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Diabetic retinopathy is a leading cause of blindness in the Western world. Aberrant intercellular adhesion molecule-1 expression and leukocyte adhesion have been implicated in its pathogenesis, raising the possibility of an underlying chronic inflammatory mechanism. In the current study, the role of insulin-like growth factor (IGF)-I in these processes was investigated. We found that systemic inhibition of IGF-I signaling with a receptor-neutralizing antibody, or with inhibitors of PI-3 kinase (PI-3K), c-Jun kinase (JNK), or Akt, suppressed retinal Akt, JNK, HIF-1alpha, nuclear factor (NF)-kappaB, and AP-1 activity, vascular endothelial growth factor (VEGF) expression, as well as intercellular adhesion molecule-1 levels, leukostasis, and blood-retinal barrier breakdown, in a relevant animal model. Intravitreous administration of IGF-I increased retinal Akt, JNK, HIF-1alpha, NF-kappaB, and AP-1 activity, and VEGF levels. IGF-I stimulated VEGF promoter activity in vitro, mainly via HIF-1alpha, and secondarily via NF-kappaB and AP-1. In conclusion, IGF-I participates in the pathophysiology of diabetic retinopathy by inducing retinal VEGF expression via PI-3K/Akt, HIF-1alpha, NF-kappaB, and secondarily, JNK/AP-1 activation. Taken together, these in vitro and in vivo signaling studies thus identify potential targets for pharmacological intervention to preserve vision in patients with diabetes.

Purpose: Vascular endothelial growth factor (VEGF) induces angiogenesis and vascular permeability and is thought to be operative in several ocular vascular diseases. The VEGF isoforms are highly conserved among species; however, little is known about their differential biological functions in adult tissue. In the current study, the inflammatory potential of two prevalent VEGF isoform splice variants, VEGF(120(121)) and VEGF(164(165)), was studied in the transparent and avascular adult mouse cornea., Methods: Controlled-release pellets containing equimolar amounts of VEGF(120) and VEGF(164) were implanted in corneas. The mechanisms underlying this differential response of VEGF isoforms were explored. The response of VEGF in cultured endothelial cells was determined by Western blot analysis. The response of VEGF isoforms in leukocytes was also investigated., Results: VEGF(164) was found to be significantly more potent at inducing inflammation. In vivo blockade of VEGF receptor (VEGFR)-1 significantly suppressed VEGF(164)-induced corneal inflammation. In vitro, VEGF(165) more potently stimulated intracellular adhesion molecule (ICAM)-1 expression on endothelial cells, an effect that was mediated by VEGFR2. VEGF(164) was also more potent at inducing the chemotaxis of monocytes, an effect that was mediated by VEGFR1. In an immortalized human leukocyte cell line, VEGF(165) was found to induce tyrosine phosphorylation of VEGFR1 more efficiently., Conclusions: Taken together, these data identify VEGF(164(165)) as a proinflammatory isoform and identify multiple mechanisms underlying its proinflammatory biology.

Purpose: A critical event in the pathogenesis of diabetic retinopathy is the inappropriate adherence of leukocytes to the retinal capillaries. Advanced glycation end-products (AGEs) are known to play a role in chronic inflammatory processes, and the authors postulated that these adducts may play a role in promoting pathogenic increases in proinflammatory pathways within the retinal microvasculature., Methods: Retinal microvascular endothelial cells (RMECs) were treated with glycoaldehyde-modified albumin (AGE-Alb) or unmodified albumin (Alb). NFkappaB DNA binding was measured by electromobility shift assay (EMSA) and quantified with an ELISA: In addition, the effect of AGEs on leukocyte adhesion to endothelial cell monolayers was investigated. Further studies were performed in an attempt to confirm that this was AGE-induced adhesion by co-incubation of AGE-treated cells with soluble receptor for AGE (sRAGE). Parallel in vivo studies of nondiabetic mice assessed the effect of intraperitoneal delivery of AGE-Alb on ICAM-1 mRNA expression, NFkappaB DNA-binding activity, leukostasis, and blood-retinal barrier breakdown., Results: Treatment with AGE-Alb significantly enhanced the DNA-binding activity of NFkappaB (P = 0.0045) in retinal endothelial cells (RMECs) and increased the adhesion of leukocytes to RMEC monolayers (P = 0.04). The latter was significantly reduced by co-incubation with sRAGE (P < 0.01). Mice infused with AGE-Alb demonstrated a 1.8-fold increase in ICAM-1 mRNA when compared with control animals (P < 0.001, n = 20) as early as 48 hours, and this response remained for 7 days of treatment. Quantification of retinal NFkappaB demonstrated a threefold increase with AGE-Alb infusion in comparison to control levels (AGE Alb versus Alb, 0.23 vs. 0.076, P < 0.001, n = 10 mice). AGE-Alb treatment of mice also caused a significant increase in leukostasis in the retina (AGE-Alb versus Alb, 6.89 vs. 2.53, n = 12, P < 0.05) and a statistically significant increase in breakdown of the blood-retinal barrier (AGE Alb versus Alb, 8.2 vs. 1.6 n = 10, P < 0.001)., Conclusions: AGEs caused upregulation of NFkappaB in the retinal microvascular endothelium and an AGE-specific increase in leukocyte adhesion in vitro was also observed. In addition, increased leukocyte adherence in vivo was demonstrated that was accompanied by blood-retinal barrier dysfunction. These findings add further evidence to the thinking that AGEs may play an important role in the pathogenesis of diabetic retinopathy.

Hypoxia-induced VEGF governs both physiological retinal vascular development and pathological retinal neovascularization. In the current paper, the mechanisms of physiological and pathological neovascularization are compared and contrasted. During pathological neovascularization, both the absolute and relative expression levels for VEGF164 increased to a greater degree than during physiological neovascularization. Furthermore, extensive leukocyte adhesion was observed at the leading edge of pathological, but not physiological, neovascularization. When a VEGF164-specific neutralizing aptamer was administered, it potently suppressed the leukocyte adhesion and pathological neovascularization, whereas it had little or no effect on physiological neovascularization. In parallel experiments, genetically altered VEGF164-deficient (VEGF120/188) mice exhibited no difference in physiological neovascularization when compared with wild-type (VEGF+/+) controls. In contrast, administration of a VEGFR-1/Fc fusion protein, which blocks all VEGF isoforms, led to significant suppression of both pathological and physiological neovascularization. In addition, the targeted inactivation of monocyte lineage cells with clodronate-liposomes led to the suppression of pathological neovascularization. Conversely, the blockade of T lymphocyte-mediated immune responses with an anti-CD2 antibody exacerbated pathological neovascularization. These data highlight important molecular and cellular differences between physiological and pathological retinal neovascularization. During pathological neovascularization, VEGF164 selectively induces inflammation and cellular immunity. These processes provide positive and negative angiogenic regulation, respectively. Together, new therapeutic approaches for selectively targeting pathological, but not physiological, retinal neovascularization are outlined.

Platelet microthrombi are present in the diabetic retinal vasculature of humans and rodents; however, the mechanisms and consequences of their presence have not been defined. The current study demonstrates that platelet containing microthrombi accumulate in the retinal vasculature of the rat within 2 weeks of experimental diabetes, a timepoint at which leukocyte-mediated endothelial cell injury and death are known to occur. Platelet accumulation increased with the duration of diabetes, and crossover experiments revealed that maximal platelet accumulation required both diabetic platelets and a diabetic endothelium. Platelet accumulation also coincided with the expression of Fas and FasL in the diabetic retina. When endothelial cell apoptosis was inhibited with an anti-FasL neutralizing antibody, platelet accumulation was effectively suppressed. When platelets were depleted from the systemic circulation with an anti-platelet antibody, blood-retinal barrier breakdown worsened in the diabetic animals. These findings suggest that platelet accumulation in the diabetic retinal vasculature is secondary to endothelial cell death and serves, in part, to suppress blood-retinal barrier breakdown.

Purpose: To determine if genetic ablation of the chemokine receptor CCR2 (involved in leukocyte and endothelial chemotaxis) inhibits the development of corneal neovascularization., Methods: Wild-type C57BL/6J mice, as well as species-specific counterparts with targeted homozygous disruption of the CCR2, underwent chemical and mechanical denudation of corneal and limbal epithelium. Corneas were harvested 2 weeks after injury. Neovascularization was quantified by CD31 immunostaining., Results: The mean percentages of neovascularized corneal area in control mice and CCR2-deficient mice 2 weeks after denudation were 58.3% and 38.8% (P = 0.047), respectively., Conclusions: Development of corneal neovascularization is inhibited in CCR2-deficient mice.

