Zhongxiao Wang

Neurovascular cross talk in diabetic retinopathy: Pathophysiological roles and therapeutic implications

Diabetic retinopathy (DR) is the leading cause of blindness in the working-age population in developed countries, and its prevalence will increase as the global incidence of diabetes grows exponentially. DR begins with an early nonproliferative stage in which retinal blood vessels and neurons degenerate as a consequence of chronic hyperglycemia, resulting in vasoregression and persistent retinal ischemia, metabolic disequilibrium, and inflammation. This is conducive to overcompensatory pathological neovascularization associated with advanced proliferative DR. Although DR is considered a microvascular complication, the retinal microvasculature is intimately associated with and governed by neurons and glia; neurodegeneration, neuroinflammation, and dysregulation of neurovascular cross talk are responsible in part for vascular abnormalities in both early nonproliferative DR and advanced proliferative DR. Neuronal activity directly regulates microvascular dilation and blood flow in the process of neurovascular coupling. Retinal neurons also secrete guidance cues in response to injury, ischemia, or metabolic stress that may either promote or suppress vascular outgrowth, either alleviating or exacerbating DR, contingent on the stage of disease and retinal microenvironment. Neurodegeneration, impaired neurovascular coupling, and dysregulation of neuronal guidance cues are key events in the pathogenesis of DR, and correcting these events may prevent or delay development of advanced DR. The review discusses the mechanisms of neurovascular cross talk and its dysregulation in DR, and their potential therapeutic implications.

Fenofibrate Inhibits Cytochrome P450 Epoxygenase 2C Activity to Suppress Pathological Ocular Angiogenesis

Neovascular eye diseases including retinopathy of prematurity, diabetic retinopathy and age-related-macular-degeneration are major causes of blindness. Fenofibrate treatment in type 2 diabetes patients reduces progression of diabetic retinopathy independent of its peroxisome proliferator-activated receptor (PPAR)α agonist lipid lowering effect. The mechanism is unknown. Fenofibrate binds to and inhibits cytochrome P450 epoxygenase (CYP)2C with higher affinity than to PPARα. CYP2C metabolizes ω-3 long-chain polyunsaturated fatty acids (LCPUFAs). While ω-3 LCPUFA products from other metabolizing pathways decrease retinal and choroidal neovascularization, CYP2C products of both ω-3 and ω-6 LCPUFAs promote angiogenesis. We hypothesized that fenofibrate inhibits retinopathy by reducing CYP2C ω-3 LCPUFA (and ω-6 LCPUFA) pro-angiogenic metabolites. Fenofibrate reduced retinal and choroidal neovascularization in PPARα-/-mice and augmented ω-3 LCPUFA protection via CYP2C inhibition. Fenofibrate suppressed retinal and choroidal neovascularization in mice overexpressing human CYP2C8 in endothelial cells and reduced plasma levels of the pro-angiogenic ω-3 LCPUFA CYP2C8 product, 19,20-epoxydocosapentaenoic acid. 19,20-epoxydocosapentaenoic acid reversed fenofibrate-induced suppression of angiogenesis ex vivo and suppression of endothelial cell functions in vitro. In summary fenofibrate suppressed retinal and choroidal neovascularization via CYP2C inhibition as well as by acting as an agonist of PPARα. Fenofibrate augmented the overall protective effects of ω-3 LCPUFAs on neovascular eye diseases.

Cytochrome P450 Oxidase 2C Inhibition Adds to ω-3 Long-Chain Polyunsaturated Fatty Acids Protection Against Retinal and Choroidal Neovascularization

Objective—Pathological ocular neovascularization is a major cause of blindness. Increased dietary intake of &ohgr;-3 long-chain polyunsaturated fatty acids (LCPUFA) reduces retinal neovascularization and choroidal neovascularization (CNV), but &ohgr;-3 LCPUFA metabolites of a major metabolizing pathway, cytochrome P450 oxidase (CYP) 2C, promote ocular pathological angiogenesis. We hypothesized that inhibition of CYP2C activity will add to the protective effects of &ohgr;-3 LCPUFA on neovascular eye diseases. Approach and Results—The mouse models of oxygen-induced retinopathy and laser-induced CNV were used to investigate pathological angiogenesis in the retina and choroid, respectively. The plasma levels of &ohgr;-3 LCPUFA metabolites of CYP2C were determined by mass spectroscopy. Aortic ring and choroidal explant sprouting assays were used to investigate the effects of CYP2C inhibition and &ohgr;-3 LCPUFA–derived CYP2C metabolic products on angiogenesis ex vivo. We found that inhibition of CYP2C activity by montelukast added to the protective effects of &ohgr;-3 LCPUFA on retinal neovascularization and CNV by 30% and 20%, respectively. In CYP2C8-overexpressing mice fed a &ohgr;-3 LCPUFA diet, montelukast suppressed retinal neovascularization and CNV by 36% and 39% and reduced the plasma levels of CYP2C8 products. Soluble epoxide hydrolase inhibition, which blocks breakdown and inactivation of CYP2C &ohgr;-3 LCPUFA–derived active metabolites, increased oxygen-induced retinopathy and CNV in vivo. Exposure to selected &ohgr;-3 LCPUFA metabolites of CYP2C significantly reversed the suppression of both angiogenesis ex vivo and endothelial cell functions in vitro by the CYP2C inhibitor montelukast. Conclusions—Inhibition of CYP2C activity adds to the protective effects of &ohgr;-3 LCPUFA on pathological retinal neovascularization and CNV.

