Influence of diabetes and diabetes type on anatomic and visual outcomes following central rein vein occlusion.

PURPOSE: To determine the influence of diabetes and diabetes type on ocular outcomes following central retinal vein occlusion (CRVO). METHODS: Retrospective chart review of all patients evaluated over a 4-year period in a tertiary diabetes eye care center. Ophthalmic findings were recorded including visual acuity and the presence of retinal neovascularization at presentation, after 3-6 months, and at last follow-up. RESULTS: The records of 19,648 patients (13,571 diabetic; 6077 nondiabetic) were reviewed. The prevalence of CRVO in diabetic patients (N=72) and nondiabetic patients (N=27) were 0.5 and 0.4%, respectively. Disc neovascularization (21.3 vs 0.0%, P=0.05) and panretinal photocoagulation (PRP) (48.7 vs 21.4%, P=0.01) were more common in diabetic patients compared with nondiabetic patients. Compared with type 2 diabetic patients, retinal neovascularization (28.6 vs 3.7%, P=0.004) and subsequent PRP (78.6 vs 41.9%, P=0.01) were more likely in type 1 patients. Optic nerve head collateral vessels (CVs) were observed less than half as often (21.4 vs 56.5%, P=0.04) in patients with type 1 diabetes. Presence of optic nerve head CVs at baseline was associated with less likelihood of PRP (14.3 vs 46.1%, P=0.03). CONCLUSIONS: In this cohort, the rates of CRVO in diabetic and nondiabetic patients were similar to previously published population-based studies. Following CRVO, diabetic patients had higher rates of disc neovascularization and were more likely to require subsequent PRP than nondiabetic patients. As compared with CRVO patients with type 2 diabetes, patients with type 1 diabetes and CRVO had worse anatomic outcomes with substantially increased risks of retinal neovascularization and PRP; however, final visual acuity outcomes were similar.

Regression activity that is naturally present in vitreous becomes ineffective as patients develop proliferative diabetic retinopathy.

AIMS/HYPOTHESIS: The realisation that targeting agents in the vitreous is an effective approach to treating patients with diabetic retinopathy (DR) has increased awareness that changes in the composition/bioactivity of the vitreous is a contributor to the pathogenesis of DR. The overall goal of this study was to test the hypothesis that the vitreous has regression activity, and that lysophosphatidic acid (LPA) contributes to such activity. LPA is a bioactive phospholipid present in many biological fluids, and has been recently appreciated for its ability to promote regression of blood vessels. METHODS: Vitreous-mediated regression was monitored on tubes organised from primary retinal endothelial cells or neovessels that sprouted from retinal explants. LPA was quantified radioenzymatically. RESULTS: Bovine and human vitreous promoted regression of retinal explant vessels and of tubes organised from primary retinal endothelial cells. LPA was a substantial component of this regression activity. Comparing the regression activities of vitreous from patients with different stages of DR revealed that, as patients developed proliferative diabetic retinopathy (PDR), vitreous lost its ability to promote regression, even though the amount of LPA did not change. The underlying mechanism was a PDR-vitreous-mediated insensitivity to LPA, which could be overcome pharmacologically. CONCLUSIONS/INTERPRETATION: Our findings suggest that a decline in the responsiveness to regression factors such as LPA, which are naturally present in the vitreous, contributes to the pathogenesis of PDR.

Possible contribution of hyalocytes to idiopathic epiretinal membrane formation and its contraction.

