Vascular progenitors from cord blood-derived induced pluripotent stem cells possess augmented capacity for regenerating ischemic retinal vasculature.

BACKGROUND: The generation of vascular progenitors (VPs) from human induced pluripotent stem cells (hiPSCs) has great potential for treating vascular disorders such as ischemic retinopathies. However, long-term in vivo engraftment of hiPSC-derived VPs into the retina has not yet been reported. This goal may be limited by the low differentiation yield, greater senescence, and poor proliferation of hiPSC-derived vascular cells. To evaluate the potential of hiPSCs for treating ischemic retinopathies, we generated VPs from a repertoire of viral-integrated and nonintegrated fibroblast and cord blood (CB)-derived hiPSC lines and tested their capacity for homing and engrafting into murine retina in an ischemia-reperfusion model. METHODS AND RESULTS: VPs from human embryonic stem cells and hiPSCs were generated with an optimized vascular differentiation system. Fluorescence-activated cell sorting purification of human embryoid body cells differentially expressing endothelial/pericytic markers identified a CD31(+)CD146(+) VP population with high vascular potency. Episomal CB-induced pluripotent stem cells (iPSCs) generated these VPs with higher efficiencies than fibroblast-iPSC. Moreover, in contrast to fibroblast-iPSC-VPs, CB-iPSC-VPs maintained expression signatures more comparable to human embryonic stem cell VPs, expressed higher levels of immature vascular markers, demonstrated less culture senescence and sensitivity to DNA damage, and possessed fewer transmitted reprogramming errors. Luciferase transgene-marked VPs from human embryonic stem cells, CB-iPSCs, and fibroblast-iPSCs were injected systemically or directly into the vitreous of retinal ischemia-reperfusion-injured adult nonobese diabetic-severe combined immunodeficient mice. Only human embryonic stem cell- and CB-iPSC-derived VPs reliably homed and engrafted into injured retinal capillaries, with incorporation into damaged vessels for up to 45 days. CONCLUSIONS: VPs generated from CB-iPSCs possessed augmented capacity to home, integrate into, and repair damaged retinal vasculature.

VEGF 165 b in the developing vasculatures of the fetal human eye.

VEGF(165) b is an anti-angiogenic form of VEGF(165) produced by alternative splicing. The localization of pro-angiogenic VEGF(165) and anti-angiogenic VEGF(165) b was investigated during development of the vasculatures in fetal human eyes from 7 to 21 weeks gestation (WG). The fetal vasculature of vitreous, which includes tunica vasculosa lentis (TVL), had moderate VEGF(165) immunoreactivity at 7WG and very little VEGF(165) b. Both forms were elevated at 12WG. VEGF(165) then decreased around 17WG when the TVL regresses but VEGF(165) b remained elevated. In choroid, VEGF(165) was present in forming choriocapillaris (CC) and retinal pigment epithelium (RPE) at 7WG while VEGF165b was present in CC and mesenchymal precursors within the choroidal stroma. By 21WG, both forms were elevated in RPE and choroidal blood vessels but VEGF(165) b was apical and VEGF(165) basal in RPE. Diffuse VEGF(165) immunoreactivity was prominent in 12WG innermost retina where blood vessels will form while VEGF(165) b was present in most CXCR4(+) progenitors in the inner neuroblastic layer and migrating angioblasts in the putative nerve fiber layer. By 21WG, VEGF(165) was present in nerve fibers and VEGF(165) b in the inner Muller cell process. The localization of VEGF(165) b was distinctly different from VEGF(165) both spatially and temporally and it was often associated with nucleus in progenitors.

A rat model for choroidal neovascularization using subretinal lipid hydroperoxide injection.

