OBJECTIVE— The mechanism underlying pericyte loss during incipient diabetic retinopathy remains controversial. Hyperglycemia induces angiopoietin-2 (Ang-2) transcription, which modulates capillary pericyte coverage. In this study, we assessed loss of pericyte subgroups and the contribution of Ang-2 to pericyte migration.
RESEARCH DESIGN AND METHODS— Numbers of total pericytes and their subgroups were quantified in retinal digest preparations of spontaneous diabetic XLacZ mice. Pericytes were divided into subgroups according to their localization, their position relative to adjacent endothelial cells, and the expression of LacZ. The contribution of Ang-2 to pericyte migration was assessed in Ang-2 overexpressing (mOpsinhAng2) and deficient (Ang2LacZ) mice.
RESULTS— Pericyte numbers were reduced by 16% (P < 0.01) in XLacZ mice after 6 months of diabetes. Reduction of pericytes was restricted to pericytes on straight capillaries (relative reduction 27%, P < 0.05) and was predominantly observed in LacZ-positive pericytes (−20%, P < 0.01). Hyperglycemia increased the numbers of migrating pericytes (69%; P < 0.05), of which the relative increase due to diabetes was exclusively in LacZ-negative pericytes, indicating reduced adherence to the capillaries (176%; P < 0.01). Overexpression of Ang-2 in nondiabetic retinas mimicked diabetic pericyte migration of wild-type animals (78%; P < 0.01). Ang-2 deficient mice completely lacked hyperglycemia-induced increase in pericyte migration compared with wild-type littermates.
CONCLUSIONS— Diabetic pericyte loss is the result of pericyte migration, and this process is modulated by the Ang-Tie system.
Diabetes is associated with a high incidence of macrovascular disease (MVD), including peripheral and coronary artery disease. Circulating soluble-Klotho (sKlotho) is produced in the kidney and is a putative anti-aging and vasculoprotective hormone. Reduced Klotho levels may therefore increase cardiovascular risk in diabetes. We investigated if sKlotho levels are decreased in type 2 diabetes and associate with MVD in the absence of diabetic nephropathy, and whether hyperglycemia affects renal Klotho production in vitro and in vivo.
sKlotho levels were determined with ELISA in diabetic and non-diabetic patients with and without MVD, and healthy control subjects. Human renal tubular epithelial cells (TECs) were isolated and exposed to high glucose levels (15 and 30 mM) in vitro and Klotho levels were measured with qPCR and quantitative immunofluorescence. Klotho mRNA expression was quantified in kidneys obtained from long term (3 and 8 months) diabetic Ins2Akita mice and normoglycemic control mice.
No significant differences in sKlotho levels were observed between diabetic patients with and without MVD (527 (433–704) pg/mL, n = 35), non-diabetic MVD patients (517 (349–571) pg/mL, n = 27), and healthy control subjects (435 (346–663) pg/mL, n = 15). High glucose (15 and 30 mM) did not alter Klotho expression in TECs. Long-term hyperglycemia in diabetic Ins2Akita mice (characterized by increased HbA1c levels [12.9 ± 0.3% (3 months) and 11.3 ± 2.0% (8 months)], p < 0.05 vs. non-diabetic mice) did not affect renal Klotho mRNA expression.
These data indicate that sKlotho levels are not affected in type 2 diabetes patients with and without MVD. Furthermore, hyperglycemia per se does not affect renal Klotho production. As type 2 diabetes does not alter sKlotho levels, sKlotho does not seem to play a major role in the pathogenesis of MVD in type 2 diabetes.
Atherosclerosis; Coronary artery disease; Klotho; Macrovascular disease; Peripheral artery disease; Type 2 diabetes
DHA diols produced by Müller cells suppress Notch activation in endothelial cells, thereby promoting retinal angiogenesis.