Purpose: To investigate the role of the leukocyte adhesion molecules CD18 and intercellular adhesion molecule (ICAM)-1 in the development of choroidal neovascularization (CNV)., Methods: Laser photocoagulation was used to induce CNV in wild-type C57BL/6J mice and species-specific counterparts with targeted homozygous disruption of the CD18 or ICAM-1 gene. Expression of CD18 and ICAM-1 after laser injury was assessed by immunostaining. CNV responses were compared on the basis of en masse volumetric measurements obtained by confocal microscopy 2 weeks after laser injury and by determination of fluorescein angiographic leakage at 1, 2, and 4 weeks after laser injury., Results: The site of laser injury showed upregulation of ICAM-1 and invasion by CD18-positive leukocytes within 1 day of laser injury. Significantly fewer lesions exhibited fluorescein leakage defined to be pathologically significant in CD18-deficient mice at weeks 1, 2, and 4 weeks and in ICAM-1-deficient mice at 1 and 4 weeks, compared with the control. There were a significantly greater number of lesions without fluorescein leakage in CD18-deficient mice than in the other two groups at all time points. The volume of CNV in CD18- and ICAM-1-deficient mice was significantly less than in wild type., Conclusions: These data suggest a nonredundant role for leukocyte adhesion to vascular endothelium in the development of laser-induced choroidal neovascularization.

Retinal ischemia can cause vision-threatening pathological neovascularization. The mechanisms of retinal ischemia are not fully understood, however. Here we have shown that leukocytes prune the retinal vasculature during normal development and obliterate it in disease. Beginning at postnatal day 5 (P5) in the normal rat, vascular pruning began centrally and extended peripherally, leaving behind a less dense, smaller-caliber vasculature. The pruning was correlated with retinal vascular expression of intercellular adhesion molecule-1 (ICAM-1) and coincided with an outward-moving wave of adherent leukocytes composed in part of cytotoxic T lymphocytes. The leukocytes adhered to the vasculature through CD18 and remodeled it through Fas ligand (FasL)-mediated endothelial cell apoptosis. In a model of oxygen-induced ischemic retinopathy, this process was exaggerated. Leukocytes used CD18 and FasL to obliterate the retinal vasculature, leaving behind large areas of ischemic retina. In vitro, T lymphocytes isolated from oxygen-exposed neonates induced a FasL-mediated apoptosis of hyperoxygenated endothelial cells. Targeting these pathways may prove useful in the treatment of retinal ischemia, a leading cause of vision loss and blindness.

Purpose: The objectives of this study were to characterize the differential potency of two major VEGF isoforms, VEGF(120) and VEGF(164), for inducing leukocyte stasis (leukostasis) within the retinal vasculature and blood-retinal barrier (BRB) breakdown and to determine whether endogenous VEGF(164) mediates retinal leukostasis and BRB breakdown in early and established diabetes., Methods: Retinal leukostasis and BRB breakdown were simultaneously quantified by combining concanavalin A lectin (ConA) perfusion labeling with a fluorophotometric dextran leakage assay. CD45 immunohistochemistry was performed to confirm that ConA-stained cells within the vasculature were leukocytes. Retinal leukostasis and BRB breakdown were compared in nondiabetic rats receiving intravitreous injections of VEGF(120) or VEGF(164). Retinal intercellular adhesion molecule (ICAM)-1 and VEGF protein levels were studied by Western blot and ELISA, respectively. An anti-VEGF(164(165)) aptamer (EYE001) was administered by intravitreous injection to 2-week and 3-month diabetic rats, and the effect on retinal leukostasis and BRB breakdown was quantified., Results: Compared with VEGF(120), VEGF(164) more potently increased retinal ICAM-1 levels (2.2-fold), leukostasis (1.9-fold), and BRB breakdown (2.1-fold, P < 0.01 for all), despite negligible differences in vitreoretinal VEGF levels at the time of evaluation (P > 0.05). Retinal leukostasis and leakage increased with the duration of diabetes (P < 0.01) and correlated closely (P < 0.01, r = 0.889). The isoform-specific blockade of endogenous VEGF(164) with EYE001 resulted in a significant suppression of retinal leukostasis and BRB breakdown in both early (72.4% and 82.6%, respectively) and established (48.5% and 55.0%, respectively) diabetes (P < 0.01)., Conclusions: On an equimolar basis, VEGF(164) is at least twice as potent as VEGF(120) at inducing ICAM-1-mediated retinal leukostasis and BRB breakdown in vivo. The inhibition of diabetic retinal leukostasis and BRB breakdown with EYE001 in early and established diabetes indicates that VEGF(164) is an important isoform in the pathogenesis of early diabetic retinopathy.

Age-related macular degeneration is the principal cause of registered legal blindness among those aged over 65 in the United States, western Europe, Australia, and Japan. Despite intensive research, the precise etiology of molecular events that underlie age-related macular degeneration is poorly understood. However, investigations on parallel fronts are addressing this prevalent public health problem. Sophisticated biochemical and biophysical techniques have refined our understanding of the pathobiology of drusen, geographic atrophy, and retinal pigment epithelial detachments. Epidemiological identification of risk factors has facilitated an intelligent search for underlying mechanisms and fueled clinical investigation of behavior modification. Gene searches have not only brought us to the cusp of identifying the culpable gene loci in age-related macular degeneration, but also localized genes responsible for other macular dystrophies. Recent and ongoing investigations, often cued by tumor biology, have revealed an important role for various growth factors, particularly in the neovascular form of the condition. Transgenic and knockout studies have provided important mechanistic insights into the development of choroidal neovascularization, the principal cause of vision loss in age-related macular degeneration. This in turn has culminated in preclinical and clinical trials of directed molecular interventions.

Objective: To determine the role of the microvascular endothelium in the regulation of regenerating liver mass after partial hepatectomy., Summary Background Data: Angiogenesis is critical for both pathologic and physiologic processes. The ability of certain tissues, such as the liver, kidney, and spleen, to regenerate after injury is poorly understood. The liver will regenerate to its normal mass within 8 days of surgical excision. Because the authors have previously shown that the endothelial cell regulates tumor mass, we hypothesized that normal adult organ mass is also controlled by the endothelial cell., Methods: Two-thirds partial hepatectomy was performed in 7- to 8-week-old C57 BL/6 mice, followed by systemic treatment with either the angiogenesis stimulator basic fibroblast growth factor (bFGF) (1 microg/g/d intraperitoneal) or the angiogenesis inhibitor TNP-470 (30 mg/kg/qod subcutaneous). Groups of three mice were then euthanized on postoperative days 2, 4, 6, and 8, and the livers were weighed and analyzed by immunohistochemistry., Results: bFGF accelerated hepatic regeneration by 42%, 19%, 16%, and 16% on postoperative days 2, 4, 6, and 8, respectively. TNP-470 inhibited hepatic regeneration by 46%, 74%, 67%, and 64% on postoperative days 2, 4, 6, and 8, respectively. Immunohistochemistry revealed that bFGF and TNP-470 primarily affected the endothelial compartment. Specifically, bFGF increased endothelial proliferation and decreased endothelial apoptosis. TNP-470, in contrast, inhibited endothelial cell proliferation. The cessation of the regenerative process correlated with a decrease in endothelial proliferation and an increase in endothelial apoptosis., Conclusions: The systemic administration of angiogenesis agents modulates the regeneration of hepatic mass primarily by affecting endothelial cell proliferation or apoptosis. Endothelial cell apoptosis is associated with the cessation of the regenerative process in control mice. These results suggest that the endothelial cell is one of the key mediators of regenerating adult tissue mass in this partial hepatectomy model.

Increases in leukostasis/monocyte adhesion to the capillary endothelium (leukostasis) and decreases in retinal blood flow may be causally associated and are implicated in the pathogenesis of diabetic retinopathy. In this study, we demonstrate that increases in leukostasis are observed in insulin-resistant states without diabetes, whereas decreases in retinal blood flow require diabetes and hyperglycemia. Microimpaction studies using beads mimicking retinal capillary obstruction by leukocytes did not affect retinal blood flow. In diabetic rats, treatment with the antioxidant alpha-lipoic acid normalized the amount of leukostasis but not retinal blood flow. In contrast, treatment with D-alpha-tocopherol and protein kinase-C beta-isoform inhibition (LY333531) prevented the increases in leukostasis and decreases in retinal blood flow in diabetic rats. Serum hydroxyperoxide, a marker of oxidative stress, was increased in diabetic rats, but normalized by treatment with antioxidants alpha-lipoic acid and D-alpha-tocopherol and, surprisingly, PKC beta-isoform inhibition. These findings suggest that leukostasis is associated with endothelial dysfunction, insulin resistance, and oxidative stress but is not related to retinal blood flow and is not sufficient to cause diabetic-like retinopathy. Moreover, treatment with PKC beta inhibition is effective to normalize diabetes or hyperglycemia-induced PKC beta-isoform activation and oxidative stress.