The purpose of this study was to develop and characterize a rat model of choroidal neovascularization (CNV) as occurs in age-related macular degeneration. The lipid hydroperoxide 13(S)-hydroperoxy-9Z,11E-octadecadienoic acid (HpODE) is found in submacular Bruch's membrane in aged humans and has been reported to generate neovascularization in a rabbit model. Three weeks after a single subretinal injection of 30 microg of HpODE, eyes of Sprague-Dawley rats were harvested. Follow-up fluorescein angiography was done on other animals until 5 weeks postinjection. Histological studies, immunohistochemical staining, and flatmount choroids for CNV measurements were performed. In addition, we used murine neuronal, bovine endothelial, and human ARPE19 cells for testing the in vitro effects of HpODE. CNV developed in 85.7% of HpODE-injected eyes. The neovascular areas were significantly greater in HpODE-injected eyes compared with those in control eyes (P = 0.023). The CNV had maximum dye leakage at 3 weeks, which subsided by the 5th week. Histologically, CNV extended from the choriocapillaris into the subretinal space. ED1-positive macrophages were recruited to the site. In vitro assays demonstrated that only 30 ng/ml HpODE induced cell proliferation and migration of endothelial cells. HpODE-induced CNV was highly reproducible, and its natural course seems to be ideal for evaluating therapeutic modalities. Because HpODE has been isolated from aged humans, the HpODE-induced rat model seems to be a relevant experimental model for CNV in age-related macular degeneration.

Low nitric oxide synthases (NOSs) in eyes with age-related macular degeneration (AMD).

Nitric oxide (NO) production by vascular endothelium is important in regulation of blood flow. Reduced production of NO can adversely affect blood flow and other vascular functions. We investigated the expression of three nitric oxide synthase (NOS) isoforms in retina and choroid of aged human eyes and eyes with AMD. Alkaline phosphatase immunohistochemistry was performed using antibodies against inducible (iNOS), neuronal (nNOS), and endothelial (eNOS) NOSs on cryopreserved sections from aged control donor eyes (n = 13) and eyes with AMD (n = 22). CD34 antibody was used as an endothelial cell (EC) marker. Three independent masked observers scored the intensity of the immunohistochemical reaction product. Mean scores from the aged control and AMD eyes were statistically compared. In aged control retinas, nNOS was in ganglion cells (RGCs) and neurons of both nuclear layers. In choroid, perivascular nerve fibers and retinal pigment epithelial (RPE) cells were nNOS+. eNOS and iNOS were confined to the retinal and choroidal vascular ECs. Some cells presumably melanocytes or dendritic cells in choroid were also eNOS+. In AMD eyes, nNOS was significantly lower in RGCs, neurons, retinal vessels and RPE (p < or = 0.05) compared to the aged control eyes. iNOS and eNOS showed no significant differences between aged control and AMD eyes except that there was significantly less eNOS in choroidal arteries (p = 0.006) and choroidal cells (p = 0.03) of AMD eyes. Although NO was not measured directly, these findings suggest that there is less NO produced in AMD eyes. The decrease in retinal nNOS in AMD eyes is probably related to neuronal degeneration. The decrease in nNOS and eNOS in AMD choroid could be associated with vasoconstriction and hemodynamic changes.

Vascular precursors in developing human retina.

PURPOSE: Prior investigation has demonstrated that angioblasts are present in the inner retinas of human embryos and fetuses and that they differentiate and organize to form the primordial retinal vasculature. The purpose of this study was to characterize these angioblasts further and examine ligands that might control their migration and differentiation. METHODS: Immunohistochemistry was used to localize stroma-derived factor-1 (SDF-1), its receptor CXCR4, stem cell factor (SCF), and its receptor c-Kit on sections obtained from human eyes at from 6 to 23 weeks' gestation (WG). Coexpression of CD39 (marker for retinal angioblasts and endothelial cells) and CXCR4 or c-Kit was investigated by confocal microscopy. RESULTS: SDF-1 was prominent in inner retina with the greatest reaction product near the internal limiting membrane (ILM). SCF immunoreactivity was also confined to the inner retina and increased significantly between 7 and 12 WG. The level of both ligands declined by 22 WG. A layer of CXCR4(+) and c-Kit(+) precursors, some of which coexpressed CD39, existed in the inner retina from 7 to 12 WG. With migration, c-Kit was downregulated, whereas CD39(+) cells continued to express CXCR4 as they formed cords. With canalization, CXCR4 expression was downregulated. CONCLUSIONS: Embryonic human retina has a pool of precursors (CXCR4(+) and c-Kit(+)) that enlarged centrifugally during fetal development. From this pool emerges angioblasts, which migrate anteriorly into the nerve fiber layer where SDF-1 and SCF levels are highest. c-Kit expression declines with apparent migration, and CXCR4 expression declines with canalization of new vessels. Both SCF and SDF-1 are associated with the differentiation of retinal precursors into angioblasts and their migration to sites of vessel assembly.

