Biomedical subjects

Gerard A Lutty

Publications and source records attributed to Gerard A Lutty.

At least 19 recordsLinked to original sources

PubMed2006

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.

Animals↗

PubMed2006

Proceedings of the Third International Symposium on Retinopathy of Prematurity: an update on ROP from the lab to the nursery (November 2003, Anaheim, California).

The Third International Symposium on Retinopathy of Prematurity (ROP) was convened with the aim of cross fertilizing the horizons of basic and clinical scientists with an interest in the pathogenesis and management of infants with ROP. Ten speakers in the clinical sciences and ten speakers in the basic sciences were recruited on the basis of their research to provide state of the art talks. The meeting was held November 9, 2003 immediately prior to the American Academy of Ophthalmology meeting; scholarships were provided for outreach to developing countries and young investigators. This review contain the summaries of the 20 platform presentations prepared by the authors and the abstracts of presented posters. Each author was asked to encapsulate the current state of understanding, identify areas of controversy, and make recommendations for future research. The basic science presentations included insights into the development of the human retinal vasculature, animal models for ROP, growth factors that affect normal development and ROP, and promising new therapeutic approaches to treating ROP like VEGF targeting, inhibition of proteases, stem cells, ribozymes to silence genes, and gene therapy to deliver antiangiogenic agents. The clinical presentations included new insights into oxygen management, updates on the CRYO-ROP and ETROP studies, visual function in childhood following ROP, the neural retina in ROP, screening for ROP, management of stage 3 and 4 ROP, ROP in the third world, and the complications of ROP in adult life. The meeting resulted in a penetrating exchange between clinicians and basic scientists, which provided great insights for conference attendees. The effect of preterm delivery on the normal cross-talk of neuroretinal and retinal vascular development is a fertile ground for discovering new understanding of the processes involved both in normal development and in retinal neovascular disorders. The meeting also suggested promising potential therapeutic interventions on the horizon for ROP.

Humans↗

PubMed2006

Canine retinal angioblasts are multipotent.

The purpose of this study was to culture and characterize endothelial cells and angioblasts, vascular precursors, from adult and neonatal dog retina and determine if angioblasts are committed to endothelial cell lineage or have the potential to be multipotent, i.e. express phenotypic characteristics of other vascular cell types. Endothelial cells were established from adult dog retina (ADREC) by the technique of Gitlin and D'Amore. For angioblasts, pieces of neonatal day 2 (P2) avascular peripheral retina were placed under coverslips until sufficient cells had explanted. All cells were maintained initially on hyaluronic acid (HA)/fibronectin (FN) substratum. Neonatal canine retinal angioblasts (NCRA) were maintained initially on retinal-derived growth factor with alpha-amino adipic acid to inhibit growth of Muller cells. Cell lines were characterized by enzyme histochemistry [menadione-dependent alpha glycerophosphate dehydrogenase (alphaGPDH), marker for angioblasts] and immunocytochemistry. Once characterized, cells were grown on FN, or collagens I or IV substrata and fed platelet-derived growth factor-BB (PDGF-BB) or fibroblast growth factor-2 (FGF-2). The phenotypic expression of a marker for endothelial cells [acetylated LDL (acLDL) uptake] or a marker for pericytes and smooth muscle cells, production of alpha smooth muscle actin (alphaSMA), was evaluated under those conditions. The canine retinal cell lines that were established had the following characteristics when maintained on serum and a retinal extract. Angioblasts had low expression of vWf and VEGF-R2 (two markers for canine endothelial cells), and very low uptake of acLDL but high expression of alphaGPDH and adenosine A2a receptors (A2aR) (two markers for canine angioblasts in vivo). ADREC had high expression of endothelial cell markers (vWf, VEGF-R2, and acLDL uptake) but minimal expression of alphaGPDH and A2aR. Both angioblasts and endothelial cells expressed CXCR4, a marker for hemangioblasts. Angioblasts grown on any of the substrata in the presence of FGF-2 had high uptake of acLDL and low expression of alphaSMA, while those grown in the presence of PDGF-BB had high expression of alphaSMA and low uptake of acLDL. In conclusion, angioblasts cultured from peripheral vascular retina have low expression of endothelial cell markers and high alphaGPDH and A2aR, markers for canine angioblasts in vivo. Angioblasts will internalize acLDL when maintained on FGF-2 and express alphaSMA when maintained on PDGF-BB, suggesting that they have the potential to become endothelial cells or pericytes, i.e. are multipotent.