Purpose: The corneal endothelium is a target of the aging process. This study was undertaken to reveal the relationship between corneal endothelial cell (CEC) death and the accumulation of advanced glycation end products (AGEs), by investigating the possible mechanism of accumulation of AGE in CECs and its effects on CEC death., Methods: First, the in vivo expression of the receptor was investigated for AGE (RAGE) and galectin-3, both receptors for AGE, at both the mRNA and protein levels. Second, AGEs were added to the culture media of the cultured CECs, and the uptake of AGEs, the generation of reactive oxygen species, and the induction of apoptosis were investigated., Results: Immunohistochemistry and RT-PCR demonstrated that both RAGE and galectin-3 were expressed in bovine CECs. After administration of AGE-modified bovine serum albumin to the culture medium, uptake of AGE was observed in the cytoplasm of the cultured bovine CECs. In addition, with increasing concentration of AGEs, the generation of reactive oxygen and the number of apoptotic cells also increased., Conclusions: These results show that the accumulation of AGEs in CECs induced apoptosis, in part, by increasing cellular oxidative stress. The accumulation of AGEs in the CECs of elderly patients may be involved in the loss of CECs during the aging process.

Purpose: To determine whether genetic ablation of the CC chemokine receptor CCR5 (involved in leukocyte and endothelial chemotaxis) inhibits the development of corneal neovascularization., Methods: Wild-type C57BL/6J mice and species-specific counterparts with targeted homozygous disruption of the CCR5 gene underwent chemical and mechanical denudation of corneal and limbal epithelium. Corneas were harvested 2 and 4 weeks after injury. Neovascularization was quantified by CD31 immunostaining. Expression of VEGF protein was quantified by ELISA., Results: The mean percentages of neovascularized corneal area in control mice and CCR5-deficient mice 2 weeks after denudation were 58.3% and 38.5% (P = 0.05), respectively. At 4 weeks after denudation, the corresponding percentages were 67.6% and 44.0% (P = 0.028). In CCR5-deficient mice, VEGF protein levels were reduced 51.1% at 2 weeks (P = 0.05) after injury and 37.3% at 4 weeks (P = 0.03)., Conclusions: CCR5-deficient mice showed a persistent 34% to 35% inhibition of corneal neovascularization for up to 4 weeks. This inhibition correlates with reduced expression of VEGF. These data implicate CCR5 as one essential component in the development of corneal neovascularization.

Purpose: To investigate the mechanisms governing corneal neovascularization and the appearance of goblet cells in a murine model of limbal insufficiency., Methods: The spatial and time-dependent relationship between corneal neovascularization and goblet cell density was analyzed in corneal flatmounts. Immunohistochemical detection of the vascular endothelial growth factor (VEGF) receptor Flt-1 (VEGFR1) was performed in paraffin-embedded sections. A transgenic mouse that expresses the reporter gene lacZ targeted to the Flt-1 locus through homologous recombination was used to analyze corneal expression of Flt-1. The presence of soluble and membranous goblet cell Flt-1 mRNA and protein content was assessed with Northern and Western blot analyses, respectively. Finally, systemic adenoviral expression of a soluble Flt-1/Fc construct was used to study the effect of inhibition of VEGF bioactivity on the appearance of goblet cells and neovascularization., Results: Corneal neovascularization preceded the appearance of goblet cells, although both processes overlapped temporally. Flt-1 was abundant in the conjunctiva-like epithelium covering the cornea, as well as in the goblet cells, invading leukocytes, and vasculature. A similar expression pattern was observed in the transgenic mice expressing the lacZ gene downstream from the Flt-1 promoter. Isolated human and rat goblet cells in culture expressed Flt-1 mRNA and protein, as did freshly isolated human conjunctiva. The systemic inhibition of VEGF bioactivity potently suppressed both corneal neovascularization (8.3% +/- 8.1% vs. 41.1% +/- 15.3% corneal area; P < 0.001) and corneal goblet cell density (1.6% +/- 2.5% vs. 12.2% +/- 2.4% corneal area; P < 0.001)., Conclusions: Two important features of corneal conjunctivalization, the appearance of goblet cells and neovascularization, are regulated by VEGF. Both processes are probably mediated, in part, through the Flt-1 receptor. Taken together, these data indicate that an anti-VEGF therapeutic approach may limit the visual loss associated with conjunctivalization of the corneal surface.

Purpose: To develop a controlled-drug delivery system for the long-term inhibition of vascular endothelial growth factor (VEGF) and its mediated responses., Methods: Poly(lactic-co-glycolic)acid (PLGA) microspheres containing anti-VEGF RNA aptamer (EYE001) formulations in the solid-state were developed by an oil-in-oil solvent evaporation process. In vitro experiments were performed to characterize the release profiles. Stability and bioactivity of the released drug were assayed by monitoring the RNA aptamer's ability to inhibit VEGF-induced cell proliferation in human umbilical vein endothelial cells (HUVECs). Cell proliferation experiments were conducted with aptamer aliquots collected after short-, mid-, and long-term release time points. To investigate the feasibility of this polymer device as a potential transscleral delivery device, an in vitro apparatus was developed to assess polymer hydration and degradation through rabbit sclera and subsequent delivery through it., Results: PLGA microspheres were able to deliver EYE001 in a sustained manner, with an average rate of 2 micro g/d over a period of 20 days. Solid-state stabilization of the aptamer with disaccharide trehalose before lyophilization and encapsulation in PLGA rendered the drug more stable after release. Cell proliferation experiments demonstrated that the bioactivity of the aptamer was preserved after release, as indicated by inhibition of endothelial cell proliferation after incubation with VEGF. Microspheres packed into a sealed chamber and placed onto the "orbital" part of a rabbit sclera for a period of 6 days became hydrated and started to degrade, as shown by scanning electron microscopy (SEM). As a result, the aptamer was delivered from the microspheres through the sclera, as determined spectrophotometrically., Conclusions: The loading of aptamer-containing microspheres into a device and placing it on the orbital surface of the sclera was assessed and shown to be feasible. RNA aptamer EYE001 encapsulated in PLGA was delivered over a period of 20 days with retained activity. This method represents a promising approach for the transscleral delivery of drugs and the treatment of choroidal and retinal diseases.

Diabetic macular edema, resulting from increased microvascular permeability, is the most prevalent cause of vision loss in diabetes. The mechanisms underlying this complication remain poorly understood. In the current study, diabetic vascular permeability (blood-retinal barrier breakdown) is demonstrated to result from a leukocyte-mediated Fas-FasL-dependent apoptosis of the retinal vasculature. Following the onset of streptozotocin-induced diabetes, FasL expression was increased in rat neutrophils (P<0.005) and was accompanied by a simultaneous increase in Fas expression in the retinal vasculature. Static adhesion assays demonstrated that neutrophils from diabetic, but not control, rats induced endothelial cell apoptosis in vitro (P<0.005). The latter was inhibited via an antibody-based FasL blockade (P<0.005). In vivo, the inhibition of FasL potently reduced retinal vascular endothelial cell injury, apoptosis, and blood-retinal barrier breakdown (P<0.0001) but did not diminish leukocyte adhesion to the diabetic retinal vasculature. Taken together, these data are the first to identify leukocyte-mediated Fas-FasL-dependent retinal endothelial cell apoptosis as a major cause of blood-retinal barrier breakdown in early diabetes. These data imply that the targeting of the Fas-FasL pathway may prove beneficial in the treatment of diabetic retinopathy.