Cytochrome P450 (CYP) epoxygenases generate bioactive lipid epoxides which can be further metabolized to supposedly less active diols by the soluble epoxide hydrolase (sEH). As the role of epoxides and diols in angiogenesis is unclear, we compared retinal vasculature development in wild-type and sEH−/− mice. Deletion of the sEH significantly delayed angiogenesis, tip cell, and filopodia formation, a phenomenon associated with activation of the Notch signaling pathway. In the retina, sEH was localized in Müller glia cells, and Müller cell–specific sEH deletion reproduced the sEH−/− retinal phenotype. Lipid profiling revealed that sEH deletion decreased retinal and Müller cell levels of 19,20–dihydroxydocosapentaenoic acid (DHDP), a diol of docosahexenoic acid (DHA). 19,20-DHDP suppressed endothelial Notch signaling in vitro via inhibition of the γ-secretase and the redistribution of presenilin 1 from lipid rafts. Moreover, 19,20-DHDP, but not the parent epoxide, was able to rescue the defective angiogenesis in sEH−/− mice as well as in animals lacking the Fbxw7 ubiquitin ligase, which demonstrate strong basal activity of the Notch signaling cascade. These studies demonstrate that retinal angiogenesis is regulated by a novel form of neuroretina–vascular interaction involving the sEH-dependent generation of a diol of DHA in Müller cells.
Vasoregression is a hallmark of vascular eye diseases but the mechanisms involved are still largely unknown. We have recently characterized a rat ciliopathy model which develops primary photoreceptor degeneration and secondary vasoregression. To improve the understanding of secondary vasoregression in retinal neurodegeneration, we used microarray techniques to compare gene expression profiles in this new model before and after retinal vasoregression. Differential gene expression was validated by quantitative RT-PCR, Western blot and immunofluorescence. Of the 157 genes regulated more than twofold, the MHC class II invariant chain CD74 yielded the strongest upregulation, and was allocated to activated microglial cells close to the vessels undergoing vasoregression. Pathway clustering identified genes of the immune system including inflammatory signaling, and components of the complement cascade upregulated during vasoregression. Together, our data suggest that microglial cells involved in retinal immune response participate in the initiation of vasoregression in the retina.
Rats expressing a transgenic polycystic kidney disease (PKD) gene develop photoreceptor degeneration and subsequent vasoregression, as well as activation of retinal microglia and macroglia. To target the whole neuroglialvascular unit, neuro- and vasoprotective Erythropoietin (EPO) was intraperitoneally injected into four –week old male heterozygous PKD rats three times a week at a dose of 256 IU/kg body weight. For comparison EPO-like peptide, lacking unwanted side effects of EPO treatment, was given five times a week at a dose of 10 µg/kg body weight. Matched EPO treated Sprague Dawley and water-injected PKD rats were held as controls. After four weeks of treatment the animals were sacrificed and analysis of the neurovascular morphology, glial cell activity and pAkt localization was performed. The number of endothelial cells and pericytes did not change after treatment with EPO or EPO-like peptide. There was a nonsignificant reduction of migrating pericytes by 23% and 49%, respectively. Formation of acellular capillaries was significantly reduced by 49% (p<0.001) or 40% (p<0.05). EPO-treatment protected against thinning of the central retina by 10% (p<0.05), a composite of an increase of the outer nuclear layer by 12% (p<0.01) and in the outer segments of photoreceptors by 26% (p<0.001). Quantification of cell nuclei revealed no difference. Microglial activity, shown by gene expression of CD74, decreased by 67% (p<0.01) after EPO and 36% (n.s.) after EPO-like peptide treatment. In conclusion, EPO safeguards the neuroglialvascular unit in a model of retinal neurodegeneration and secondary vasoregression. This finding strengthens EPO in its protective capability for the whole neuroglialvascular unit.
Neuronal damage is correlated with vascular dysfunction in the diseased retina, but the underlying mechanisms remain controversial because of the lack of suitable models in which vasoregression related to neuronal damage initiates in the mature retinal vasculature. The aim of this study was to assess the temporal link between neuronal damage and vascular patency in a transgenic rat (TGR) with overexpression of a mutant cilia gene polycystin-2.
Vasoregression, neuroglial changes and expression of neurotrophic factors were assessed in TGR and control rats in a time course. Determination of neuronal changes was performed by quantitative morphometry of paraffin-embedded vertical sections. Vascular cell composition and patency were assessed by quantitative retinal morphometry of digest preparations. Glial activation was assessed by western blot and immunofluorescence. Expression of neurotrophic factors was detected by quantitative PCR.