Actins↗

PubMed2005

The expression of advanced glycation endproduct receptors in rpe cells associated with basal deposits in human maculas.

Basal deposits within Bruch's membrane are associated with aging and age-related macular degeneration (AMD) although the factors causing their formation are incompletely understood. Advanced glycation endproducts (AGEs) accumulate in Bruch's membrane including basal deposits and drusen with aging. One mechanism by which AGEs alter a cell's phenotype is via AGE receptors. The purpose of this study was to immunolocalize and quantify the expression of AGE receptors by RPE cells associated with basal deposits or normal Bruch's membrane that were microdissected from human maculas. Postmortem eyes from 14 aged control donors and five donors with non-neovascular AMD were cryopreserved. RPE cells associated with normal Bruch's membrane or basal deposits were laser capture microdissected. The RNA was extracted and used for RT-qPCR to quantify the expression of RAGE, AGE R1, AGE R2, and AGE R3. Streptavidin alkaline phosphatase immunohistochemistry for these receptors was also performed and sections were bleached from 14 normal and nine AMD donors. RT-qPCR showed significant upregulation of RAGE, AGE R1, and AGE R3 in RPE cells overlying basal deposits compared to cells attached to morphologically normal Bruch's membrane. Immunohistochemical analysis for RAGE, AGER1, R2, and R3 showed diffuse, light staining of RPE cells and strong choriocapillaris staining in areas of normal Bruch's membrane. In areas of basal deposits, the RPE had more intense staining for RAGE and AGER1 compared to regions of normal Bruch's membrane. These results suggest that AGE receptors could influence the formation of basal deposits during aging and AMD.

Adult↗

PubMed2005

Phosphatase enzyme histochemistry for studying vascular hierarchy, pathology, and endothelial cell dysfunction in retina and choroid.

Phosphatase enzymes cleave an inorganic phosphate from a substrate. Phosphatase enzyme histochemistry followed by flat-embedding in glycol methacrylate is extremely useful in studying retinal and choroidal vascular development and loss, since only viable blood vessels have these enzyme activities. Sites of occlusion and remodeling can be identified and analysed, resulting in new insights into the cause of occlusion. The phosphatase activities are elevated in neovascularization making possible high resolution analysis of neovascularization, the feeder vessels, and the retinal milieu in which angiogenesis occurs. Adenosine diphosphatase (ADPase) catalyzes ADP to an inorganic phosphate plus adenosine monophosphate, preventing accumulation of ADP, one of the most potent stimuli for platelet aggregation. The ADPase technique can be used in any species but this report highlights its use in dog and human retinas. The ADPase technique has yielded important insights into vaso-occlusive and vasoproliferative processes in retinopathy of prematurity, sickle cell and diabetic retinopathies. The alkaline phosphatase flatembedding technique is useful in evaluating dog, cat, and human choroidal vasculatures. It has permitted quantification of the loss of choriocapillaris in diabetic choroidopathy and of the RPE and choriocapillaris in geographic atrophy and exudative age-related macular degeneration.

Adult↗

PubMed2005

Pathology of Berkeley sickle cell mice: similarities and differences with human sickle cell disease.