Nanoparticle-delivered biosensor for reactive oxygen species in diabetes.

The cell's own antioxidant response element (ARE) can be used to evaluate the complications of diabetes mellitus. The hypothesis that a synthetic ARE could be used as a genetic switch, or biosensor, to turn on and off therapeutic genes is tested herein. Mitochondrial oxidative stress (MOS) has been hypothesized as one of the earliest insults in diabetes. Fluorescent probes used to monitor MOS revealed that the addition of glucose at physiological levels to cultures of endothelial cells was able to induce MOS above normal levels and in a dose-dependant manner. Additional data showed that increased glucose levels activated the ARE-GFP in a dose-dependant manner. These data support the hypothesis that the induction of MOS is more sensitive to hyperglycemia than the induction of the ARE. Delivery of an ARE-GFP construct with nanoparticles to the eye was successful using sub-retinal injection. This ARE-GFP/nanoparticle construct was functional and reported the activation of the ARE in diabetic rat retinal pigment epithelium (RPE). These data support the use of nanoparticle-delivered biosensors for monitoring the oxidative status of tissues in vivo.

Ocular nanoparticle toxicity and transfection of the retina and retinal pigment epithelium.

Chitosan, PCEP (poly{[(cholesteryl oxocarbonylamido ethyl) methyl bis(ethylene) ammonium iodide] ethyl phosphate}), and magnetic nanoparticles (MNPs) were evaluated for the safe delivery of genes in the eye. Rabbits were injected with nanoparticles either intravitreally (IV) or subretinally (SR) and sacrificed 7 days later. Eyes were grossly evaluated for retinal pigment epithelium abnormalities, retinal degeneration, and inflammation. All eyes were cryopreserved and sectioned for analysis of toxicity and expression of either enhanced green or red fluorescent proteins. All of the nanoparticles were able to transfect cells in vitro and in vivo. IV chitosan showed inflammation in 12/13 eyes, whereas IV PCEP and IV MNPs were not inflammatory and did not induce retinal pathology. SR PCEP was nontoxic in the majority of cases but yielded poor transfection, whereas SR MNPs were nontoxic and yielded good transfection. Therefore, we conclude that the best nanoparticle evaluated in vivo was the least toxic nanoparticle tested, the MNP.

Nanoparticle tethered antioxidant response element as a biosensor for oxygen induced toxicity in retinal endothelial cells.

PURPOSE: A novel system, based on biosensor DNA tethered to a nanoparticle, was developed for the treatment of retinopathy of prematurity. METHODS: The construction of a five-layered nanoparticle was visualized with gel electrophoresis. Transcriptionally active PCR products (TAP) containing the biosensor sequence, were bioconjugated to the surface of magnetic nanoparticles yielding biosensor tethered magnetic nanoparticles (MNP). The biosensor was based on an enhanced green fluorescent protein (EGFP) reporter gene driven by an enhanced antioxidant response element (ARE). Image analysis and flow cytometry were used to characterize MNP delivery and biosensor activity. RESULTS: The MNP penetrated dividing and migrating cells more often than quiescent endothelial cells in a wound-healing in vitro assay. Prussian blue staining demonstrated that more cells have nanoparticle cores than are transfected. When compared to naked TAP alone, MNP transfected more cells in a dose dependent manner. Both the biosensor alone and MNP induce gene expression in the presence of hyperoxia, greater than 1.5 fold over normoxic controls. These data also show that the MNP had a signal to noise ratio of 0.5 greater than the plasmid form of the biosensor as demonstrated by flow cytometry. CONCLUSIONS: This approach has the potential to allow the endothelial cells of the retinal vasculature to prevent or treat themselves after hyperoxic insult, rather than systemic treatment to protect or treat only the retina.

Localization of collagen XVIII and the endostatin portion of collagen XVIII in aged human control eyes and eyes with age-related macular degeneration.