Purpose: This study was designed to gain an insight into the inflammatory milieu into which a donor limbal graft is routinely introduced. The objective of this study was to modulate this environment by gene therapy with the anti-inflammatory cytokine interleukin-1 receptor antagonist (IL-1 RA)., Methods: In a mouse model, the ocular surface cytokine environment associated with a conjunctivalized cornea was assessed 4 weeks after injury. Total corneal epithelial and limbal debridement was performed with a combination of alkali and scrape injury. The cytokines and adhesion molecules measured included IL-1alpha, IL-1beta, IL-6, VEGF, intercellular adhesion molecule (ICAM)-1, and vascular adhesion molecule (VCAM)-1, by real-time PCR or ELISA. Injured corneas were transfected with IL-1 RA by injection of naked plasmid vector pIRES-EGFP-IL-1 RA immediately after injury. Corneas transfected with pIRES-EGFP served as the control. Expression of corneal IL-1 RA after transfection with pIRES-EGFP-IL1-RA was assessed over a 2-week period by real-time PCR and Western blot analysis. In addition, limbal stem cell grafts transfected with IL-1 RA were assessed for leukocyte influx., Results: Conjunctivalized corneas showed increased expression of IL-1alpha, IL-1beta, IL-1 RA, IL-6, VEGF, ICAM-1, and VCAM-1, compared with normal cornea. Transfection-efficiency experiments indicated that corneal expression of IL-1 RA peaked between 12 and 24 hours and lasted up to 2 weeks after the initial transfection. IL-1 RA corneal gene therapy resulted in a downregulation of IL-1beta and VCAM-1 expression at 4 weeks after injury, whereas downregulation of IL-6 was evident only at 1 week after injury. Corneal neovascularization was also reduced. In addition, corneal limbal stem cell grafts transfected with IL-1 RA showed a decreased leukocyte influx compared with control grafts., Conclusions: Transfection of a cornea with IL-1 RA immediately after epithelial injury selectively altered the cytokine profile of the resultant conjunctivalized cornea and suppressed corneal neovascularization. Transfection of corneal limbal donor tissue with IL-1 RA before engraftment can reduce leukocyte influx into the graft. The findings demonstrate the feasibility of using transient cytokine gene expression, either in donor or recipient corneal tissue, to alter the ocular surface environment beneficially.

Objective: To determine the ability of angiostatin and the angiostatin-producing low-metastatic (LM) clone of Lewis lung carcinoma (LLC) to inhibit and regress corneal neovascularization, as compared with the non-angiostatin-producing high-metastatic (HM) clone., Methods: Three groups of C57BL6/J mice underwent chemical and mechanical denudation of corneal and limbal epithelium. One group remained tumor free while the other 2 were implanted with LLC cells (either the HM or LM clones) subcutaneously the day before, 2 weeks after, or 4 weeks after denudation. Corneas were harvested 2 weeks after tumor implantation (at 2, 4, and 6 weeks after denudation for tumor-free mice). Neovascularization was quantified by CD31 immunostaining. In a second experiment, recombinant angiostatin was delivered continuously for 2 weeks via an osmotic pump in mice with established corneal neovascularization., Results: The mean percentages of neovascularized corneal area in mice 2 weeks after LM-LLC implantation were 4.6%, 3.7%, and 37.0%, at 2, 4, and 6 weeks after scraping, respectively. In contrast, in the mice implanted with HM-LLC, the corresponding values were 45.4% (P =.01), 90.1% (P =.03), and 80.3% (P =.005). For tumor-free mice, the corresponding values were 62.0% (P =.003), 68.9% (P =.03), and 59.3% (P =.06). Mice implanted with angiostatin pumps had a 37.7% neovascularized corneal area 2 weeks after implantation and 4 weeks after scraping while mice implanted with sham pumps had 60.5% (P =.007)., Conclusion: Angiostatin inhibits and regresses corneal neovascularization induced by mechanical and alkali corneal injury., Clinical Relevance: This appears to be the first evidence of biologically induced regression of corneal neovascularization, and the first direct demonstration of angiostatin-induced regression of neovascularization in any tissue.

The aim of this paper was to develop a GFP-expressing transgenic mouse model for the keratoepithelioplasty and to use this to follow the outcome of this form of graft, when placed on an inflamed corneal surface. Further aims were to characterize both the graft and the epithelial surface of the mouse and rat cornea using putative stem cell markers (P63 and Telomerase) and marker of cell differentiation (14-3-3 sigma). Keratepithelioplasty was carried out using a GFP transgenic mouse cornea as donor tissue. Fluorescent epithelial outgrowth from each keratepithelioplasty was scored and quantified. Donor corneal graft tissue was obtained from the paracentral region or the anatomical limbal region of murine corneas. Paracentral donor grafts (n = 20) consistently demonstrated a significant increase in proliferative potential compared to grafts obtained from the anatomical limbal region of the mouse cornea (n = 25) (P = 0.000, Mann-Whitney U). Correspondingly, P63 expression was maximal in the paracentral region of the mouse cornea, in keeping with the demonstrated increased proliferative potential of donor grafts harvested from this region of the cornea. The murine corneal epithelium demonstrated decreased rather than increased cellular layers at the limbal region, in contrast to that of the rat or human epithelium. In addition, as a general finding in all species tested, there was an apparent increase noted in P63 expression in basal corneal epithelial cells in regions that had increased cellular layers (limbus in humans and rats and the paracentral corneal region in the mouse). Epithelium, which had migrated from donor grafts onto recipient corneas, retained P63 expression for the period of time examined (up to 3 days postengraftment). In addition, the conjunctival surface of an injured conjunctivalized displayed an abnormal pattern of P63 expression. Telomerase expression was widespread throughout many layers of both the murine and rat corneal epithelium. In the mouse and rat corneal epithelium P63 expression was maximal in areas of increased proliferative potential. Its expression, however, was not confined to stem cells alone. Migrating cells from transplanted keratoepithelial grafts retained P63 expression at least in the early stages post-transplantation. Finally, damaged conjunctivalized corneas displayed an abnormal P63 expression pattern when compared to either normal conjunctiva or normal cornea.

Diabetic retinopathy remains a leading cause of irreversible blindness. A critical early pathology in the disease is the adhesion of leukocytes to the retinal vasculature, a process that occurs, in part, via intercellular adhesion molecule-1. Once leukocyte adhesion occurs, endothelial cell injury ensues, as does blood-retinal barrier breakdown. Here we show that angiopoietin-1 can prevent and reverse these diabetic retinal vascular changes in both new and established diabetes. Angiopoietin-1, when given intravitreally to newly diabetic rats, normalized retinal vascular endothelial growth factor (VEGF) and intercellular adhesion molecule-1 mRNA and protein levels, leading to reductions in leukocyte adhesion, endothelial cell injury, and blood-retinal barrier breakdown. When an adenovirus coding for angiopoietin-1 was given systemically to mice with established diabetes, it similarly inhibited leukocyte adhesion and endothelial cell injury and blood-retinal barrier breakdown. These changes coincided with reductions in retinal eNOS, nitric oxide, Akt (protein kinase B), and MAP kinase activity, known mediators of VEGF bioactivity and leukocyte adhesion. When endogenous VEGF bioactivity was inhibited with a soluble Flt-1/Fc chimera, retinal Akt kinase activity was significantly reduced in vivo. Taken together, these data document new vascular and anti-inflammatory bioactivities for angiopoietin-1 and identify it as the first naturally occurring protein that directly protects the retinal vasculature in diabetes.

Purpose: Leukocyte endothelial interactions are a key feature of ocular angiogenesis but also play a role in nonproliferative vascular alterations as are found in early diabetes or uveitis. The adhesion of leukocytes to endothelial cells during inflammation is a multistep process that involves leukocyte rolling, adhesion, and extravasation mediated by selectins, cell adhesion molecules (CAMs), integrins, and chemokines. Heparan sulfate (HS) is known to bind to and modify the function of these molecules under physiological conditions. In this study, the role of the HS proteoglycan syndecan-1 in mediating leukocyte-endothelial interactions in the ocular vasculature was investigated., Methods: Mice carrying a deletion in the gene encoding the cell surface HS proteoglycan syndecan-1 (sdc1) were used to study the interactions of leukocytes and endothelial cells in vivo, using a perfusion technique with FITC-coupled ConA and intravital microscopy., Results: In a retina perfusion model, Sdc1(-/-) mice showed increased leukocyte adhesion that was largely attributable to the leukocytes. Intravital microscopy studies revealed a dramatic increase in adhesion after tumor necrosis factor (TNF)-alpha treatment of sdc1(-/-) mice compared with similarly treated wild-type mice. The higher degree of leukocyte adhesion may account for the increase in inflammation-mediated corneal angiogenesis observed in sdc1(-/-) mice., Conclusions: The results indicate a role for syndecan-1 as a negative regulator of leukocyte-mediated inflammatory responses. Thus, syndecan-1 could have use as a target for prevention of pathologic leukocyte-endothelial interactions in angiogenesis and inflammation.