At one month, number and thickness of the outer nuclear cell layers (ONL) in TGR rats were reduced by 31% (p<0.001) and 17% (p<0.05), respectively, compared to age-matched control rats. Furthermore, the reduction progressed from 1 to 7 months in TGR rats. Apoptosis was selectively detected in the photoreceptor in the ONL, starting after one month. Nevertheless, TGR and control rats showed normal responses in electroretinogram at one month. From the second month onwards, TGR retinas had significantly increased acellular capillaries (p<0.001), and a reduction of endothelial cells (p<0.01) and pericytes (p<0.01). Upregulation of GFAP was first detected in TGR retinas after 1 month in glial cells, in parallel with an increase of FGF2 (fourfold) and CNTF (60 %), followed by upregulation of NGF (40 %) at 3 months.
Our data suggest that TGR is an appropriate animal model for vasoregression related to neuronal damage. Similarities to experimental diabetic retinopathy render this model suitable to understand general mechanisms of maturity-onset vasoregression.
This study aimed to analyze the effect of HbA1c variability on the occurrence of diabetic retinopathy in type 1 diabetes patients.
Patients and Methods
35,891 patients with childhood, adolescent or adult onset of type 1 diabetes from a large multicentre survey, the German/Austrian prospective documentation system (DPV), were analysed. Cox proportional hazard models were used to examine whether intra-individual HbA1c variability expressed as variation coefficient is an independent risk factor for the occurrence of diabetic retinopathy.
Kaplan-Meier curves stratified by median HbA1c and variation coefficient revealed that retinopathy-free survival probability is lower when both median HbA1c and HbA1c variability are above the 50th percentile. Cox regression models confirmed this finding: After adjustment for age at diabetes onset, gender and median HbA1c, HbA1c variability was independently associated with the occurrence of diabetic retinopathy. Time-covariate interactions used to model non-proportionality indicated an effect decreasing with duration of diabetes for both median HbA1c and HbA1c variability. Predictive accuracy increased significantly when adding HbA1c variability to the Cox regression model.
In patients with type 1 diabetes, HbA1c variability adds to the risk of diabetic retinopathy independently of average metabolic control.
Obese Zucker diabetic fatty (ZDF) rats are used as a type-2 diabetes model for microvascular complications. In order to study retinopathy in this model, changes in retinal vasculature were analyzed by quantitative morphometry and related to retinal expression of 46 selected genes that were analyzed by microfluidic card PCR technology. At 3 months of age, obese animals had developed stable hyperglycemia (20.7 ± 1.3 mmol/L plasma glucose vs. 6.5 ± 0.1 mmol/L in lean). Hyperinsulinemia initially presented in obese rats at 2 months (10.5 ± 0.7 μg/L plasma insulin vs. 0.2 ± 0.04 μg/L in lean) and decreased at 3 months (3.9 ± 0.6 vs. 0.5 ± 0.09 μg/ml in lean). At 8 months of age, animals had developed microvascular complications. An increased number of acellular capillaries in obese (24 ± 5/mm2) versus lean (15 ± 4/mm2) and a decreased number of retinal pericytes in obese (2,270 ± 250/mm2) versus lean animals (1,620 ± 243/mm2) could be observed. VEGFa, MIF, and HIF-1α were the most abundantly expressed and inflammatory genes such as TNFα and IL-6 are the least abundantly expressed genes. None of these genes were differentially regulated. Surprisingly, specific growth factors such as bFGF (FGF2) and placental growth factor, and adhesion molecules such as ICAM-1 were abundantly expressed and up-regulated in diabetic versus non-diabetic ZDF rats. In summary, we observed in type-2 diabetic ZDF rats retinopathy with retinal vasoregression along with a simultaneous up-regulation of specific growth factors such as bFGF and adhesion molecules, but only minor changes in key inflammatory genes.
Electronic supplementary material
The online version of this article (doi:10.1007/s00592-013-0550-2) contains supplementary material, which is available to authorized users.
Zucker diabetic fatty rats; Diabetic retinopathy; Preclinical model; Growth factors; Inflammation; Adhesion
Retinal degeneration in transgenic rats that express a mutant cilia gene polycystin-2 (CMV-PKD2(1/703)HA) is characterized by initial photoreceptor degeneration and glial activation, followed by vasoregression and neuronal degeneration (Feng et al., 2009, PLoS One 4: e7328). It is unknown whether glial activation contributes to neurovascular degeneration after photoreceptor degeneration. We characterized the reactivity of Müller glial cells in retinas of rats that express defective polycystin-2.