Because Berkeley sickle cell mice are used as an animal model for human sickle cell disease, we investigated the progression of the histopathology in these animals over 6 months and compared these findings to those published in humans with sickle cell disease. The murine study groups were composed of wild-type mixed C57Bl/6-SV129 (control) mice and sickle cell (SS) mice (alpha-/-, beta-/-, transgene +) of both sexes and between 1 and 6 months of age. SS mice were similar to humans with sickle cell disease in having erythrocytic sickling, vascular ectasia, intravascular hemolysis, exuberant hematopoiesis, cardiomegaly, glomerulosclerosis, visceral congestion, hemorrhages, multiorgan infarcts, pyknotic neurons, and progressive siderosis. Cerebral perfusion studies demonstrated increased blood-brain barrier permeability in SS mice. SS mice differed from humans with sickle cell disease in having splenomegaly, splenic hematopoiesis, more severe hepatic infarcts, less severe pulmonary manifestations, no significant vascular intimal hyperplasia, and only a trend toward vascular medial hypertrophy. Early retinal degeneration caused by a homozygous mutation (rd1) independent from that causing sickle hemoglobin was an incidental finding in some Berkeley mice. While our study reinforces the fundamental strength of this model, the notable differences warrant careful consideration when drawing parallels to human sickle cell disease.

Anemia, Sickle Cell↗

PubMed2005

Pigment epithelium-derived factor (PEDF) and vascular endothelial growth factor (VEGF) in aged human choroid and eyes with age-related macular degeneration.

The purpose of this study was to examine the localization and relative levels of vascular endothelial growth factor (VEGF; an angiogenic factor) and pigment epithelium-derived factor (PEDF; an antiangiogenic factor) in aged human choroid and to determine if the localization or their relative levels changed in age-related macular degeneration (AMD). Ocular tissues were obtained from eight aged control donors (age range, 75-86 years; mean age, 79.8 years) with no evidence or history of chorioretinal disease and from 12 donors diagnosed with AMD (age range, 61-105 years; mean age, 83.9 years). Tissues were cryopreserved and streptavidin alkaline phosphatase immunohistochemistry was performed with rabbit polyclonal anti-human VEGF and rabbit polyclonal anti-human PEDF antibodies. Binding of the antibodies was blocked by preincubation of the antibody with an excess of recombinant human PEDF or VEGF peptide. Choroidal blood vessels were identified with mouse anti-human CD-34 antibody in adjacent tissue sections. Three independent observers graded the immunohistochemical reaction product. The most prominent sites of VEGF and PEDF localization in aged control choroid were RPE-Bruch's membrane-choriocapillaris complex including RPE basal lamina, intercapillary septa, and choroidal stroma. There was no significant difference in immunostaining intensity and localization of VEGF and PEDF in aged control choroids. The most intense VEGF immunoreactivity was observed in leukocytes within blood vessels. AMD choroid had a similar pattern and intensity of VEGF immunostaining to that observed in aged controls. However, PEDF immunoreactivity was significantly lower in RPE cells (p=0.0073), RPE basal lamina (p=0.0141), Bruch's membrane (p<0.0001), and choroidal stroma (p=0.0161) of AMD choroids. The most intense PEDF immunoreactivity was observed in disciform scars. Drusen and basal laminar deposits (BLDs) were positive for VEGF and PEDF. In aged control subjects, VEGF and PEDF immunostaining was the most intense in RPE-Bruch's membrane-choriocapillaris complex. In AMD, PEDF was significantly lower in RPE cells, RPE basal lamina, Bruch's membrane and choroidal stroma. These data suggest that a critical balance exists between PEDF and VEGF, and PEDF may counteract the angiogenic potential of VEGF. The decrease in PEDF may disrupt the balance and be permissive for the formation of choroidal neovascularization (CNV) in AMD.

Aged↗

PubMed2005

Transgenic expression of leukemia inhibitory factor (LIF) blocks normal vascular development but not pathological neovascularization in the eye.