PURPOSE: Endostatin, a C-terminal fragment of collagen XVIII (coll XVIII) formed by proteolysis, specifically inhibits endothelial cell migration and proliferation in vitro and potently inhibits angiogenesis and tumor growth in vivo. The purpose of this study was to examine the immunolocalization of endostatin and coll XVIII in the retina and choroid of human donor tissue sections from aged control donor eyes and to determine whether the localization or relative levels are changed in age-related macular degeneration (AMD). METHODS: Ocular tissues were obtained from six aged control donors (age range, 75-86 years; mean age, 80.5 years) without evidence or history of chorioretinal disease and from nine donors with AMD (age range, 74-105 years; mean age, 88.6 years). Tissues were cryopreserved, and streptavidin alkaline phosphatase immunohistochemistry was performed with goat anti-human and mouse anti-human endostatin antibodies and rabbit anti-mouse coll XVIII. Blood vessels were identified with mouse anti-human CD-34 antibody in adjacent sections. Pigment in RPE and choroidal melanocytes was bleached. Three independent observers scored the immunohistochemical reaction product. RESULTS: In aged control eyes, coll XVIII and endostatin (the endostatin portion of coll XVIII) immunoreactivity was observed in large retinal blood vessels and in capillaries in some individuals, but the internal limiting membrane (ILM) had the most intense retinal immunostaining. There was no significant difference in immunoreactivity to both antibodies in retinal blood vessels in aged control eyes. In the choroid, endostatin and coll XVIII were localized to blood vessels, Bruch's membrane, and RPE basal lamina. AMD retina and choroid had a similar pattern and intensity of coll XVIII immunostaining, as observed in control eyes but reaction product was more diffuse in the choroid. Endostatin immunoreactivity was significantly higher in ILM (P = 0.037) in AMD retina and significantly lower in the choriocapillaris, Bruch's membrane, and RPE basal lamina of AMD choroids (P < 0.05) and completely negative in some areas of AMD choroids. CONCLUSIONS: These data suggest that reduced levels of the endostatin portion of coll XVIII in Bruch's membrane, RPE basal lamina, intercapillary septa, and choriocapillaris in eyes with AMD may be permissive for choroidal neovascularization.

Caspase activation and amyloid precursor protein cleavage in rat ocular hypertension.

PURPOSE: Retinal ganglion cell (RGC) death in glaucoma involves apoptosis. Activation of caspases and abnormal processing of amyloid precursor protein (APP) are important events in other chronic neurodegenerations, such as Alzheimer's disease (AD). The retinal expression and activation of caspases and the patterns of caspase-3-mediated APP processing in ocular hypertensive models of rat glaucoma were investigated. METHODS: RGC death was produced in one eye by chronic exposure to increased intraocular pressure (IOP) or by optic nerve transection. Elevated IOP was produced by obstruction of aqueous humor outflow with laser coagulation or limbal hypertonic saline injection. Caspase activity and APP processing in the retina were examined by RNase protection assay (RPA), immunocytochemistry, immunoblot assay, and colorimetric assay. RESULTS: RPA revealed elevations of caspase-3 mRNA, as well as other apoptosis-related mRNAs. Immunocytochemistry showed caspase-3 activation in RGCs damaged by ocular hypertension. The generation of the caspase-3-mediated APP cleavage product (DeltaC-APP) was also increased in ocular hypertensive RGCs. Western immunoblot assay and colorimetry revealed significantly more activated caspase-3 in ocular hypertensive retinas than in control retinas. The activated form of caspase-8, an initiator caspase, and amyloid-beta, a product of APP proteolysis and a component of senile plaques in AD, were detected in RGCs by immunohistochemistry significantly more often in ocular hypertensive than in control retinas. The amounts of full-length APP were reduced and amyloid-beta-containing fragments were increased in ocular hypertensive retinas by Western immunoblot assay. CONCLUSIONS: Rat RGCs subjected to chronic ocular hypertension demonstrate caspase activation and abnormal processing of APP, which may contribute to the pathophysiology of glaucoma.

Mechanisms for sickle red blood cell retention in choroid.