Age-matched Sprague-Dawley rats served as control. Retinal slices were immunostained for intermediate filaments, the potassium channel Kir4.1, and aquaporins 1 and 4. The potassium conductance of isolated Müller cells was recorded by whole-cell patch clamping. The osmotic swelling characteristics of Müller cells were determined by superfusion of retinal slices with a hypoosmotic solution.
Müller cells in retinas of transgenic rats displayed upregulation of GFAP and nestin which was not observed in control cells. Whereas aquaporin-1 labeling of photoreceptor cells disappeared along with the degeneration of the cells, aquaporin-1 emerged in glial cells in the inner retina of transgenic rats. Aquaporin-4 was upregulated around degenerating photoreceptor cells. There was an age-dependent redistribution of Kir4.1 in retinas of transgenic rats, with a more even distribution along glial membranes and a downregulation of perivascular Kir4.1. Müller cells of transgenic rats displayed a slight decrease in their Kir conductance as compared to control. Müller cells in retinal tissues from transgenic rats swelled immediately under hypoosmotic stress; this was not observed in control cells. Osmotic swelling was induced by oxidative-nitrosative stress, mitochondrial dysfunction, and inflammatory lipid mediators.
Cellular swelling suggests that the rapid water transport through Müller cells in response to osmotic stress is altered as compared to control. The dislocation of Kir4.1 will disturb the retinal potassium and water homeostasis, and osmotic generation of free radicals and inflammatory lipids may contribute to neurovascular injury.
Diabetic retinopathy (DRP) is a common complication caused by multiple biochemical abnormalities of the underlying metabolic disease. While the incidence of DRP appears to decline due to evidence-based changes in diabetes management, the predicted increase in patients affected in particular by type 2 diabetes may outweigh the positive trend. The diagnosis is based on the alterations of the vessels, usually indicating abnormalities of the blood–retinal barrier and increased vasoregression, but the neuroglial elements appear equally vulnerable to the diabetic condition. Control of blood glucose, blood pressure and timely identification of coincident nephropathy are important to prevent progression to vision-threatening stages. Guidelines give specific indications for laser photocoagulation, in particular when euglycemia is no longer effective in preventing progression to advanced stages. Intravitreal administration of antibodies directed against the single best characterized propagator of clinically significant macular edema, vascular endothelial growth factor (VEGF), has become popular despite uncertainty about the patient subgroups which benefit best and the optimum administration schedule. Multifactorial intervention beyond glycemic control includes antihypertensive, lipid-lowering and antiaggregatory and is effective in type 2 diabetic patients with high-risk profiles, in particular coincident nephropathy.
biomarkers; cardiovascular risk; diabetic retinopathy; treatment
Dipeptidyl peptidase 4 (DPP4) and angiotensin-converting enzyme (ACE) are important target enzymes in glycemic control and renovascular protection. Here, we studied the effect of NWT-03, an egg protein hydrolysate with DPP4- and ACE-inhibitory activity, on renovascular damage in Zucker diabetic fatty (ZDF) rats. Comparisons were made to rats treated with vildagliptin (VIL), included as a positive control for the effect of DPP4 inhibition.
ZDF rats received NWT-03 (1 g/kg/day) or VIL (3 mg/kg/day) from 10 to 25 weeks of age. Metabolic and renal functions were assessed; the kidney was removed for histological analysis of glomerulosclerosis and expression of pro-inflammatory/fibrotic markers (RT-PCR and Western blotting); and the aorta was removed for studies of endothelium-dependent relaxation (EDR).
Hyperinsulinemic ZDF rats typically developed signs of type-2 diabetes and renovascular damage, as evidenced by albuminuria, glomerulosclerosis, and impaired EDR. Neither NWT-03 nor VIL improved metabolic parameters; for VIL, this was despite a 5-fold increase in glucagon-like peptide (GLP)-1 levels. NWT-03 and VIL both reduced renal interleukin (Il)-1β/Il-13 mRNA expression and glomerulosclerosis. However, only NWT-03 additionally decreased renal tumor necrosis factor (TNF)-α mRNA and P22phox protein expression, reduced albuminuria, and restored aortic EDR. Indomethacin added to the organ bath instantly improved aortic EDR, indicating a role for cyclooxygenase (COX)-derived contractile prostanoids in opposing relaxation in ZDF rats. This indomethacin effect was reduced by NWT-03, but not by VIL, and coincided with decreased renal COX-1/2 protein expression.