PURPOSE: Vascular development in the eye has been described as a complex process involving both vasculogenesis and angiogenesis. Multiple cell types are involved in the process including angioblasts, vascular endothelial cells, astrocytes, pericytes, and Muller glial cells. This suggests that multiple growth factors and cytokines are required to regulate retinal vascular development. Leukemia inhibitory factor (LIF) is a member of the interleukin 6 family of cytokines. LIF is expressed during inflammation and has been reported to affect vascular development in culture; however, its effects in vivo have not been demonstrated. The purpose of this study was to determine how LIF could regulate ocular vascular development. METHODS: We have analyzed ocular vascular development in transgenic mice that express LIF in the ocular lens from embryonic day 11. RESULTS: In transgenic mice, LIF reduced development of embryonic vasculature in the eye, and inhibited retinal vascular development. Inhibition in vivo was independent of vascular endothelial cell growth factor (VEGF) expression. In older transgenic mice, the absence of a retinal vasculature resulted in retinal ischemia and elevated VEGF levels. The upregulation of VEGF resulted in the proliferation of pathological vascular membranes in the vitreous and neovascularization penetrating the retina, which in turn resulted in tractional retinal detachment. CONCLUSIONS: LIF is a potent inhibitor of retinal vascular development. These transgenic mice will be useful as a model of persistent fetal vasculature and to study the mechanism for the development of neovascular membranes in the vitreous and could be used to develop inhibitors of tractional detachment.

Animals↗

PubMed2005

Effects of sickle cell disease on the eye: clinical features and treatment.

Ocular complications of the sickling disorders are multiple and require continuing assessment to detect lesions early enough for effective prophylactic therapy to maximize visual functioning. The disease requires close monitoring of the patient for progression or regression of lesions in determining the selection of therapeutic options.

Anemia, Sickle Cell↗

PubMed2005

Neutrophils are associated with capillary closure in spontaneously diabetic monkey retinas.

Type 2 diabetes develops spontaneously in obese aging rhesus monkeys (Macaca mulatta). This study investigates the association between polymorphonuclear leukocytes and development of retinopathy. Blood pressure and plasma glucose levels were determined in 15 diabetic and 6 nondiabetic monkeys. The plasma levels of total cholesterol, LDL cholesterol, HDL cholesterol, and triglycerides were determined just before the start of the animal's final decline and elective necropsy. Retinas were incubated for ADPase (labels viable retinal blood vessels) and nonspecific esterase (labels neutrophils) activities. Polymorphonuclear leukocytes were counted per millimeter squared of retina. After the retina was flat-embedded in glycol methacrylate, tissue sections were taken through areas of interest and observed microscopically. Elevated numbers of intravascular polymorphonuclear leukocytes were present adjacent to areas with retinal capillary nonperfusion. There were significantly more polymorphonuclear leukocytes per millimeter squared in diabetic retinas (6.91 +/- 5.01) compared with normal retinas (1.45 +/- 1.62, P = 0.018). Severity of hypertension in diabetes was also significantly associated with greater numbers of polymorphonuclear leukocytes (P = 0.02). There was a significant positive exponential correlation between the number of polymorphonuclear leukocytes per millimeter squared and the level of total cholesterol (R = 0.907), LDL cholesterol (R = 0.875), the total cholesterol-to-HDL cholesterol ratio (R = 0.86), and total triglycerides (R = 0.888). This study demonstrates that severity of diabetes and the development of retinopathy are associated with increased numbers of polymorphonuclear leukocytes in the retina of diabetic monkeys. Hypertension, high plasma levels of LDL cholesterol and triglycerides, and low plasma levels of HDL cholesterol also are associated with increased polymorphonuclear leukocytes in retina.

Aneurysm↗

PubMed2004

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.

Aged↗

PubMed2004

Astrocyte-endothelial cell relationships during human retinal vascular development.