AIM: To address the cellular components and the contractile mechanisms of the idiopathic epiretinal membrane (ERM). METHODS: Ten surgically removed ERMs were fixed in 4% paraformaldehyde and analysed by whole-mount immunohistochemistry with anti-glial fibrillar acidic protein (GFAP) and alpha smooth-muscle actin (alphaSMA) antibodies. Type I collagen gel contraction assay, an established wound-healing assay in vitro, was performed using cultured bovine hyalocytes or normal human astrocytes (NHA) to evaluate the contractile property of the cells in the presence of tissue growth factor (TGF)-beta2. The expression of alphaSMA was also analysed by western blot analysis to examine myofibroblastic transdifferentiation of the cells. Vitreous-induced collagen gel contraction was also evaluated. RESULTS: All membranes were composed of alphaSMA immunopositive cells in contracted foci and GFAP immunopositive cells in the periphery. No apparent double positive cells were observed in any membranes examined. Cultured hyalocytes showed overexpression of alphaSMA and hypercontraction of collagen gels in response to TGF-beta2, while glial cells showed marginal change. The vitreous from ERM patients also caused overexpression of alphaSMA and hypercontraction of the gels embedding hyalocytes, which were almost completely inhibited in the presence of anti-TGF-beta2 neutralising antibody. CONCLUSIONS: Hyalocytes might be one of the critical components of ERM mediating its contractile property through the effect of TGF-beta2 in the vitreous fluid.

Sustained moderate visual loss as a predictive end point for visual loss in non-proliferative diabetic retinopathy.

PURPOSE: In PKC-DRS2, the efficacy of the oral PKC-beta inhibitor, ruboxistaurin 32 mg/day, was measured by the primary end point of sustained moderate visual loss (SMVL: a > or = 15 letter decrease from baseline on the ETDRS (Early Treatment Diabetic Retinopathy Study) chart sustained at least for the last 6 months of study participation). We now evaluate whether SMVL is more accurate than moderate visual loss (MVL: a single occurrence of a decrease from baseline of > or = 15 ETDRS letters) for predicting future visual loss. METHODS: Study eyes with moderately severe to very-severe non-proliferative diabetic retinopathy, best-corrected visual acuity of at least 45 letters on the ETDRS chart (approximately Snellen 20/125), and no prior pan retinal photocoagulation were evaluated in 506 patients (869 eyes) who completed 36 months of treatment. RESULTS: Sixty-five percentage (26/40) of study eyes with the onset of SMVL within 24 months of enrolment still had SMVL at study completion (36 months). In comparison, only 24% (30/126) with MVL within 24 months had SMVL at study completion. Analyses based on data from 6, 12, and 18 months of treatment were similar. CONCLUSIONS: SMVL is a more predictable measure of subsequent visual loss than is a single time point measure of MVL.

Relation of optical coherence tomography and unusual angiographic leakage patterns in central serous chorioretinopathy.

PURPOSE: To investigate the anatomic basis of atypical angiographic leaks in central serous chorioretinopathy (CSC) with optical coherence tomography (OCT). METHODS: Fluorescein angiography (FA) and OCT were performed in three eyes of three patients (two men, one woman) with CSC. The angiographic leaks were treated with transpupillary thermotherapy (TTT) in two patients with long-standing CSC. The investigations were repeated in the treated patients during follow-up visits. RESULTS: Clinically, all patients demonstrated typical CSC; the female patient had subretinal fibrin under the detachment. FA showed unusual leakage patterns and OCT revealed bridging tissue connecting the pigment epithelial detachment (PED) to the overlying detached retina in all patients. CSC resolved completely in the two patients who underwent TTT along with normalization of the OCT findings. In one patient re-evaluated before complete resolution of CSC, OCT showed a flattened PED with disappearance of the bridging tissue and persistent serous detachment. FA demonstrated conversion of the previously atypical leak into a classic 'smokestack' configuration. Over the next month, leakage resolved completely. CSC and the anatomical findings persisted in the untreated patient. CONCLUSION: OCT identified a potential anatomic basis for unusual angiographic leakage pattern in all three cases of CSC evaluated.

Randomized trial of peribulbar triamcinolone acetonide with and without focal photocoagulation for mild diabetic macular edema: a pilot study.