PURPOSE: Although sickle (SS) red cell-mediated vaso-occlusion in retina and resultant retinopathy is well documented, the effects of SS red cells on choroidal vasculature are poorly understood. The intent of this study was to determine, using a rat model, the conditions under which retention of sickle erythrocytes in choroid occur and if that retention can be inhibited. METHODS: Sickle red cells were density separated into high density (SS4) or normal density, reticulocyte-enriched fractions (SS2). Red cells were labeled with FITC and administered IV to anesthetized Sprague Dawley rats. Rats were made either hypoxic or were given TNF-alpha intraperitoneally 5 hours before intravenous administration of red cells. Five minutes after administration of red cells, rats were exsanguinated, the retinas removed, and choroids prepared as flatmounts. The number of red cells retained in five high power fields of choroid was then determined. In other experiments, SS red cells were preincubated with the cyclic peptide TBC772 [inhibits binding of alpha4beta1 (VLA-4) and alpha4beta7 to their ligands], a control peptide TBC1194, or a VLA-4 neutralizing antibody before administration to the rat or antibodies against VLA-4 ligands were delivered IV before administration of SS red cells. RESULTS: Hypoxic conditions before administration of SS red cells significantly stimulated retention of SS4 cells (P = 0.0003), but did not significantly increase retention of SS2 cells. Administration of TNF-alpha significantly increased retention of all types of SS red cells (P < 0.001). Preincubation of cells with anti-VLA-4 or TBC 772 inhibited retention of SS red cells in choriocapillaris of TNF-alpha-treated rats (P < 0.0001). Complete inhibition of cytokine-stimulated retention was also accomplished by IV administration of monoclonal antibodies against fibronectin or its CS-1 domain, a ligand for VLA-4. CONCLUSIONS: The mechanisms for retention of SS red cells in retina and choroid appear identical: hypoxia-mediated retention of dense red cells and adherence of red cells in reticulocyte-rich fractions after cytokine stimulation. TNF-alpha-stimulated retention of SS red cells in choroid appears to be mediated by VLA-4, presumably on the surface of some reticulocytes. This increased retention was inhibited by a VLA-4 antagonist (TBC772), a VLA-4 neutralizing antibody or by blocking one of VLA-4's ligands, the CS-1 portion of fibronectin.

From blood islands to blood vessels: morphologic observations and expression of key molecules during hyaloid vascular system development.

PURPOSE: The mode of development of the human hyaloid vascular system (HVS) remains unclear. Early studies suggested that these blood vessels formed by vasculogenesis, while the current concept seems to favor angiogenesis as the mode of development. We examined embryonic and fetal human HVS using a variety of techniques to gain new insights into formation of this vasculature. METHODS: Embryonic and fetal human eyes from 5.5 to 12 weeks gestation (WG) were prepared for immunohistochemical analysis or for light and electron microscopy. Immunolabeling of sections with a panel of antibodies directed at growth factors, transcription factors, and hematopoietic stem cell markers was employed. RESULTS: Light microscopic examination revealed free blood islands (BI) in the embryonic vitreous cavity (5.5-7 WG). Giemsa stain revealed that BI were aggregates of mesenchymal cells and primitive nucleated erythroblasts. Free cells were also observed. Immunolabeling demonstrated that BI were composed of mesenchymal cells that expressed hemangioblast markers (CD31, CD34, C-kit, CXCR4, Runx1, and VEGFR2), erythroblasts that expressed embryonic hemoglobin (Hb-ε), and cells that expressed both. Few cells were proliferating as determined by lack of Ki67 antigen. As development progressed (12 WG), blood vessels became more mature structurally with pericyte investment and basement membrane formation. Concomitantly, Hb-ε and CXCR4 expression was down-regulated and von Willebrand factor expression was increased with the formation of Weibel-Palade bodies. CONCLUSIONS: Our results support the view that the human HVS, like the choriocapillaris, develops by hemo-vasculogenesis, the process by which vasculogenesis, erythropoiesis, and hematopoiesis occur simultaneously from common precursors, hemangioblasts.

C-reactive protein and complement factor H in aged human eyes and eyes with age-related macular degeneration.