Retinal expression of small non-coding RNAs in a murine model of proliferative retinopathy

Ocular neovascularization is a leading cause of blindness in proliferative retinopathy. Small non-coding RNAs (sncRNAs) play critical roles in both vascular and neuronal development of the retina through post-transcriptional regulation of target gene expression. To identify the function and therapeutic potential of sncRNAs in retinopathy, we assessed the expression profile of retinal sncRNAs in a mouse model of oxygen-induced retinopathy (OIR) with pathologic proliferation of neovessels. Approximately 2% of all analyzed sncRNAs were significantly altered in OIR retinas compared with normoxic controls. Twenty three microRNAs with substantial up- or down-regulation were identified, including miR-351, -762, -210, 145, -155, -129-5p, -150, -203, and -375, which were further analyzed for their potential target genes in angiogenic, hypoxic, and immune response-related pathways. In addition, nineteen small nucleolar RNAs also revealed differential expression in OIR retinas compared with control retinas. A decrease of overall microRNA expression in OIR retinas was consistent with reduced microRNA processing enzyme Dicer, and increased expression of Alu element in OIR. Together, our findings elucidated a group of differentially expressed sncRNAs in a murine model of proliferative retinopathy. These sncRNAs may exert critical post-transcriptional regulatory roles in regulating pathological neovascularization in eye diseases.

Animal models of ocular angiogenesis: from development to pathologies

Pathological angiogenesis in the eye is an important feature in the pathophysiology of many vision‐threatening diseases, including retinopathy of prematurity, diabetic retinopathy, and age‐related macular degeneration, as well as corneal diseases with abnormal angiogenesis. Development of reproducible and reliable animal models of ocular angiogenesis has advanced our understanding of both the normal development and the pathobiology of ocular neovascularization. These models have also proven to be valuable experimental tools with which to easily evaluate potential antiangiogenic therapies beyond eye research. This review summarizes the current available animal models of ocular angiogenesis. Models of retinal and choroidal angiogenesis, including oxygen‐induced retinopathy, laser‐induced choroidal neovascularization, and transgenic mouse models with deficient or spontaneous retinal/choroidal neovascularization, as well as models with induced corneal angiogenesis, are widely used to investigate the molecular and cellular basis of angiogenic mechanisms. Theoretical concepts and experimental protocols of these models are outlined, as well as their advantages and potential limitations, which may help researchers choose the most suitable models for their investigative work.—Liu, C.‐H., Wang, Z., Sun, Y., Chen, J. Animal models of ocular angiogenesis: from development to pathologies. FASEB J. 31, 4665–4681 (2017). www.fasebj.org

Pharmacologic Activation of Wnt Signaling by Lithium Normalizes Retinal Vasculature in a Murine Model of Familial Exudative Vitreoretinopathy

Familial exudative vitreoretinopathy (FEVR) is characterized by delayed retinal vascular development, which promotes hypoxia-induced pathologic vessels. In severe cases FEVR may lead to retinal detachment and visual impairment. Genetic studies linked FEVR with mutations in Wnt signaling ligand or receptors, including low-density lipoprotein receptor-related protein 5 (LRP5) gene. Here, we investigated ocular pathologies in a Lrp5 knockout (Lrp5(-/-)) mouse model of FEVR and explored whether treatment with a pharmacologic Wnt activator lithium could bypass the genetic defects, thereby protecting against eye pathologies. Lrp5(-/-) mice displayed significantly delayed retinal vascular development, absence of deep layer retinal vessels, leading to increased levels of vascular endothelial growth factor and subsequent pathologic glomeruloid vessels, as well as decreased inner retinal visual function. Lithium treatment in Lrp5(-/-) mice significantly restored the delayed development of retinal vasculature and the intralaminar capillary networks, suppressed formation of pathologic glomeruloid structures, and promoted hyaloid vessel regression. Moreover, lithium treatment partially rescued inner-retinal visual function and increased retinal thickness. These protective effects of lithium were largely mediated through restoration of canonical Wnt signaling in Lrp5(-/-) retina. Lithium treatment also substantially increased vascular tubular formation in LRP5-deficient endothelial cells. These findings suggest that pharmacologic activation of Wnt signaling may help treat ocular pathologies in FEVR and potentially other defective Wnt signaling-related diseases.

