cells, and that the structural determinants for these properties are within the peptide region 34-mer of the PEDF polypeptide. The data strongly suggest a role for the interaction between the 34-mer region of PEDF and tumor cell-surface ATP synthase in promoting tumor cell death.

Opposing effects of pigment epithelium-derived factor on breast cancer cell versus neuronal survival: implication for brain metastasis and metastasis-induced brain damage.

Brain metastases are a significant cause of morbidity and mortality for patients with cancer, yet preventative and therapeutic options remain an unmet need. The cytokine pigment epithelium-derived factor (PEDF) is downregulated in resected human brain metastases of breast cancer compared with primary breast tumors, suggesting that restoring its expression might limit metastatic spread. Here, we show that outgrowth of large experimental brain metastases from human 231-BR or murine 4T1-BR breast cancer cells was suppressed by PEDF expression, as supported by in vitro analyses as well as direct intracranial implantation. Notably, the suppressive effects of PEDF were not only rapid but independent of the effects of this factor on angiogenesis. Paralleling its cytotoxic effects on breast cancer cells, PEDF also exerted a prosurvival effect on neurons that shielded the brain from tumor-induced damage, as indicated by a relative 3.5-fold reduction in the number of dying neurons adjacent to tumors expressing PEDF. Our findings establish PEDF as both a metastatic suppressor and a neuroprotectant in the brain, highlighting its role as a double agent in limiting brain metastasis and its local consequences.