PURPOSE: To evaluate evidence for the presence of vascular precursor cells (angioblasts) and astrocyte precursor cells (APCs) in the developing human retina and determine their relationship. METHODS: Pax-2/GFAP/CD-34 triple-label immunohistochemistry was applied to four retinas aged 12, 14, 16, and 20 weeks of gestation (WG) to label APCs, astrocytes, and patent blood vessels. APCs are Pax-2(+)/GFAP(-), whereas astrocytes are Pax-2(+)/GFAP(+). Adenosine diphosphatase (ADPase) enzyme histochemistry, which identifies endothelial cells and vascular precursors, was applied to human retinas aged 12, 16, 17, and 19 WG. Nissl stain, a nonspecific cell soma marker, was applied to 14.5-, 18-, and 21-WG retinas. Established blood vessels were visualized with CD34 and ADPase. RESULTS: Topographical analysis of the distribution of Nissl-stained spindle cells and ADPase(+) vascular cells showed that these two populations have similar distributions at corresponding ages. ADPase(+) vascular precursor cells preceded the leading edge of patent vessels by more than 1 millimeter. In contrast, Pax-2(+)/GFAP(-) APCs preceded the leading edge of CD34(+) blood vessels by a very small margin, and committed astrocytes (Pax-2(+)/GFAP(+)) were associated with formed vessels and nerve fiber bundles. Two populations of ADPase(+) cells were evident, a spindle-shaped population located superficially and a deeper spherical population. The outer limits of these populations remain static with maturation. CONCLUSIONS: A combination of Pax-2/GFAP/CD34 immunohistochemistry, Nissl staining, and ADPase histochemistry showed that the vascular precursor cells (angioblasts), identified using ADPase and Nissl, represent a population distinct from Pax-2(+)/GFAP(-) APCs in the human retina. These results lead to the conclusion that formation of the initial human retinal vasculature takes place through vasculogenesis from the prior invasion of vascular precursor cells.

Antigens, CD34↗

PubMed2004

Retinopathy in monkeys with spontaneous type 2 diabetes.

PURPOSE: Type 2 diabetes occurs spontaneously in rhesus monkeys and shows an extraordinary similarity to human diabetes in clinical features and relative time course. The purpose of this study was to investigate clinically and histopathologically the ocular changes in these monkeys. METHODS: Ophthalmoscopic examinations were performed on aged normal and diabetic monkeys. Retinas from 16 diabetic monkeys and 6 nondiabetic monkeys were incubated postmortem for adenosine diphosphatase (ADPase) activity (labels viable retinal blood vessels) and flat-embedded in JB-4. Tissue sections were cut through areas of interest. RESULTS: Cotton-wool spots, intraretinal hemorrhages, and hard exudates in the macula were observed by ophthalmoscopy in some diabetic monkeys. Dot/blot hemorrhages, cotton-wool spots, and small nonperfused areas were the earliest histologically documented changes in the retinas. Large nonperfused areas extending from optic disc to midfovea were observed in four diabetic monkeys. Formation of small intraretinal microvascular abnormalities (IRMAs) and microaneurysms were associated with the areas of nonperfusion. There were apparent fluid-filled spaces in the outer plexiform layer in three of these maculas, suggesting macular edema. There was a significant correlation between the occurrence of retinopathy and hypertension (P = 0.037 for systolic pressure; P = 0.019 for diastolic pressure). In elastase-digested retinas, the ratio of pericytes to endothelial cells was 0.66:1 in diabetic and 0.64:1 in nondiabetic (P = 0.75) retinas. CONCLUSIONS: This is the first detailed analysis of retinopathy in a colony of spontaneous type 2 diabetic monkeys. Monkeys with type 2 diabetes have many of the angiopathic changes associated with human diabetic retinopathy. Hypertension correlates with the severity of the diabetic retinopathy.

Aneurysm↗

PubMed2003

Expression of pigment epithelium-derived factor (PEDF) and vascular endothelial growth factor (VEGF) in sickle cell retina and choroid.