BACKGROUND: There is increasing evidence that inflammation and immune-mediated processes (complement activation) play an important role in age-related macular degeneration (AMD) pathogenesis. A genetic variation in the gene encoding complement factor H (CFH) and plasma levels of C-reactive protein (CRP), a systemic marker of subclinical inflammation, have consistently been shown to be associated with an increased risk for AMD. In the present study, we examined the immunolocalisation of CRP and CFH in aged control human donor eyes (n=10; mean age 79 years) and eyes with AMD (n=18; mean age 83 years). METHODS: Alkaline phosphatase immunohistochemistry was performed using polyclonal antibodies against CRP and CFH on cryopreserved tissue sections from disc/macular blocks. Three independent masked observers scored the reaction product (0-8). RESULTS: In aged control eyes, the retinal pigment epithelium/Bruch's membrane/choriocapillaris (RPE/BrM/CC) complex including intercapillary septa (ICS) had the most prominent immunostaining for CRP and CFH. CRP was significantly higher than controls in BrM/CC/ICS and choroidal stroma in early and wet AMD eyes (p<0.05). In contrast, CFH was significantly lower in BrM/CC/ICS complex of AMD choroids than in controls (p<0.05). Interestingly, CRP and CFH were significantly reduced in BrM/CC/ICS complex in atrophic area of macula in geographical atrophy (p<0.05). Drusen and basal laminar deposits were intensely positive for CRP and CFH. CONCLUSION: These immunohistochemical findings show that changes in distribution and relative levels of CRP and CFH were evident in early and late AMD eyes. This suggests that high levels of CRP and insufficient CFH at the retina/choroid interface may lead to uncontrolled complement activation with associated cell and tissue damage. This study supports the hypothesis that inflammation and immune-mediated mechanisms are involved in the pathogenesis of AMD.

Maturation of the fetal human choriocapillaris.

PURPOSE: The purpose of this study was to examine the structural and functional maturation of the choriocapillaris (CC) and to determine when fenestrations form, the capillaries are invested with pericytes, and the endothelial cells (ECs) became functional. METHODS: Immunohistochemistry was performed on cryopreserved sections of embryonic/fetal human eyes from 7 to 22 weeks' gestation (WG), using antibodies against PAL-E, PV-1 (fenestrations), carbonic anhydrase IV (CA IV), eNOS, and alpha-smooth muscle actin (alphaSMA) and NG2 (two pericyte markers) and the EC marker (CD31). Alkaline phosphatase (APase) enzymatic activity was demonstrated by enzyme histochemistry. Transmission electron microscopy (TEM) was performed on eyes at 11, 14, 16, and 22 WG. Adult human eyes were used as the positive control. RESULTS: All EC markers were present in the CC by 7 WG. PAL-E, CA IV, and eNOS immunoreactivities and APase activity were present in the CC by 7 to 9 WG. TEM analysis demonstrated how structurally immature this vasculature was, even at 11 WG: no basement membrane, absence of pericytes, and poorly formed lumens that were filled with filopodia. The few fenestrations that were observed were often present within the luminal space in the filopodia. Contiguous fenestrations and significant PV-1 were not observed until 21 to 22 WG. alphaSMA was prominent at 22 WG, and the maturation of pericytes was confirmed by TEM. CONCLUSIONS: It appears that ECs and their precursors express enzymes present in adult CC well before they are structurally mature. Although ECs make tight junctions early in development, contiguous fenestrations and mature pericytes occur much later in development.

Relationship between RPE and choriocapillaris in age-related macular degeneration.