OBJECTIVE: To provide pilot data on the safety and efficacy of anterior and posterior sub-Tenon injections of triamcinolone either alone or in combination with focal photocoagulation in the treatment of mild diabetic macular edema (DME). DESIGN: Prospective, phase II, multicenter, randomized clinical trial. PARTICIPANTS: One hundred nine patients (129 eyes) with mild DME and visual acuity 20/40 or better. METHODS: The participants were assigned randomly to receive either focal photocoagulation (n = 38), a 20-mg anterior sub-Tenon injection of triamcinolone (n = 23), a 20-mg anterior sub-Tenon injection followed by focal photocoagulation after 4 weeks (n = 25), a 40-mg posterior sub-Tenon injection of triamcinolone (n = 21), or a 40-mg posterior sub-Tenon injection followed by focal photocoagulation after 4 weeks (n = 22). Follow-up visits were performed at 4, 8, 17, and 34 weeks. MAIN OUTCOME MEASURES: Change in visual acuity and retinal thickness measured with optical coherence tomography (OCT). RESULTS: At baseline, mean visual acuity in the study eyes was 20/25 and mean OCT central subfield thickness was 328 mum. Changes in retinal thickening and in visual acuity were not significantly different among the 5 groups at 34 weeks (P = 0.46 and P = 0.94, respectively). There was a suggestion of a greater proportion of eyes having a central subfield thickness less than 250 mum at 17 weeks when the peribulbar triamcinolone was combined with focal photocoagulation. Elevated intraocular pressure and ptosis were adverse effects attributable to the injections. CONCLUSIONS: In cases of DME with good visual acuity, peribulbar triamcinolone, with or without focal photocoagulation, is unlikely to be of substantial benefit. Based on these results, a phase III trial to evaluate the benefit of these treatments for mild DME is not warranted.

Growth factors and protein kinase C inhibitors as novel therapies for the medical management diabetic retinopathy.

Diabetic retinopathy is a leading cause of acquired visual loss. Current treatment modalities are not effective in all cases and may have side effects. Investigation of the biochemical basis of diabetic retinopathy suggests that future treatments may reverse or halt the progression of diabetic retinopathy, or actually prevent the development of diabetic retinopathy. Pharmacological manipulation of protein kinase C and various growth factors may form the basis of future treatments for diabetic retinopathy.

Systemic considerations in the management of diabetic retinopathy.

PURPOSE: To highlight the systemic factors which affect onset and/or progression of diabetic retinopathy (DR) and to emphasize the role and responsibilities of ophthalmologists and other eye care providers to ensure that appropriate systemic medical evaluation of the patient with diabetes is being pursued. DESIGN: Literature review of publications relevant to diabetic retinopathy, blood glucose control, diabetes mellitus type, hypertension, renal disease, elevated serum lipids, exercise, pregnancy, anticoagulation, thrombolysis, smoking, anemia and antioxidant ingestion. FINDINGS: Intensive blood glucose control and control of systemic hypertension reduce the risk of new onset DR and slow the progression of existing DR. Severe DR may be an indicator of renal disease while severe renal disease and its treatment can affect the progression of DR. Elevated serum lipids are associated with macular exudate and moderate visual loss. Certain types of excessive exercise in patients with advanced stages of retinopathy may aggravate vitreous hemorrhage. During pregnancy, DR should be monitored closely as transient progression of DR can occur. Therapeutic anticoagulation and thrombolysis are not contraindicated at any stage of DR. Anemia can result in progression of DR, smoking in general should be discouraged, and the role of antioxidant therapy requires further study. CONCLUSIONS: Blindness from diabetic retinopathy is now largely preventable with timely detection and appropriate interventional therapy. Routine, repetitive, lifelong, expert clinical retinal examination is essential for the fundamental ophthalmic care of the patient with diabetes. However, diabetes mellitus is a systemic disease and thus optimal ophthalmic care must include diligent evaluation and treatment of concomitant systemic disorders that influence the development, progression and ultimate outcome of diabetic retinopathy. Optimization of these systemic considerations through an intensive, multi-disciplinary, healthcare team-based approach will maximize the ophthalmic and general health of these patients. Ophthalmologists and other eye care providers are critical members of this team with unique responsibilities to ensure that appropriate systemic medical evaluation is being pursued.

Circulating plasma vascular endothelial growth factor and microvascular complications of type 1 diabetes mellitus: the influence of ACE inhibition.