Conclusion and Interpretation
Long-term supplementation with the egg protein hydrolysate NWT-03 attenuated renovascular damage in this preclinical rat model of type 2 diabetes. A comparison to the DPP4-inhibitor VIL suggests that the effects of NWT-03 were related to both ACE- and DPP4-inhibitory properties. The development of protein hydrolysates with a multiple-targeting strategy may be of benefit to functional food formulations.
In established tumors, angiogenic endothelial cells (ECs) coexist next to “quiescent” EC in matured vessels. We hypothesized that angio-gene expression of B16.F10 melanoma would differ depending on the growth stage. Unraveling the spatiotemporal nature thereof is essential for drug regimen design aimed to affect multiple neovascularization stages. We determined the angiogenic phenotype—represented by 52 angio-genes—and vascular morphology of small, intermediate, and large s.c. growing mouse B16.F10 tumors and demonstrated that expression of these genes did not differ between the different growth stages. Yet vascular morphology changed dramatically from small vessels without lumen in small to larger vessels with increased lumen size in intermediate/large tumors. Separate analysis of these vascular morphologies revealed a significant difference in αSMA expression in relation to vessel morphology, while no relation with VEGF, HIF-1α, nor Dll4 expression levels was observed. We conclude that the tumor vasculature remains actively engaged in angiogenesis during B16.F10 melanoma outgrowth and that the major change in tumor vascular morphology does not follow molecular concepts generated in other angiogenesis models.
The ubiquitin-proteasome system is the main degradation machinery for intracellularly altered proteins. Hyperglycemia has been shown to increase intracellular levels of the reactive dicarbonyl methylglyoxal (MGO) in cells damaged by diabetes, resulting in modification of proteins and alterations of their function. In this study, the influence of MGO-derived advanced glycation end product (AGE) formation on the activity of the proteasome was investigated in vitro and in vivo.
RESEARCH DESIGN AND METHODS
MGO-derived AGE modification of proteasome subunits was analyzed by mass spectrometry, immunoprecipitation, and Western blots. Proteasome activity was analyzed using proteasome-specific fluorogenic substrates. Experimental models included bovine retinal endothelial cells, diabetic Ins2Akita mice, glyoxalase 1 (GLO1) knockdown mice, and streptozotocin (STZ)-injected diabetic mice.
In vitro incubation with MGO caused adduct formation on several 20S proteasomal subunit proteins. In cultured endothelial cells, the expression level of the catalytic 20S proteasome subunit was not altered but proteasomal chymotrypsin-like activity was significantly reduced. In contrast, levels of regulatory 19S proteasomal proteins were decreased. In diabetic Ins2Akita, STZ diabetic, and nondiabetic and diabetic G101 knockdown mice, chymotrypsin-like activity was also reduced and MGO modification of the 20S-β2 subunit was increased.
Hyperglycemia-induced formation of MGO covalently modifies the 20S proteasome, decreasing its activity in the diabetic kidney and reducing the polyubiquitin receptor 19S-S5a. The results indicate a new link between hyperglycemia and impairment of cell functions.
Diabetes mellitus, as well as subsequent ocular complications such as cystoid macular edema (CME), are of fundametal socio-economic relevance. Therefore, we evaluated the influence of internal limiting membrane (ILM) removal on longterm morphological and functional outcome in patients with diabetes mellitus (DM) type 2 and chronic CME without evident vitreomacular traction.
Forty eyes with attached posterior hyaloid were included in this prospective trial and randomized intraoperatively. Prior focal (n = 31) or panretinal (n = 25) laser coagulation was permitted. Group I (n = 19 patients) underwent surgical induction of posterior vitreous detachment (PVD), group II (n = 20 patients) PVD and removal of the ILM. Eleven patients with detached posterior hyaloid (group III) were not randomized, and ILM removal was performed. One eye had to be excluded from further analysis. Examinations included ETDRS best-corrected visual acuity (BCVA), fluorescein angiography (FLA) and OCT at baseline, 3 and 6 months postoperatively. Main outcome measure was BCVA at 6 months, secondary was foveal thickness.