Inflammatory signals from photoreceptor modulate pathological retinal angiogenesis via c-Fos

Pathological neovessels growing into the normally avascular photoreceptors cause vision loss in many eye diseases, such as age-related macular degeneration and macular telangiectasia. Ocular neovascularization is strongly associated with inflammation, but the source of inflammatory signals and the mechanisms by which these signals regulate the disruption of avascular privilege in photoreceptors are unknown. In this study, we found that c-Fos, a master inflammatory regulator, was increased in photoreceptors in a model of pathological blood vessels invading photoreceptors: the very low-density lipoprotein receptor–deficient (Vldlr−/−) mouse. Increased c-Fos induced inflammatory cytokines interleukin 6 (IL-6) and tumor necrosis factor (TNF), leading to activation of signal transducer and activator of transcription 3 (STAT3) and increased TNF&agr;–induced protein 3 (TNFAIP3) in Vldlr−/− photoreceptors. IL-6 activated the STAT3/vascular endothelial growth factor A (VEGFA) pathway directly, and elevated TNFAIP3 suppressed SOCS3 (suppressor of cytokine signaling 3)–activated STAT3/VEGFA indirectly. Inhibition of c-Fos using photoreceptor-specific AAV (adeno-associated virus)-hRK (human rhodopsin kinase)–sh_c-fos or a chemical inhibitor substantially reduced the pathological neovascularization and rescued visual function in Vldlr−/− mice. These findings suggested that the photoreceptor c-Fos controls blood vessel growth into the normally avascular photoreceptor layer through the inflammatory signal–induced STAT3/VEGFA pathway.

FGF21 Administration Suppresses Retinal and Choroidal Neovascularization in Mice

Pathological neovascularization, a leading cause of blindness, is seen in retinopathy of prematurity, diabetic retinopathy, and age-related macular degeneration. Using a mouse model of hypoxia-driven retinal neovascularization, we find that fibroblast growth factor 21 (FGF21) administration suppresses, and FGF21 deficiency worsens, retinal neovessel growth. The protective effect of FGF21 against neovessel growth was abolished in adiponectin (APN)-deficient mice. FGF21 administration also decreased neovascular lesions in two models of neovascular age-related macular degeneration: very-low-density lipoprotein-receptor-deficient mice with retinal angiomatous proliferation and laser-induced choroidal neovascularization. FGF21 inhibited tumor necrosis α (TNF-α) expression but did not alter Vegfa expression in neovascular eyes. These data suggest that FGF21 may be a therapeutic target for pathologic vessel growth in patients with neovascular eye diseases, including retinopathy of prematurity, diabetic retinopathy, and age-related macular degeneration.

Photoreceptor glucose metabolism determines normal retinal vascular growth

The neural cells and factors determining normal vascular growth are not well defined even though vision‐threatening neovessel growth, a major cause of blindness in retinopathy of prematurity (ROP) (and diabetic retinopathy), is driven by delayed normal vascular growth. We here examined whether hyperglycemia and low adiponectin (APN) levels delayed normal retinal vascularization, driven primarily by dysregulated photoreceptor metabolism. In premature infants, low APN levels correlated with hyperglycemia and delayed retinal vascular formation. Experimentally in a neonatal mouse model of postnatal hyperglycemia modeling early ROP, hyperglycemia caused photoreceptor dysfunction and delayed neurovascular maturation associated with changes in the APN pathway; recombinant mouse APN or APN receptor agonist AdipoRon treatment normalized vascular growth. APN deficiency decreased retinal mitochondrial metabolic enzyme levels particularly in photoreceptors, suppressed retinal vascular development, and decreased photoreceptor platelet‐derived growth factor (Pdgfb). APN pathway activation reversed these effects. Blockade of mitochondrial respiration abolished AdipoRon‐induced Pdgfb increase in photoreceptors. Photoreceptor knockdown of Pdgfb delayed retinal vascular formation. Stimulation of the APN pathway might prevent hyperglycemia‐associated retinal abnormalities and suppress phase I ROP in premature infants.

Fibroblast Growth Factor 21 Protects Photoreceptor Function in Type 1 Diabetic Mice

Retinal neuronal abnormalities occur before vascular changes in diabetic retinopathy. Accumulating experimental evidence suggests that neurons control vascular pathology in diabetic and other neovascular retinal diseases. Therefore, normalizing neuronal activity in diabetes may prevent vascular pathology. We investigated whether fibroblast growth factor 21 (FGF21) prevented retinal neuronal dysfunction in insulin-deficient diabetic mice. We found that in diabetic neural retina, photoreceptor rather than inner retinal function was most affected and administration of the long-acting FGF21 analog PF-05231023 restored the retinal neuronal functional deficits detected by electroretinography. PF-05231023 administration protected against diabetes-induced disorganization of photoreceptor segments seen in retinal cross section with immunohistochemistry and attenuated the reduction in the thickness of photoreceptor segments measured by optical coherence tomography. PF-05231023, independent of its downstream metabolic modulator adiponectin, reduced inflammatory marker interleukin-1β (IL-1β) mRNA levels. PF-05231023 activated the AKT-nuclear factor erythroid 2–related factor 2 pathway and reduced IL-1β expression in stressed photoreceptors. PF-05231023 administration did not change retinal expression of vascular endothelial growth factor A, suggesting a novel therapeutic approach for the prevention of early diabetic retinopathy by protecting photoreceptor function in diabetes.