Person:

Stahl, Andreas

Objective: Diabetic retinopathy (DR) is associated with hyperglycemia-driven microvascular pathology and neuronal compromise in the retina. However, DR is also linked to dyslipidemia. As omega-3 (ω-3) polyunsaturated fatty acids (PUFAs) are protective in proliferative retinopathy, we investigated the capacity of ω-3PUFAs to preserve retinal function in a mouse model of type 2 diabetes mellitus (T2DM). Design: Male leptin-receptor-deficient (db/db) mice were maintained for 22 weeks (4 weeks–26 weeks of life) on calorically and compositionally matched diets, except for 2% enrichment in either ω-3 or ω-6PUFAs. Visual function was assessed at 9, 14 and 26 weeks by electroretinography. Retinal capillary and neuronal integrity, as well as glucose challenge responses, were assessed on each diet. Results: The ω-3PUFA diet significantly preserved retinal function in the mouse model of T2DM to levels similar to those observed in nondiabetic control mice on normal chow. Conversely, retinal function gradually deteriorated in db/db mice on a ω-6PUFA-rich diet. There was also an enhanced ability of ω-3PUFA-fed mice to respond to glucose challenge. The protection of visual function appeared to be independent of cytoprotective or anti-inflammatory effects of ω-3PUFAs. Conclusion: This study identifies beneficial effects of dietary ω-3PUFAs on visual function in T2DM. The data are consistent with dyslipidemia negatively impacting retinal function. As ω-3PUFA lipid dietary interventions are readily available, safe and inexpensive, increasing ω-3PUFA intake in diabetic patients may slow the progression of vision loss in T2DM.

Mutations in low-density lipoprotein receptor-related protein 5 (Lrp5) impair retinal angiogenesis in patients with familial exudative vitreoretinopathy (FEVR), a rare type of blinding vascular eye disease. The defective retinal vasculature phenotype in human FEVR patients is recapitulated in Lrp5 knockout ((Lrp5^{-/-})) mouse with delayed and incomplete development of retinal vessels. In this study we examined gene expression changes in the developing (Lrp5^{−/−}) mouse retina to gain insight into the molecular mechanisms that underlie the pathology of FEVR in humans. Gene expression levels were assessed with an Illumina microarray on total RNA from (Lrp5^{−/−}) and WT retinas isolated on postnatal day (P) 8. Regulated genes were confirmed using RT-qPCR analysis. Consistent with a role in vascular development, we identified expression changes in genes involved in cell-cell adhesion, blood vessel morphogenesis and membrane transport in (Lrp5^{−/−}) retina compared to WT retina. In particular, tight junction protein claudin5 and amino acid transporter slc38a5 are both highly down-regulated in (Lrp5^{−/−}) retina. Similarly, several Wnt ligands including Wnt7b show decreased expression levels. Plasmalemma vesicle associated protein (plvap), an endothelial permeability marker, in contrast, is up-regulated consistent with increased permeability in (Lrp5^{−/−}) retinas. Together these data suggest that Lrp5 regulates multiple groups of genes that influence retinal angiogenesis and may contribute to the pathogenesis of FEVR.

BACKGROUND: Ischemic proliferative retinopathy, characterized by pathological retinal neovascularization, is a major cause of blindness in working-age adults and children. Defining the molecular pathways distinguishing pathological neovascularization from normal vessels is critical to controlling these blinding diseases with targeted therapy. Because mutations in Wnt signaling cause defective retinal vasculature in humans with some characteristics of the pathological vessels in retinopathy, we investigated the potential role of Wnt signaling in pathological retinal vascular growth in proliferative retinopathy. METHODS AND RESULTS: In this study, we show that Wnt receptors (Frizzled4 and low-density lipoprotein receptor-related protein5 [Lrp5]) and activity are significantly increased in pathological neovascularization in a mouse model of oxygen-induced proliferative retinopathy. Loss of Wnt coreceptor Lrp5 and downstream signaling molecule dishevelled2 significantly decreases the formation of pathological retinal neovascularization in retinopathy. Loss of Lrp5 also affects retinal angiogenesis during development and formation of the blood-retinal barrier, which is linked to significant downregulation of tight junction protein claudin5 in Lrp5(-/-) vessels. Blocking claudin5 significantly suppresses Wnt pathway-driven endothelial cell sprouting in vitro and developmental and pathological vascular growth in retinopathy in vivo. CONCLUSIONS: These results demonstrate an important role of Wnt signaling in pathological vascular development in retinopathy and show a novel function of Cln5 in promoting angiogenesis.