Pigment epithelium-derived factor (PEDF) has been shown to be an inhibitor of angiogenesis as well as a multipotent neurotrophic factor in the mammalian eye. Changes in PEDF levels have been correlated with development of retinal neovascularization in oxygen-induced retinopathy. The purpose of this study was to determine the localization and relative level of PEDF in human retinas and choroids using immunohistochemistry and evaluate the changes in PEDF and vascular endothelial growth factor (VEGF) localization and their relation to the progression of proliferative sickle cell retinopathy. Cryopreserved tissues from eyes of normal subjects and subjects with non-proliferative or proliferative sickle cell retinopathy were used with streptavidin peroxidase immunohistochemistry. A rabbit polyclonal antibody was made against recombinant human PEDF. Binding of the antibody was blocked by preincubation of the antibody with excess human recombinant PEDF. Relative levels of immunoreactivity were scored with a seven-point grading system and by microdensitometric analysis.The most prominent sites of PEDF localization in the normal eye were the vitreous condensed at the internal limiting membrane and RPE-Bruch's membrane-choriocapillaris complex. PEDF was also prominent in choroidal stroma. There was limited immunoreactivity in some cells of the neural retinas, in blood vessels and in the interphotoreceptor matrix (IPM). There was no difference in ratio (1.47 vs. 1.44) of PEDF/VEGF or the relative levels of either growth factor in the retinal vasculatures of the control subjects and perfused area of non-proliferative sickle cell retinas. The ratio was increased in the non-perfused area of the non-proliferative sickle cell retinas (2.24). In eyes with proliferative sickle cell retinopathy, elevated PEDF and VEGF immunostaining was present in viable vessels of sea fan neovascular formations as well as feeder vessels of sea fans. The PEDF/VEGF ratio in sea fans was 1.0. Immunoreactivity for PEDF was prominent in retinal vessels in non-perfused regions and in atrophic sea fans, while VEGF immunoreactivity was weak or absent in these structures. In conclusion, PEDF and VEGF were both significantly elevated in viable sea fan formations in sickle cell disease (p<0.05) but only PEDF was present in non-viable sea fans. The highest levels of PEDF in all eyes were associated with extracellular matrices (vitreous, choroidal stroma, IPM, and walls of blood vessels). PEDF might play an important role in inhibiting angiogenesis and inducing the regression of sea fans. Progression of angiogenesis may be dependent on the ratio of PEDF/VEGF.

Adolescent↗

PubMed2003

Retinal vascular development and oxygen-induced retinopathy: a role for adenosine.

Adenosine is a ubiquitous molecule that is produced predominantly by catabolism of adenosine triphosphate. Levels of this nucleoside increase dramatically with ischemia and elevated tissue activity. Adenosine levels are high in inner retina during retinal vascular development in postnatal dog. The source appears to be the ectoenzyme 5' nucleotidase, which is prominent at this time in the innermost process of Muller cells. One of the adenosine receptors, A(2A), is present on endothelial cell precursors, angioblasts, and endothelial cells in formed blood vessels in neonatal dog. These observations suggest that adenosine is important in retinal vascular development. Oxygen-induced retinopathy (OIR) is a model for human retinopathy of prematurity (ROP). The initial event in OIR is induced by exposure of the developing retina to high oxygen. Vascular development is halted and over 60% of the retinal vasculature is lost during this stage of the disease in dog, which is called vaso-obliteration. 5' nucleotidase is dramatically reduced during vaso-obliteration, resulting in a sharp decline in adenosine. When animals are returned to room air, the retina is hypoxic because of the lack of blood vessels, oxygen consumption is increased due to neuronal development, and systemic levels of oxygen have returned to normal. At this time, 5' nucleotidase activity and adenosine levels are elevated well beyond normal levels. This stage of OIR is the vasoproliferative stage and A(2A) expression and endothelial cell proliferation are very elevated compared to control animals. Florid preretinal neovascularization forms, which has high levels of adenosine and A(2A) receptors. Therefore, adenosine and its A(2A) receptor appear to be important in canine OIR. This work suggests that adenosine and its receptors may be a therapeutic target in OIR. This hypothesis is supported by recent studies in mouse (Mino et al., Invest. Ophthalmol. Vis. Sci. 42(13) (2001) 3320), which demonstrated that targeting one of the A(2) receptors can inhibit formation of neovascularization in OIR.

Adenosine↗

PubMed2002

Pathologic features of vascular endothelial growth factor-induced retinopathy in the nonhuman primate.