Objective: To evaluate the safety and efficacy of intravitreal injections of an antigen-binding fragment of a recombinant humanized monoclonal antibody directed toward vascular endothelial growth factor (rhuFab VEGF) in a monkey model of choroidal neovascularization (CNV)., Methods: In phase 1 of the study, each animal received intravitreal injections, 500 microg per eye, of rhuFab VEGF in one eye (prevention eye), while the contralateral eye received rhuFab VEGF vehicle (control eye) at 2-week intervals. On day 21, laser photocoagulation was performed to induce CNV. In phase 2, the vehicle-treated eye was crossed over and both eyes received 500 microg of rhuFab VEGF beginning 21 days following laser-induced injury at days 42 and 56. The eyes were monitored by ophthalmic examinations, color photographs, and fluorescein angiography., Results: rhuFab VEGF did not cause any ocular hemorrhages. All eyes treated with rhuFab VEGF developed acute anterior chamber inflammation within 24 hours of the first injection that resolved within 1 week, and this inflammation was less severe with subsequent injections. The incidence of CNV, defined angiographically, was significantly lower in the prevention eyes than the control eyes (P<.001). Subsequent treatments were associated with less leakage in eyes with established CNV that were crossed over from the control eyes to the treatment eyes (P =.001)., Conclusions: Intravitreal rhuFab VEGF injections prevented formation of clinically significant CNV in cynomolgus monkeys and decreased leakage of already formed CNV with no significant toxic effects., Clinical Relevance: This study provides the nonclinical proof of principle for ongoing clinical studies of intravitreally injected rhuFab VEGF in patients with neovascular age-related macular degeneration.

Leukocyte adhesion to the diabetic retinal vasculature results in blood-retinal barrier breakdown, capillary nonperfusion, and endothelial cell injury and death. Intercellular adhesion molecule-1 (ICAM-1) and the leukocyte integrin CD18 are required for these processes. Diabetes was induced in Long Evans rats, resulting in a two- to threefold increase in retinal leukocyte adhesion. Following one week of diabetes, neutrophil CD11a, CD11b, and CD18 expression was increased significantly, as were retinal ICAM-1 levels. Animals were treated with aspirin, a cyclooxygenase 2 (COX-2) inhibitor (meloxicam), or a soluble tumor necrosis factor alpha (TNF-alpha) receptor/Fc construct (TNFR-Fc, etanercept). High-dose aspirin, etanercept, and high-dose meloxicam each reduced leukocyte adhesion and suppressed blood-retinal barrier breakdown. High-dose aspirin also reduced the expression of CD11a, CD11b, and CD18, whereas meloxicam and etanercept did not. High-dose aspirin, etanercept, and high-dose meloxicam each reduced retinal ICAM-1 expression. Aspirin and meloxicam both lowered retinal TNF-alpha levels. Notably, aspirin, meloxicam, and etanercept did not change retinal vascular endothelial growth factor levels. High-dose aspirin, etanercept and high-dose meloxicam, each suppressed the retinal expression of eNOS and the DNA-binding capacity of retinal nuclear factor-kappaB. High-dose aspirin also suppressed Erk kinase activity, which is involved in CD18 up-regulation. Taken together, these data identify COX-2 and TNF-alpha as operative in the early signature pathologies of diabetic retinopathy, a newly recognized inflammatory disease.

It is conceivable that VEGF inhibition may prevent edema formation at the early stages of diabetic retinopathy. Once the retina is irreversibly ischemic or new vessels have formed, however, antagonizing VEGF may lead to retinal necrosis due to chronic ischemia. An alternative approach would be the induction of neovascular maturation. Once the new vessels become mature, retina ischemia resolves. There would be no edema, hemorrhage, or retinal detachment. Acute administration of an angiogenic molecule called angiopoietin-1 protects vasculature from leaking [103]. Angiopoietins bind to the endothelial cell-specific receptor Tie 2 and play an important role is vascular development, especially vessel maturation. The proposed mechanisms include recruiting pericytes and organizing vascular matrix [103]. Since VEGF is constitutively expressed at low levels in normal eyes [46], it may contribute to the maintenance of vascular integrity. Thus, oversuppression of VEGF expression may be harmful to the retinal vasculature. Inhibiting VEGF action may need to be delivered in a tightly regulated manner such that complete inhibition may be avoided both to maintain basal levels and to provide rapid reversal of inhibition when acute angiogenic responses are desired [72]. VEGF is involved in normal angiogenic processes in adults such as cardiac collateral circulation, wound healing and menstrual cycle [27]. Local drug delivery seems to be more appealing than systemic administration to avoid the side effects. Some VEGF antagonists, such as VEGF receptor chimeric protein and the VEGF neutralizing antibodies are large molecules with poor diffusion into tissues. Repetitive invasive procedures such as intravitreal injection seem to be impractical due to potential complications of retinal detachment and bacterial infection. Recent progress on transscleral delivery of bioactive proteins and DNAs to the choroid and retina provides promising future on local delivery of therapeutic agents [12,13].

Rapid advances in the molecular pathogenesis of retinal and choroidal disorders have highlighted the urgent need for innovative drug delivery modalities to the loci of pathology. In vitro and in vivo studies suggest that the transscleral route may offer a means to achieve the goal of sustained, targeted drug delivery to the posterior segment. Potentially therapeutic concentrations of macromolecules with retention of bioactivity can be attained in the choroid and retina via minimally invasive transscleral delivery.

AMD is a poorly understood disease at this time. Since it is the leading cause of blindness in the elderly in the developed world, there is a pressing need for better treatment. Therefore, there is extensive ongoing research in both pathogenesis and therapy of AMD. Epidemiological studies have shown significant risk associated with increasing age and cigarette smoking, future studies may identify environmental risk factors though at present studies have been inconclusive. Genetic studies may identify subgroups of disease and thus help provide a selective approach to treatment. The vascular model of AMD may provide better understanding of the blood flow and post capillary resistance and help in early and newer intervention in the disease. Vascular endothelial growth factor has been extensively studied in choroidal neovascularization. It has been demonstrated in human and animal models of CNV and VEGF antagonists are currently in clinical trial. Extensive work is ongoing to prevent and treat CNV with antiangiogenic agents.

Acute intensive insulin therapy is an independent risk factor for diabetic retinopathy. Here we demonstrate that acute intensive insulin therapy markedly increases VEGF mRNA and protein levels in the retinae of diabetic rats. Retinal nuclear extracts from insulin-treated rats contain higher hypoxia-inducible factor-1alpha (HIF-1alpha) levels and demonstrate increased HIF-1alpha-dependent binding to hypoxia-responsive elements in the VEGF promoter. Blood-retinal barrier breakdown is markedly increased with acute intensive insulin therapy but can be reversed by treating animals with a fusion protein containing a soluble form of the VEGF receptor Flt; a control fusion protein has no such protective effect. The insulin-induced retinal HIF-1alpha and VEGF increases and the related blood-retinal barrier breakdown are suppressed by inhibitors of p38 mitogen-activated protein kinase (MAPK) and phosphatidylinositol (PI) 3-kinase, but not inhibitors of p42/p44 MAPK or protein kinase C. Taken together, these findings indicate that acute intensive insulin therapy produces a transient worsening of diabetic blood-retinal barrier breakdown via an HIF-1alpha-mediated increase in retinal VEGF expression. Insulin-induced VEGF expression requires p38 MAPK and PI 3-kinase, whereas hyperglycemia-induced VEGF expression is HIF-1alpha-independent and requires PKC and p42/p44 MAPK. To our knowledge, these data are the first to identify a specific mechanism for the transient worsening of diabetic retinopathy, specifically blood-retinal barrier breakdown, that follows the institution of intensive insulin therapy.

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.

Leukocyte adhesion to the diabetic retinal vasculature results in early blood-retinal barrier breakdown, capillary nonperfusion, and endothelial cell injury and death. Previous work has shown that intercellular adhesion molecule-1 (ICAM-1) and CD18 are required for these processes. However the relevant in vivo stimuli for ICAM-1 and CD18 expression in diabetes remain unknown. The current study investigated the causal role of endogenous vascular endothelial growth factor (VEGF) and nitric oxide in initiating these events. Diabetes was induced in Long-Evans rats with streptozotocin, resulting in a two- to threefold increase in retinal leukocyte adhesion. Confirmed diabetic animals were treated with a highly specific VEGF-neutralizing Flt-Fc construct (VEGF TrapA(40)). Retinal ICAM-1 mRNA levels in VEGF TrapA(40)-treated diabetic animals were reduced by 83.5% compared to diabetic controls (n = 5, P < 0.0001). VEGF TrapA(40) also potently suppressed diabetic leukocyte adhesion in retinal arterioles (47%, n = 11, P < 0.0001), venules (36%, n = 11, P < 0.0005), and capillaries (36%, n = 11, P < 0.001). The expression of endothelial nitric oxide synthase (eNOS), a downstream mediator of VEGF activity, was increased in diabetic retina, and was potently suppressed with VEGF TrapA(40) treatment (n = 8, P < 0.005). Further, VEGF TrapA(40) reduced the diabetes-related nitric oxide increases in the retinae of diabetic animals. The inhibition of eNOS with N-omega-nitro-L-arginine methyl ester also potently reduced retinal leukocyte adhesion. Although neutrophil CD11a, CD11b, and CD18 levels were increased in 1-week diabetic animals, VEGF TrapA(40) did not alter the expression of these integrin adhesion molecules. Taken together, these data demonstrate that VEGF induces retinal ICAM-1 and eNOS expression and initiates early diabetic retinal leukocyte adhesion in vivo. The inhibition of VEGF bioactivity may prove useful in the treatment of the early diabetic retinopathy.