PURPOSE: Vascular degeneration and the ensuing abnormal vascular proliferation are central to proliferative retinopathies. Given the metabolic discordance associated with these diseases, the authors explored the role of ghrelin and its growth hormone secretagogue receptor 1a (GHSR-1a) in proliferative retinopathy. METHODS: In a rat model of oxygen-induced retinopathy (OIR), the contribution of ghrelin and GHSR-1a was investigated using the stable ghrelin analogs [Dap3]-ghrelin and GHRP6 and the GSHR-1a antagonists JMV-2959 and [D-Lys3]-GHRP-6. Plasma and retinal levels of ghrelin were analyzed by ELISA, whereas retinal expression and localization of GHSR-1a were examined by immunohistochemistry and Western blot analysis. The angiogenic and vasoprotective properties of ghrelin and its receptor were further confirmed in aortic explants and in models of vaso-obliteration. RESULTS: Ghrelin is produced locally in the retina, whereas GHSR-1a is abundantly expressed in retinal endothelial cells. Ghrelin levels decrease during the vaso-obliterative phase and rise during the proliferative phase of OIR. Intravitreal delivery of [Dap3]-ghrelin during OIR significantly reduces retinal vessel loss when administered during the hyperoxic phase. Conversely, during the neovascular phase, ghrelin promotes pathologic angiogenesis through the activation of GHSR-1a. These angiogenic effects were confirmed ex vivo in aortic explants. CONCLUSIONS: New roles were disclosed for the ghrelin-GHSR-1a pathway in the preservation of retinal vasculature during the vaso-obliterative phase of OIR and during the angiogenic phase of OIR. These findings suggest that the ghrelin-GHSR-1a pathway can exert opposing effects on retinal vasculature, depending on the phase of retinopathy, and thus holds therapeutic potential for proliferative retinopathies.

PURPOSE OF REVIEW: Despite current treatments, retinopathy of prematurity (ROP) remains a major cause of blindness in premature infants and the incidence is increasing with increased survival of infants born at very early gestational ages. This review summarizes the recent literature on ROP with a special focus on recent advances in treatment options as well as newly developed methods for disease screening. RECENT FINDINGS: Genetic studies find a genetic predisposition to ROP-linking genes in the Wnt pathway with development of severe ROP. With regard to diagnosis, a new screening method has been developed that allows prediction of ROP risk based on postnatal body weight gain alone. Formerly weight gain postnatally in combination with insulin-like growth factor levels was found to predict treatable ROP. New treatment options for severe cases of ROP have been proposed targeting vascular endothelial growth factor (VEGF). Whether anti-VEGF treatment is well tolerated in preterm infants, however, has to be further evaluated in controlled clinical trials. Finally, new reports from the early treatment ROP group suggest that early laser treatment for type 1 but not type 2 high-risk prethreshold ROP improves visual acuity outcomes at 6 years of age. SUMMARY: With the increasing survival of premature infants and increased incidence of ROP, it is important to screen for ROP risk and treat at-risk patients in a timely manner to preserve their visual function and reduce complications.

Lipids and lipid metabolites have long been known to play biological roles that go beyond energy storage and membrane structure. In age-related macular degeneration and diabetes, for example, dysregulation of lipid metabolism is closely associated with disease onset and progression. At the same time, some lipids and their metabolites can exert beneficial effects in the same disorders. This review summarises our current knowledge of the contributions of lipids to both the pathogenesis and treatment of neovascular eye disease. The clinical entities covered are exudative age-related macular degeneration, diabetic retinopathy and retinopathy of prematurity, with a special emphasis on the potential therapeutic effects of ω3- (also known as n-3) polyunsaturated fatty acids.