PURPOSE: The purpose of this study was to examine the relationships between choriocapillaris (CC) and retinal pigment epithelial changes in age-related macular degeneration (AMD). Morphologic changes in the retinal pigment epithelium (RPE)/choriocapillaris complex were quantified in dry and wet forms of AMD, and the results were compared with those in aged control eyes without maculopathy. METHODS: Postmortem choroids from three aged control subjects, five subjects with geographic atrophy (GA), and three subjects with wet AMD were analyzed using a semiquantitative computer-assisted morphometric technique developed to measure the percentages of retinal pigment epithelial and CC areas in choroidal wholemounts incubated for alkaline phosphatase activity. The tissues were subsequently embedded in methacrylate and were sectioned so that structural changes could be examined. RESULTS: There was a linear relationship between the loss of RPE and CC in GA. A 50% reduction in vascular area was found in regions of complete retinal pigment epithelial atrophy. Extreme constriction of remaining viable capillaries was found in areas devoid of RPE. Adjacent to active choroidal neovascularization (CNV) in wet AMD, CC dropout was evident in the absence of retinal pigment epithelial atrophy, resulting in a 50% decrease in vascular area. Lumenal diameters of the remaining capillaries in wet AMD eyes were similar to those in control eyes. CONCLUSIONS: The primary insult in GA appears to be at the level of the RPE, and there is an intimate relationship between retinal pigment epithelial atrophy and secondary CC degeneration. CC degeneration occurs in the presence of viable RPE in wet AMD. The RPE in regions of vascular dropout are presumably hypoxic, which may result in an increase in VEGF production by the RPE and stimulation of CNV.

Reduction of endogenous angiogenesis inhibitors in Bruch's membrane of the submacular region in eyes with age-related macular degeneration.

OBJECTIVES: To determine the relative levels of 3 potent inhibitors of angiogenesis (endostatin, pigment epithelium-derived factor, and thrombospondin 1) in the retinal pigment epithelium-Bruch's membrane-choriocapillaris complex in the submacular region in aged control eyes and eyes with age-related macular degeneration (AMD). METHODS: Immunohistochemical analysis with antibodies against endostatin, pigment epithelium-derived factor, and thrombospondin 1 was performed on the macular region of aged control donor eyes (n = 8; mean age, 79.8 years) and eyes with AMD (n = 12; mean age, 83.9 years). Three independent masked observers scored the reaction product (scored from 0-7). Mean scores from the control eyes and the eyes with AMD were analyzed using 1-way analysis of variance and unpaired t test. RESULTS: In control eyes, strong immunoreactivity of all 3 inhibitors was observed in the retinal pigment epithelium-Bruch's membrane-choriocapillaris complex. Immunoreactivity for endostatin, pigment epithelium-derived factor, and thrombospondin 1 in Bruch's membrane was significantly lower in eyes with AMD compared with aged control eyes (analysis of variance, P = .003, P = .009, and P < .001, respectively). In the choriocapillaris, a significant reduction was observed in endostatin (analysis of variance, P = .02) and thrombospondin 1 (analysis of variance, P = .005) in eyes with AMD. CONCLUSIONS: These findings suggest that endogenous angiogenesis inhibitors in the retinal pigment epithelium-Bruch's membrane-choriocapillaris complex may provide a biochemical barrier for choroidal neovascular invasion. CLINICAL RELEVANCE: Decreased levels of angiogenic inhibitors at the retinal pigment epithelium-Bruch's membrane-choriocapillaris complex in eyes with AMD make Bruch's membrane vulnerable to choroidal neovascularization.

Developmental basis of nanophthalmos: MFRP Is required for both prenatal ocular growth and postnatal emmetropization.

BACKGROUND: Nanophthalmos is a genetic disorder characterized by very small, hyperopic eyes that are without gross structural defects. Recessive nanophthalmos is caused by severe mutations in the MFRP gene, which encodes a Frizzled-related transmembrane protein that is selectively expressed in the retinal pigment epithelium (RPE) and ciliary body. RESULTS: For two MFRP -/- adults, we have obtained records of refraction that begin in early childhood. At the age of 6 months, one patient's eyes already had a refractive error of +12.25 D, and over the next 20 years this slowly increased to +17.50 D. Adults homozygous for null mutations in MFRP have eyes with axial lengths shorter than those of normal newborns. Furthermore, the unusually high curvature of their corneas is consistent with eyes that had been smaller than normal during late fetal development. MFRP protein was first detected at 14 weeks of gestation, when it was restricted to the posterior pole RPE. By 20 weeks gestation, MFRP expression had spread laterally, and was found throughout the RPE. MFRP protein was detected in both posterior and lateral RPE of the adult eye. CONCLUSIONS: Embryonic function of the MFRP gene appears necessary for the eye to reach its full size at birth. Its onset of expression in the RPE during mid-gestation suggests that MFRP does not participate in early formation of the optic cup, and is consistent with a role in later growth and development of the eye. Patients without MFRP gene function exhibit no correction of refractive error during childhood, which suggests that this gene is essential for emmetropization, a complex process by which vision regulates axial growth of the eye.