Purpose: Studies have demonstrated a causal role for specific molecules in the pathogenesis of diabetic retinopathy. Among the implicated mediators are growth factors such as vascular endothelial growth factor (VEGF) as well as adhesion molecules and proliferation- and apoptosis-related genes. However, a coordinated large-scale investigation of gene expression in the diabetic retina has not yet been reported. Here the retinal gene expression profile of diabetic and nondiabetic animals using cDNA microarrays were analyzed and compared., Methods: Long-Evans rats were made diabetic with streptozotocin. Retinal gene expression was analyzed over 3 weeks using high-density nylon filter-based cDNA arrays. Genes were sorted into clusters according to their temporal expression profiles. They were also grouped according to their potential pathophysiological significance. The in vivo gene expression profiles of selected genes were verified via RNase protection assay., Results: The rat GeneFilter contains a total of 5147 genes, of which 1691 are known genes and 3456 are expressed sequence tags (ESTs). On day 3, the expression of 27 known genes was increased by more than twofold. On days 7 and 21, the corresponding numbers were 60 and 12, respectively. A transient upregulation (>2-fold) in expression was seen in 627 of 5147 total genes. A subset of 926 genes exhibited a modest (<2-fold) decrease in expression. No genes showed a greater than twofold decrease in expression. Overall, the identity of the genes that were upregulated suggests that the response of the retina to the diabetic challenge contains an inflammatory component. Moreover, most regulatory activity occurs during the first week of diabetes., Conclusions: The development of a rational therapy for diabetic retinopathy will be assisted by detailed knowledge regarding the molecular pathophysiology of the disease. Here, an expression profile of an underlying retinal inflammatory process in early diabetes was extracted. Beyond providing insight into the general nature of the response to a pathogenic challenge, gene expression profiling may also allow the efficient identification of potential drug targets and markers for monitoring the course of disease.

Purpose: The objectives of this study were to (1) determine whether endogenous vascular endothelial growth factor (VEGF) triggers diabetic blood-retinal barrier breakdown, and (2) identify the site as well as phenotype of the hyperpermeable diabetic retinal vessels., Methods: Retinal VEGF mRNA levels were quantified in 1-week diabetic rats using the RNase protection assay. VEGF bioactivity was blocked via the systemic administration of a highly specific VEGF-neutralizing soluble Flt/F(c) construct (VEGF TrapA(40)). An inactive IL6 receptor/F(c) construct (IL6R Trap) was used as an isotype control. Blood-retinal barrier breakdown was quantified using the Evans blue technique and was spatially localized with fluorescent microspheres., Results: Retinal VEGF mRNA levels in 1-week diabetic animals were 3.2-fold higher than in nondiabetic controls (P < 0.0001). Similarly, retinal vascular permeability in 8-day diabetic animals was 1.8-fold higher than in normal nondiabetic controls (P < 0.05). Diabetes-induced blood-retinal barrier breakdown was dose-dependently inhibited with VEGF TrapA(40), with 25 mg/kg producing complete inhibition of the diabetes-induced increases (P < 0.05). Blood-retinal barrier breakdown in diabetic animals treated with solvent alone or IL6R Trap did not differ significantly from untreated diabetic animals (P > 0.05). Spatially, early blood-retinal barrier breakdown was localized to the retinal venules and capillaries of the superficial retinal vasculature., Conclusions: Early blood-retinal barrier breakdown in experimental diabetes is VEGF dependent and is restricted, in part, to the venules and capillaries of the superficial inner retinal vasculature. VEGF inhibition should prove a useful therapeutic approach in the treatment of early diabetic blood-retinal barrier breakdown.

Purpose: To determine the efficacy and safety of naked plasmid gene therapy to the corneal stroma and epithelium., Methods: Naked plasmid DNA was injected under pressure into the cornea of mice. The expression of genes coding for beta galactosidase (beta-gal), enhanced green fluorescent protein (EGFP), vascular endothelial growth factor (VEGF), and soluble Flt-1 (s-Flt) was recorded and measured with regard to dose, time course, and bioactivity., Results: LacZ gene expression of the protein beta-gal was demonstrated as early as 1 hour, with expression persisting for 10 days. Plasmid-injected corneas remained clear and free of inflammation. EGFP was bicistronically expressed with VEGF to demonstrate the practicality of simultaneous in vivo analysis of gene expression and growth factor bioactivity. Corneal injection of a plasmid containing VEGF cDNA induced corneal and anterior chamber neovascularization. Moreover, corneal injection of plasmid containing the cDNA for the soluble form of the VEGF receptor Flt-1 effectively prevented corneal neovascularization., Conclusions: The cornea is readily accessible for gene therapy in the laboratory and in the clinic. The method described is safe, effective, titratable, and easily monitored. Naked DNA delivery to the cornea has the potential to alter the treatment of a wide variety of corneal and anterior segment diseases.

Choroidal neovascularization in age-related macular degeneration is a frequent and poorly treatable cause of vision loss in elderly Caucasians. This choroidal neovascularization has been associated with the expression of vascular endothelial growth factor (VEGF). In current animal models choroidal neovascularization is induced by subretinal injection of growth factors or vectors encoding growth factors such as VEGF, or by disruption of the Bruch's membrane/retinal pigment epithelium complex with laser treatment. We wished to establish a transgenic murine model of age-related macular degeneration, in which the overexpression of VEGF by the retinal pigment epithelium induces choroidal neovascularization. A construct consisting of a tissue-specific murine retinal pigment epithelium promoter (RPE(65) promoter) coupled to murine VEGF(164) cDNA with a rabbit beta-globin-3' UTR was introduced into the genome of albino mice. Transgene mRNA was expressed in the retinal pigment epithelium at all ages peaking at 4 months. The expression of VEGF protein was increased in both the retinal pigment epithelium and choroid. An increase of intravascular adherent leukocytes and vessel leakage was observed. Histopathology revealed intrachoroidal neovascularization that did not penetrate through an intact Bruch's membrane. These results support the hypothesis that additional insults to the integrity of Bruch's membrane are required to induce growth of choroidal vessels into the subretinal space as seen in age-related macular degeneration. This model may be useful to screen for inhibitors of choroidal vessel growth.

Purpose: This study investigated whether a nonradioactive dye, Evans blue, can be adapted as a safe alternative to the isotope-dilution method for quantitating blood-retinal barrier breakdown., Methods: Blood-retinal barrier breakdown was induced in rats with vascular endothelial growth factor (VEGF) or through the induction of diabetes. After allowing Evans blue to circulate in the vasculature, the dye was cleared from the bloodstream with saline, citrate, or citrate-buffered paraformaldehyde, and the efficacies of the perfusion solutions were compared. Extravasated dye was detected at 620 nm and was normalized against the time-averaged Evans blue plasma concentration, the circulation time, and also against wet and dry retina weights., Results: Evans blue leakage from retinas treated with VEGF was 4.0-fold higher than that of contralateral untreated eyes (n = 6 rats, P: < 0.05). Retinal Evans blue leakage of eyes from 1-week diabetic animals (n = 11 retinas) was 1.7-fold higher (P: < 0.05) than that of nondiabetic controls (n = 10 retinas). Intra-animal, inter-retina weights showed significantly less variability (P: < 0.05) with the use of dry weights (11.2%, n = 74 retina pairs) than with wet weights (20.5%, n = 93 retina pairs)., Conclusions: The Evans blue dye technique can be modified to be as sensitive and quantitative as the isotope-dilution method for measuring blood-retinal barrier breakdown. The advantages of the Evans blue technique are its safety, relative simplicity, and economy.

Endothelial cell death is a hallmark of diabetic retinopathy. Its occurrence is required for the formation of acellular (devitalized) capillaries, lesions that produce irreversible retinal ischemia through their inability to support blood flow. The mechanisms underlying diabetic retinal endothelial cell injury and death remain largely unknown. The current study demonstrates that adherent leukocytes are temporally and spatially associated with retinal endothelial cell injury and death within 1 week of streptozotocin-induced experimental diabetes in rats. Moreover, the antibody-based neutralization of intercellular adhesion molecule-1 and CD18 is shown to prevent both leukocyte adhesion and retinal endothelial cell injury and death. These data highlight the central and causal role of adherent leukocytes in the pathogenesis of diabetic retinopathy. They also underscore the potential utility of anti-intercellular adhesion molecule1- and anti-CD18-based therapies in the treatment of diabetic retinopathy, a newly recognized inflammatory disease.