Mean BCVA over 6 months remained unchanged in 85% of patients of group II, and decreased in 53% of patients of group I. Results were not statistically significant different [group I: mean decrease log MAR 95% CI (0.06; 0.32), group II: (−0.02; 0.11)]. OCT revealed a significantly greater reduction of foveal thickness following PVD with ILM removal [group I: mean change: 95% CI (−208.95 μm; −78.05 μm), group II: (−80.90 μm: +59.17 μm)].
Vitrectomy, PVD with or without ILM removal does not improve vision in patients with DM type 2 and cystoid diabetic macular edema without evident vitreoretinal traction. ILM delamination shows improved morphological results, and appears to be beneficial in eyes with preexisting PVD.
Vitrectomy; Posterior vitreous detachment; Internal limiting membrane; Optical coherence tomography; Cystoid diabetic macular edema
Many disabling human retinal disorders involve the central retina, particularly the macula. However, the commonly used rodent models in research, mouse and rat, do not possess a macula. The purpose of this study was to identify small laboratory rodents with a significant central region as potential new models for macular research.
Gerbillus perpallidus, Meriones unguiculatus and Phodopus campbelli, laboratory rodents less commonly used in retinal research, were subjected to confocal scanning laser ophthalmoscopy (cSLO), fluorescein and indocyanine green angiography, and spectral-domain optical coherence tomography (SD-OCT) using standard equipment (Heidelberg Engineering HRA1 and Spectralis™) adapted to small rodent eyes. The existence of a visual streak-like pattern was assessed on the basis of vascular topography, retinal thickness, and the topography of retinal ganglion cells and cone photoreceptors. All three species examined showed evidence of a significant horizontal streak-like specialization. cSLO angiography and retinal wholemounts revealed that superficial retinal blood vessels typically ramify and narrow into a sparse capillary net at the border of the respective area located dorsal to the optic nerve. Similar to the macular region, there was an absence of larger blood vessels in the streak region. Furthermore, the thickness of the photoreceptor layer and the population density of neurons in the ganglion cell layer were markedly increased in the visual streak region.
The retinal specializations of Gerbillus perpallidus, Meriones unguiculatus and Phodopus campbelli resemble features of the primate macula. Hence, the rodents reported here may serve to study aspects of macular development and diseases like age-related macular degeneration and diabetic macular edema, and the preclinical assessment of therapeutic strategies.
ADAM15 (named for a disintegrin and metalloprotease 15, metargidin) is a membrane-anchored glycoprotein that has been implicated in cell-cell or cell-matrix interactions and in the proteolysis of molecules on the cell surface or extracellular matrix. To characterize the potential roles of ADAM15 during development and in adult mice, we analyzed its expression pattern by mRNA in situ hybridization and generated mice carrying a targeted deletion of ADAM15 (adam15−/− mice). A high level of expression of ADAM15 was found in vascular cells, the endocardium, hypertrophic cells in developing bone, and specific areas of the hippocampus and cerebellum. However, despite the pronounced expression of ADAM15 in these tissues, no major developmental defects or pathological phenotypes were evident in adam15−/− mice. The elevated levels of ADAM15 in endothelial cells prompted an evaluation of its role in neovascularization. In a mouse model for retinopathy of prematurity, adam15−/− mice had a major reduction in neovascularization compared to wild-type controls. Furthermore, the size of tumors resulting from implanted B16F0 mouse melanoma cells was significantly smaller in adam15−/− mice than in wild-type controls. Since ADAM15 does not appear to be required for developmental angiogenesis or for adult homeostasis, it may represent a novel target for the design of inhibitors of pathological neovascularization.
The murine VEGF gene is alternatively transcribed to yield the VEGF120, VEGF164, and VEGF188 isoforms, which differ in their potential to bind to heparan sulfate and neuropilin-1 and to stimulate endothelial growth. Here, their role in retinal vascular development was studied in mice selectively expressing single isoforms. VEGF164/164 mice were normal, healthy, and had normal retinal angiogenesis. In contrast, VEGF120/120 mice exhibited severe defects in vascular outgrowth and patterning, whereas VEGF188/188 mice displayed normal venular outgrowth but impaired arterial development. It is noteworthy that neuropilin-1, a receptor for VEGF164, was predominantly expressed in retinal arterioles. These findings reveal distinct roles of the various VEGF isoforms in vascular patterning and arterial development in the retina.