Hyperoxia inhibits several critical aspects of vascular development.

Normal human retinal vascular development uses angiogenesis and vasculogenesis, both of which are interrupted in the vaso-obliteration phase of retinopathy of prematurity (ROP). Canine oxygen-induced retinopathy (OIR) closely resembles human ROP. Canine retinal endothelial cells (ECs) and angioblasts were used to model OIR and characterize the effects of hyperoxia on angiogenesis and vasculogenesis. Cell cycle analysis showed that hyperoxia reduced the number of G1 phase cells and showed increased arrest in S phase for both cell types. Migration of ECs was significantly inhibited in hyperoxia (P < 0.01). Hyperoxia disrupted the cytoskeleton of angioblasts but not ECs after 2 days. Differentiation of angioblasts into ECs (determined by acetylated low-density lipoprotein uptake) was evaluated after basic fibroblast growth factor treatment. Differentiation of angioblasts into pericytes was determined by smooth muscle actin expression after treatment with platelet-derived growth factor. Differentiation into ECs was significantly inhibited by hyperoxia (P < 0.0001). The percentage of CXCR4(+) cells (a marker for retinal vascular precursors) increased in both treatment groups after hyperoxia. These data show novel mechanisms of hyperoxia-induced disruption of vascular development.

The embryonic human choriocapillaris develops by hemo-vasculogenesis.

The purpose of this study was to characterize normal human choroidal vascular development from 6-23 weeks gestation (WG). Markers of endothelial cells (EC) (CD34, CD31, vWf), angioblasts and EC (CD39), leukocytes (CD45), erythroblasts (epsilon chain of hemoglobin, Hb-e), proliferating cells (Ki67), and VEGFR-2 were employed. At 6-7 WG, many erythroblasts were observed within islands of precursor cells in the choriocapillaris layer and others were independent from the islands. Many erythroblasts (Hb-epsilon(+)) were also positive for EC markers and/or VEGFR-2. By 8-12 WG, most of the Hb-epsilon cells had disappeared and vascular lumens became apparent. At 14-23 WG, some EC were proliferating on the scleral side of choriocapillaris in association with forming deeper vessels. In conclusion, embryonic choriocapillaris appears to form initially by hemo-vasculogenesis (blood vessels and blood cells form simultaneously from common precursors) while angiogenesis appears to be the mode of intermediate and large choroidal vessel development in the fetus.

The initial fetal human retinal vasculature develops by vasculogenesis.

There is increasing evidence that the hemangioblast, a common progenitor for hematopoietic cells and endothelial cells, participates in embryonic and extra-embryonic vasculogenesis in some organs. Whether resident angioblasts or endothelial progenitor cells (EPCs) contribute to human retinal vasculogenesis is still a matter of controversy. To address this controversy, fetal human retinas of 6-23 weeks gestation (WG) were examined using immunohistochemistry and a panel of antibodies against endothelial cell markers (CD34, CD31), a marker for retinal angioblasts and endothelium (CD39/ecto-ADPase), and a marker for precursors and hemangioblasts (CXCR4). Confocal microscopic spectral analysis and double labeling with Ki67 was used to identify the proliferating cell types. In the inner neuroblastic layer of the 6-8 WG retina and in the putative ganglion cell layer in avascular regions of older eyes (14 WG-20 WG), scattered CD39+ angioblasts were well in advance of forming vasculature. There was a layer of CXCR4+ cells in the inner retina that was reduced in size with development. As blood vessels formed, CD39+ cells were always well in advance of the vascular front and they expressed CXCR4. This demonstrates that a pool of resident angioblasts express CD39 and CXCR4 as they differentiate and participate in vasculogenesis in the fetal human. They retain expression of CD39 as endothelial cells in the newly formed retinal vasculature but they down-regulate CXCR4 expression.