Objectives: To search for novel mutations that cause corneal stromal dystrophies and to confirm or revise the clinical diagnosis of patients with these mutations., Patients: Through review of the records of the Cogan Eye Pathology Laboratory at the Massachusetts Eye and Ear Infirmary, Boston, and of clinical records, we ascertained 14 unrelated patients with the clinical or histopathologic diagnosis of granular (3 cases), Avellino (5 cases), lattice (5 cases), or Reis-Bücklers (1 case) corneal dystrophy., Methods: Clinical records and histopathologic findings of the index patients and their relatives were reviewed. Patients and selected relatives donated a blood sample from which leukocyte DNA was purified and assayed for mutations in the BIGH3 gene and, in 2 patients, the gelsolin gene, using the polymerase chain reaction and direct genomic sequencing., Results: All index patients with the diagnosis of granular dystrophy or Avellino dystrophy had the missense mutation Arg555Trp or Arg124His, respectively, previously reported in the BIGH3 gene. Of the 5 index patients with a prior diagnosis of lattice dystrophy, 2 had the originally reported lattice mutation (Arg124Cys) in the BIGH3 gene, 1 had a more recently reported missense mutation (His626Arg) in the same gene, 1 had the missense mutation Asp187Asn in the gelsolin gene, and 1 had no detected mutation in either gene. Affected members of the family with Reis-Bücklers dystrophy did not carry the previously reported mutations Arg555Gln or Arg124Leu but instead carried a novel missense mutation Gly623Asp in the BIGH3 gene., Conclusions: Molecular genetic analysis can improve the accuracy of diagnosis of patients with corneal dystrophies. Two patients with a prior diagnosis of lattice corneal dystrophy had their diagnosis changed to gelsolin-related amyloidosis (1 case) or secondary, nonhereditary localized amyloidosis (1 case). A novel mutation in the BIGH3 gene that causes Reis-Bücklers dystrophy was uncovered through this analysis, and another recently reported novel mutation was encountered. These findings serve to expand our knowledge of the spectrum of pathogenic mutations in BIGH3.

Extensive limbal injury is a leading cause of irreversible blindness. The destruction of corneal limbal stem cells often results in corneal neovascularization and an optically inferior epithelium. Previous work has shown that the neovascularization after limbal injury is vascular endothelial growth factor (VEGF)-dependent, with much of the VEGF emanating from the inflammatory cells that invade the cornea. Using a relevant mouse model of limbal injury, we examined the role of CD18 and intercellular adhesion molecule-1 (ICAM-1) in limbal injury-induced neovascularization. The results show that CD18- and ICAM-1-deficient mice developed 35% (n = 5, P = 0.003) and 36% (n = 5, P = 0.002) less neovascularization than strain-specific normal controls, respectively. The corneal neutrophil counts were similarly reduced by 51% (n = 5, P < 0.003) and 46% (n = 5, P < 0.006), respectively. When VEGF mRNA levels were analyzed, they were reduced by 66% (n = 3, P = 0.004) and 48% (n = 3, P = 0.024), respectively. Taken together, these data identify CD-18 and ICAM-1 as mediators of the inflammatory and VEGF-dependent corneal neovascularization that follows limbal injury. The targeting of CD18 and ICAM-1 may prove useful in the treatment of inflammation-associated neovascularization in the cornea and elsewhere.

Objective: To determine if topical ketorolac 0.5% relieves the symptoms and signs of viral conjunctivitis better than artificial tears., Design: Randomized, controlled trial., Participants: One hundred seventeen patients with a clinical diagnosis of viral conjunctivitis were randomized to the treatment group or control group., Methods: Physicians and patients were masked to treatment. Patients in the treatment group received topical ketorolac 0.5% four times daily. Patients in the control group received artificial tears four times daily. Symptom and sign scores were recorded on the day of recruitment and at the time of a follow-up examination 3 to 4 days later., Main Outcome Measures: Change in six symptoms of conjunctivitis (overall discomfort, itching, foreign body sensation, tearing, redness, and lid swelling) and four signs of conjunctivitis (conjunctival injection, conjunctival chemosis, conjunctival mucus, and lid edema). Adverse effects were also studied., Results: A total of 105 patients returned for their 3- to 4-day follow-up. Both the artificial tear and ketorolac groups showed improvement in all symptom scores at their 3- to 4-day follow-up visit. There was no statistically significant difference between the change in symptom scores between the treatment group and control group in any symptom category except redness. Patients in the control group were more likely to report improvement in redness than those in the treatment group, P = 0.012. There was no statistically significant difference between the change in sign scores between the treatment and control groups. Ketorolac 0.5% was more likely to produce stinging than artificial tears, 59.2% versus 18.8%, P < 0.001., Conclusions: Topical ketorolac 0.5% used four times daily is no better than artificial tears at relieving the symptoms or signs of viral conjunctivitis and produces more stinging than artificial tears.

Purpose: In tissues outside the brain, vascular endothelial growth factor-A (VEGF) causes vascular hyper-permeability by opening of inter-endothelial junctions and induction of fenestrations and vesiculo-vacuolar organelles (VVOs). In preliminary studies, we observed that in blood-retinal barrier endothelium, other cellular mechanisms may underlie increased permeability caused by VEGF. This was further investigated in material of an in vivo experimental model of VEGF-induced retinopathy., Methods: Two monkeys received 4 intravitreal injections of 0.5 microg VEGF in one eye and PBS in the other eye prior to sacrifice at day 9. One monkey received 12 injections of 1.25 microg VEGF in one eye and PBS in the other eye prior to sacrifice at day 24. As a control, an untreated eye of a fourth monkey was studied., Results: In the high-dose VEGF-injected eye, fluorescein angiography showed intense retinal micro-vascular leakage. This leakage was also demonstrated by immunohistochemistry demonstrating extravasation of endogenous fibrinogen and IgG. However, in these leaky blood vessels the number of pinocytotic vesicles (caveolae) at the endothelial luminal membrane were significantly higher and, only in the VEGF-injected eyes, these pinocytotic vesicles transported plasma IgG. By electron microscopy, no fenestrations or VVOs were found in the endothelial cells of the VEGF-injected eyes., Conclusion: We conclude that increased vascular permeability for plasma proteins induced by VEGF in blood-retinal barrier endothelium is predominantly caused by a mechanism involving active trans-endothelial transport via pinocytotic vesicles and not by formation of endothelial fenestrations or VVOs.

Two prominent vascular endothelial growth factor (VEGF)-induced retinal effects are vascular permeability and capillary nonperfusion. The mechanisms by which these effects occur are not completely known. Using a rat model, we show that intravitreous injections of VEGF precipitate an extensive retinal leukocyte stasis (leukostasis) that coincides with enhanced vascular permeability and capillary nonperfusion. The leukostasis is accompanied by the up-regulation of intercellular adhesion molecule-1 expression in the retina. The inhibition of intercellular adhesion molecule-1 bioactivity with a neutralizing antibody prevents the permeability and leukostasis increases by 79% and 54%, respectively. These data are the first to demonstrate that a nonendothelial cell type contributes to VEGF-induced vascular permeability. Additionally, they identify a potential mechanism for VEGF-induced retinal capillary nonperfusion.

Purpose: To investigate the feasibility of transscleral drug delivery to the choroid and retina., Methods: An osmotic pump was used to deliver IgG across the sclera of pigmented rabbits, and levels were measured in the choroid, retina, vitreous humor, aqueous humor, orbit, and plasma over 28 days. This method was then used to deliver an anti-intercellular adhesion molecule-1 (ICAM-1) monoclonal antibody (mAb), and its effect on inhibiting vascular endothelial growth factor (VEGF)-induced leukostasis in the choroid and retina was determined by measuring tissue myeloperoxidase (MPO) activity., Results: Levels of retinal and choroidal IgG were significantly higher than baseline at all points up to 28 days (P < or = 0.01). IgG levels in the orbit, vitreous humor, aqueous humor, and plasma were negligible (P > 0.05). MPO activity in the choroid of eyes treated with anti-ICAM-1 mAb was 80% less (P = 0.01) than in eyes receiving an equal rate of delivery of an isotype control antibody. Inhibition of MPO activity in the retina was 70% (P = 0.01). The plasma concentration of anti-ICAM-1 mAb was 31,000-fold less than the concentration in the osmotic pump., Conclusions: Minimally invasive transscleral delivery can be used to deliver therapeutic levels of bioactive drugs to the choroid and retina with negligible systemic absorption. This method of ocular drug delivery may be used in the treatment of a variety of chorioretinal disorders.