PURPOSE: Macular telangiectasia (MacTel) is a vision-threatening retinal disease with unknown pathogenesis and no approved treatment. Very low-density lipoprotein receptor mutant mice (Vldlr(-/-)) exhibit critical features of MacTel such as retinal neovascularization and photoreceptor degeneration. In this study, the authors evaluate the therapeutic potential of resveratrol, a plant polyphenol, in Vldlr(-/-) mice as a model for MacTel. METHODS: Vldlr(-/-) and wild-type mice at postnatal day (P) 21 to P60 or P10 to P30 were treated orally with resveratrol. The number of neovascular lesions was evaluated on retinal flatmounts, and resveratrol effects on endothelial cells were assessed by Western blot for phosphorylated ERK1/2, aortic ring, and migration assays. Vegf and Gfap expression was evaluated in laser-capture microdissected retinal layers of angiogenic lesions and nonlesion areas from Vldlr(-/-) and wild-type retinas. RESULTS: From P15 onward, Vldlr(-/-) retinas develop vascular lesions associated with the local upregulation of Vegf in photoreceptors and Gfap in the inner retina. Oral resveratrol reduces lesion formation when administered either before or after disease onset. The reduction of vascular lesions in resveratrol-treated Vldlr(-/-) mice is associated with the suppression of retinal Vegf transcription. Resveratrol also reduces endothelial ERK1/2 signaling as well as the migration and proliferation of endothelial cells. Furthermore, a trend toward increased rhodopsin mRNA in Vldlr(-/-) retinas is observed. CONCLUSIONS: Oral administration of resveratrol is protective against retinal neovascular lesions in Vldlr(-/-) mice by inhibiting Vegf expression and angiogenic activation of retinal endothelial cells. These results suggest that resveratrol might be a safe and effective intervention for treating patients with MacTel.

Purpose.: Retinopathy of prematurity (ROP) is a leading cause of blindness in children and is, in its most severe form, characterized by uncontrolled growth of vision-threatening pathologic vessels. Propranolol, a nonselective β-adrenergic receptor blocker, was reported to protect against pathologic retinal neovascularization in a mouse model of oxygen-induced retinopathy (OIR). Based on this single animal study using nonstandard evaluation of retinopathy, clinical trials are currently ongoing to evaluate propranolol treatment in stage 2 ROP patients who tend to experience spontaneous disease regression and are at low risk of blindness. Because these ROP patients are vulnerable premature infants who are still in a fragile state of incomplete development, the efficacy of propranolol treatment in retinopathy needs to be evaluated thoroughly in preclinical animal models of retinopathy and potential benefits weighed against potential adverse effects.

Methods.: Retinopathy was induced by exposing neonatal mice to 75% oxygen from postnatal day (P) 7 to P12. Three routes of propranolol treatment were assessed from P12 to P16: oral gavage, intraperitoneal injection, or subcutaneous injection, with doses varying between 2 and 60 mg/kg/day. At P17, retinal flatmounts were stained with isolectin and quantified with a standard protocol to measure vasoobliteration and pathologic neovascularization. Retinal gene expression was analyzed with qRT-PCR using RNA isolated from retinas of control and propranolol-treated pups.

Results.: None of the treatment approaches at any dose of propranolol (up to 60 mg/kg/day) were effective in preventing the development of retinopathy in a mouse model of OIR, evaluated using standard techniques. Propranolol treatment also did not change retinal expression of angiogenic factors including vascular endothelial growth factor.

Conclusions.: Propranolol treatment via three routes and up to 30 times the standard human dose failed to suppress retinopathy development in mice. These data bring into question whether propranolol through inhibition of β-adrenergic receptors is an appropriate therapeutic approach for treating ROP.