PURPOSE: Vascular endothelial growth factor (VEGF) is a potent ischemia-upregulated angiogenic protein that has been implicated in diabetic retinopathy. Intravitreal VEGF injections have not previously been shown to produce preretinal neovascularization. The purpose of this study was to further characterize the angiopathic changes that occur after intravitreal injections in a nonhuman primate and determine if preretinal neovascularization develops. DESIGN: Experimental animal study. METHODS: Vascular endothelial growth factor 165 was injected into the eyes of normal cynomolgus monkeys at regular intervals. As a control, normal eyes were injected with phosphate buffered saline. Color photography and fluorescein angiography were performed at regular intervals. The retinas were incubated for adenosine diphosphatase (ADPase) activity to visualize retinal vessels. The retinas were flat-embedded and areas of potential preretinal neovascularization were identified en bloc and serially sectioned. RESULTS: Areas of capillary nonperfusion and vessel dilation and tortuousity were seen by angiography. In serial sections, the nonperfused areas were found to be associated with endothelial cell hyperplasia in vessel lumens. Preretinal neovascularization originating only from superficial veins and venules was observed throughout peripheral retina, but was not seen in the posterior pole. Lacunae-like veins were subdivided by the process of intussusception and endothelial cell bridging. Arterioles demonstrated endothelial cell hyperplasia and microaneurysms. CONCLUSION: Intraocular injections of VEGF were sufficient to produce preretinal neovascularization in the nonhuman primate. Most vasculopathic structures were associated with endothelial cell hyperplasia. These results demonstrate that VEGF alone can produce many features of both nonproliferative and proliferative diabetic retinopathy including the previously undescribed development of preretinal neovascularization. This well-characterized VEGF-induced primate model of retinal neovascularization may be useful as a means of testing new treatments for retinal neovascularization.

Animals↗

PubMed2002

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.

Anemia, Sickle Cell↗

PubMed2002

Localization of VEGF receptor-2 (KDR/Flk-1) and effects of blocking it in oxygen-induced retinopathy.

PURPOSE: Vascular endothelial cell growth factor (VEGF) has been implicated in vascular development and in proliferative retinopathies. The goal of this study was to examine the immunohistochemical localization and relative levels of VEGF receptor-2 (KDR) in canine retina during postnatal vasculogenesis and during angiogenesis in oxygen-induced retinopathy (OIR) and to investigate the effects of neutralizing KDR on these processes. METHODS: Eyes from normal dogs ranging from 1 to 22 days of age and age-matched oxygen-treated animals were snap frozen for immunohistochemical analysis with antibodies against human KDR. To examine the effects of blocking KDR, 6-day-old air-reared control and oxygen-treated animals were surgically implanted with slow release polymer pellets containing control IgG or anti-KDR. Material eluted from pellets was assessed using a binding assay (measures binding to soluble KDR) to determine the kinetics of anti-KDR release and endothelial cell proliferation to measure bioactivity. Animals were killed at 22 days of age and tissues examined with adenosine diphosphatase (ADPase) histochemical staining of blood vessels. RESULTS: KDR immunoreactivity was only weakly associated with developing retinal vessels and was not observed in angioblasts throughout normal postnatal development. Immunoreactivity was very strong in reforming retinal vessels and intravitreal neovascularization in oxygen-treated animals. Anti-KDR had no effect on vessel morphology or growth in air-reared control animals. In oxygen-treated animals, anti-KDR significantly inhibited revascularization of the retina (P = 0.005) and formation of intravitreal neovascularization compared with control IgG pellet eyes (P < 0.04). CONCLUSIONS: KDR/Flk-1 was only weakly associated with normal developing primary retinal vessels but was strongly expressed by proliferating endothelial cells in reforming retinal vessels and intravitreal neovascularization after hyperoxic insult. Anti-KDR antibody delivered by slow-release pellets had no effect on normal vasculogenesis, but it inhibited the formation of intravitreal neovascularization and retinal vessel development in OIR. The study suggests that blocking KDR may be beneficial for treating pathologic angiogenesis in adult tissue.

Animals↗