Purpose: To determine the in vitro permeability of the sclera to high molecular weight compounds and the relationship between scleral permeability and molecular size., Methods: Fresh rabbit sclera was mounted in a two-chamber diffusion apparatus, and its permeability to sodium fluorescein, fluorescein isothiocyanate (FITC)-conjugated bovine serum albumin, FITC-IgG, and FITC dextrans ranging in molecular weight from 4 to 150 kDa was determined by fluorescence spectrophotometry. Electron microscopy was used to assess the impact of the experimental design on scleral ultrastructural integrity. The effect of the diffusion apparatus on scleral hydration was examined. Rabbit scleral permeability was compared with previously reported data for human and bovine sclera., Results: Scleral permeability decreased with increasing molecular weight and molecular radius, consistent with previous human and bovine data. Molecular radius was a better predictor of scleral permeability than molecular weight. The sclera was more permeable to globular proteins than to linear dextrans of similar molecular weight. The experimental apparatus did not alter scleral ultrastructure. Permeability of rabbit sclera was similar to human sclera but greater than bovine sclera., Conclusions: Large molecules, such as IgG, diffuse across sclera in a manner consistent with porous diffusion through a fiber matrix. Transscleral delivery of immunoglobulins and other large compounds to the choroid and retina may be feasible.

Purpose: A critical early event in the pathogenesis of diabetic retinopathy is leukocyte adhesion to the diabetic retinal vasculature. The process is mediated, in part, by intercellular adhesion molecule-1 (ICAM-1) and results in blood-retinal barrier breakdown and capillary nonperfusion. This study evaluated the expression and function of the corresponding ICAM-1-binding leukocyte beta2-integrins in experimental diabetes., Methods: Diabetes was induced in Long Evans rats with streptozotocin. The expression of the surface integrin subunits CD11a, CD11b, and CD18 on rat neutrophils isolated from peripheral blood was quantitated with flow cytometry. In vitro neutrophil adhesion was studied using quantitative endothelial cell-neutrophil adhesion assays. The adhesive role of the integrin subunits CD11a, CD11b, and CD18 was tested using specific neutralizing monoclonal antibodies. CD18 bioactivity was blocked in vivo with anti-CD18 F(ab')2 fragments, and the effect on retinal leukocyte adhesion was quantitated with acridine orange leukocyte fluorography., Results: Neutrophil CD11a, CD11b, and CD18 surface integrin levels were 62% (n = 5, P = 0.006), 54% (n = 5, P = 0.045), and 38% (n = 5, P = 0.009) greater in diabetic versus nondiabetic animals, respectively. Seventy-five percent more neutrophils from diabetic versus nondiabetic animals adhered to rat endothelial cell monolayers (n = 6, P = 0.02). Pretreatment of leukocytes with either anti-CD11b or anti-CD18 antibodies lowered the proportion of adherent diabetic neutrophils by 41% (n = 6, P = 0.01 for each treatment), whereas anti-CD11a antibodies had no significant effect (n = 6, P = 0.5). In vivo, systemic administration of anti-CD18 F(ab')2 fragments decreased diabetic retinal leukostasis by 62% (n = 5, P = 0.001)., Conclusions: Neutrophils from diabetic animals exhibit higher levels of surface integrin expression and integrin-mediated adhesion. In vivo, CD18 blockade significantly decreases leukostasis in the diabetic retinal microvasculature. Integrin adhesion molecules may serve as therapeutic targets for the treatment and/or prevention of early diabetic retinopathy.

Objective: To determine if anti-vascular endothelial growth factor antibody and a range of dextrans with varying diffusion radii and molecular weights are permeable through experimental choroidal neovascularization (CNV)., Methods: Choroidal neovascularization was induced in 10 cynomolgus monkey retinas by means of argon laser injury. Digital fundus fluorescein angiograms were performed with fluorescein sodium, fluoresceinated IgG antibodies (anti-vascular endothelial growth factor and a control antibody), and fluoresceinated dextrans with molecular weights of 4, 20, 40, 70 and 150 kd. The 40- and 70-kd dextrans straddle the effective diffusion radius of IgG. For each reagent, early and late angiograms were performed in a standardized fashion, with follow-up images obtained to monitor residual fluorescence., Results: Perfusion of retinal vessels and choroidal vasculature was seen with all reagents. Fluorescein and 4- and 20-kd dextran leaked rapidly from the CNV within the first minute. Angiography with the use of 40-kd dextran and fluoresceinated antibody, either anti-vascular endothelial growth factor or control IgG, showed fluorescence within the CNV that increased during the first 1 to 5 hours, with mild leakage from the CNV. By 24 hours, fluorescence in the CNV was minimal, although in some cases persistent fluorescence in the surrounding tissue was evident up to 2 weeks. The 70-kd dextran showed fluorescence within the CNV and leakage in 1 of 3 eyes. The 150-kd dextran showed fluorescence within the CNV but did not demonstrate leakage., Conclusions: Fluoresceinated antibodies and dextran with smaller effective diffusion radii showed CNV perfusion and leakage. Dextrans with larger effective diffusion radii (70 kd and 150 kd) perfused into CNV but did not show leakage consistently., Clinical Relevance: Determining the permeablity of antibodies and molecules of similar size through CNV can help ascertain the feasibility of using intravenously administered antibodies against angiogenic growth factors as a future treatment for choroidal neovascularization.

Purpose: To describe two cases of de novo development of corneal guttae and Fuchs' dystrophy in donor tissue following penetrating keratoplasty (PK) for unrelated conditions., Methods: Two patients underwent PK for keratoconus and a disciform scar secondary to herpes simplex virus. They were followed clinically for a period of 16 and 11 years, respectively. Specular microscopy was used in one patient., Results: Corneal guttae were first noted 10 years and 4 years following transplantation in the first and second patient, respectively. In both cases, the corneal guttae gradually increased in number, involving the central and temporal portions of the corneal graft There were no corneal guttae present in the host corneal rim or contralateral cornea of either patient., Conclusions: These cases provide evidence to suggest that some corneas may be genetically predetermined to develop corneal guttae and Fuchs' dystrophy many years before any changes can be clinically detected.

Purpose: Clinical studies have demonstrated that intensive insulin therapy causes a transient worsening of retinopathy. The mechanisms underlying the initial insulin-induced deterioration of retinal status in patients with diabetes remain unknown. Vascular endothelial growth factor (VEGF) is known to be operative in the pathogenesis of diabetic retinopathy. The current study was conducted to characterize the effect of insulin on retinal VEGF gene expression in vitro and in vivo., Methods: The effect of insulin on VEGF expression in vivo was examined by in situ hybridization studies of rat retinal VEGF transcripts. To examine the mechanisms by which insulin regulates VEGF expression, human retinal pigment epithelial (RPE) cells were exposed to insulin, and VEGF mRNA levels were quantified with RNase protection assays (RPAs). Conditioned media from insulin-treated RPE cells were assayed for VEGF protein and capillary endothelial cell proliferation. The capacity of insulin to stimulate the VEGF promoter linked to a luciferase reporter gene was characterized in transient transfection assays., Results: Insulin increased VEGF mRNA levels in the ganglion, inner nuclear, and RPE cell layers. In vitro, insulin increased VEGF mRNA levels in human RPE cells and enhanced VEGF promoter activity without affecting transcript stability. Insulin treatment also increased VEGF protein levels in conditioned RPE cell media in a dose-dependent manner with a median effective concentration of 5 nM. The insulin-conditioned RPE cell media stimulated capillary endothelial cell proliferation, an effect that was completely blocked by anti-VEGF neutralizing antibody., Conclusions: Insulin increases VEGF mRNA and secreted protein levels in RPE cells through enhanced transcription of the VEGF gene. Intensive insulin therapy may cause a transient worsening of retinopathy in patients with diabetes through increased retinal VEGF gene expression.

Diabetic retinopathy is a leading cause of adult vision loss and blindness. Much of the retinal damage that characterizes the disease results from retinal vascular leakage and nonperfusion. This study shows that diabetic retinal vascular leakage and nonperfusion are temporally and spatially associated with retinal leukocyte stasis (leukostasis) in the rat model of streptozotocin-induced diabetes. Retinal leukostasis increases within days of developing diabetes and correlates with the increased expression of retinal intercellular adhesion molecule-1 (ICAM-1). ICAM-1 blockade with a mAb prevents diabetic retinal leukostasis and vascular leakage by 48.5% and 85.6%, respectively. These data identify the causal role of leukocytes in the pathogenesis of diabetic retinopathy and establish the potential utility of ICAM-1 inhibition as a therapeutic strategy for the prevention of diabetic retinopathy.