BACKGROUND: Increased intake of ω-3 long-chain polyunsaturated fatty acids (LCPUFAs) and use of peroxisome proliferator activator receptor (PPAR)-activating drugs are associated with attenuation of pathologic retinal angiogenesis. ω-3 LCPUFAs are endogenous agonists of PPARs. We postulated that DNA sequence variation in PPAR gamma (PPARG) co-activator 1 alpha (PPARGC1A), a gene encoding a co-activator of the LCPUFA-sensing PPARG-retinoid X receptor (RXR) transcription complex, may influence neovascularization (NV) in age-related macular degeneration (AMD). METHODS: We applied exact testing methods to examine distributions of DNA sequence variants in PPARGC1A for association with NV AMD and interaction of AMD-associated loci in genes of complement, lipid metabolism, and VEGF signaling systems. Our sample contained 1858 people from 3 elderly cohorts of western European ancestry. We concurrently investigated retinal gene expression profiles in 17-day-old neonatal mice on a 2% LCPUFA feeding paradigm to identify LCPUFA-regulated genes both associated with pathologic retinal angiogenesis and known to interact with PPARs or PPARGC1A. RESULTS: A DNA coding variant (rs3736265) and a 3'UTR-resident regulatory variant (rs3774923) in PPARGC1A were independently associated with NV AMD (exact P = 0.003, both SNPs). SNP-SNP interactions existed for NV AMD (P<0.005) with rs3736265 and a AMD-associated variant in complement factor B (CFB, rs512559). PPARGC1A influences activation of the AMD-associated complement component 3 (C3) promoter fragment and CFB influences activation and proteolysis of C3. We observed interaction (P ≤ 0.003) of rs3736265 with a variant in vascular endothelial growth factor A (VEGFA, rs3025033), a key molecule in retinal angiogenesis. Another PPARGC1A coding variant (rs8192678) showed statistical interaction with a SNP in the VEGFA receptor fms-related tyrosine kinase 1 (FLT1, rs10507386; P ≤ 0.003). C3 expression was down-regulated 2-fold in retinas of ω-3 LCPUFA-fed mice - these animals also showed 70% reduction in retinal NV (P ≤ 0.001). CONCLUSION: Ligands and co-activators of the ω-3 LCPUFA sensing PPAR-RXR axis may influence retinal angiogenesis in NV AMD via the complement and VEGF signaling systems. We have linked the co-activator of a lipid-sensing transcription factor (PPARG co-activator 1 alpha, PPARGC1A) to age-related macular degeneration (AMD) and AMD-associated genes.

Regeneration of blood vessels in ischemic neuronal tissue is critical to reduce tissue damage in diseases. In proliferative retinopathy, initial vessel loss leads to retinal ischemia, which can induce either regrowth of vessels to restore normal metabolism and minimize damage, or progress to hypoxia-induced sight-threatening pathologic vaso-proliferation. It is not well understood how retinal neurons mediate regeneration of vascular growth in response to ischemic insults. In this study we aim to investigate the potential role of Sirtuin 1 (Sirt1), a metabolically-regulated protein deacetylase, in mediating the response of ischemic neurons to regulate vascular regrowth in a mouse model of oxygen-induced ischemic retinopathy (OIR). We found that Sirt1 is highly induced in the avascular ischemic retina in OIR. Conditional depletion of neuronal Sirt1 leads to significantly decreased retinal vascular regeneration into the avascular zone and increased hypoxia-induced pathologic vascular growth. This effect is likely independent of PGC-1α, a known Sirt1 target, as absence of PGC-1α in knockout mice does not impact vascular growth in retinopathy. We found that neuronal Sirt1 controls vascular regrowth in part through modulating deacetylation and stability of hypoxia-induced factor 1α and 2α, and thereby modulating expression of angiogenic factors. These results indicate that ischemic neurons induce Sirt1 to promote revascularization into ischemic neuronal areas, suggesting a novel role of neuronal Sirt1 in mediating vascular regeneration in ischemic conditions, with potential implications beyond retinopathy.