AIMS: To determine whether circulating plasma vascular endothelial growth factor (VEGF) is elevated in the presence of diabetic microvascular complications, and whether the impact of angiotensin-converting enzyme (ACE) inhibitors on these complications can be accounted for by changes in circulating VEGF. METHODS: Samples (299/354 of those with retinal photographs) from the EUCLID placebo-controlled clinical trial of the ACE inhibitor lisinopril in mainly normoalbuminuric non-hypertensive Type 1 diabetic patients were used. Albumin excretion rate (AER) was measured 6 monthly. Geometric mean VEGF levels by baseline retinopathy status, change in retinopathy over 2 years, and by treatment with lisinopril were calculated. RESULTS: No significant correlation was observed between VEGF at baseline and age, diabetes duration, glycaemic control, blood pressure, smoking, fibrinogen and von Willebrand factor. Mean VEGF concentration at baseline was 11.5 (95% confidence interval 6.0--27.9) pg/ml in those without retinopathy, 12.9 (6.0--38.9) pg/ml in those with non-proliferative retinopathy, and 16.1 (8.1--33.5) pg/ml in those with proliferative retinopathy (P = 0.06 for trend). Baseline VEGF was 15.2 pg/ml in those who progressed by at least one level of retinopathy by 2 years compared to 11.8 pg/ml in those who did not (P = 0.3). VEGF levels were not altered by lisinopril treatment. Results were similar for AER. CONCLUSIONS: Circulating plasma VEGF concentration is not strongly correlated with risk factor status or microvascular disease in Type 1 diabetes, nor is it affected by ACE inhibition. Changes in circulating VEGF cannot account for the beneficial effect of ACE inhibition on retinopathy.

Cyclic stretch and hypertension induce retinal expression of vascular endothelial growth factor and vascular endothelial growth factor receptor-2: potential mechanisms for exacerbation of diabetic retinopathy by hypertension.

Systemic hypertension exacerbates diabetic retinopathy and other coexisting ocular disorders through mechanisms that remain largely unknown. Increased vascular permeability and intraocular neovascularization characterize these conditions and are complications primarily mediated by vascular endothelial growth factor (VEGF). Because systemic hypertension increases vascular stretch, we evaluated the expression of VEGF, VEGF-R2 (kinase insert domain-containing receptor [KDR]), and VEGF-R1 (fms-like tyrosine kinase [Flt]) in bovine retinal endothelial cells (BRECs) undergoing clinically relevant cyclic stretch and in spontaneously hypertensive rat (SHR) retina. A single exposure to 20% symmetric static stretch increased KDR mRNA expression 3.9 +/- 1.1-fold after 3 h (P = 0.002), with a gradual return to baseline within 9 h. In contrast, BRECs exposed to cardiac-profile cyclic stretch at 60 cpm continuously accumulated KDR mRNA in a transcriptionally mediated, time-dependent and stretch-magnitude-dependent manner. Exposure to 9% cyclic stretch increased KDR mRNA expression 8.7 +/- 2.9-fold (P = 0.011) after 9 h and KDR protein concentration 1.8 +/- 0.3-fold (P = 0.005) after 12 h. Stretched-induced VEGF responses were similar. Scatchard binding analysis demonstrated a 180 +/- 40% (P = 0.032) increase in high-affinity VEGF receptor number with no change in affinity. Cyclic stretch increased basal thymidine uptake 60 +/- 10% (P < 0.001) and VEGF-stimulated thymidine uptake by 2.6 +/- 0.2-fold (P = 0.005). VEGF-NAb reduced cyclic stretch-induced thymidine uptake by 65%. Stretched-induced KDR expression was not inhibited by AT1 receptor blockade using candesartan. Hypertension increased retinal KDR expression 67 +/- 42% (P < 0.05) in SHR rats compared with normotensive WKY control animals. When hypertension was reduced using captopril or candesartan, retinal KDR expression returned to baseline levels. VEGF reacted similarly, but Flt expression did not change. These data suggest a novel molecular mechanism that would account for the exacerbation of diabetic retinopathy by concomitant hypertension, and may partially explain the principal clinical manifestations of hypertensive retinopathy itself. Furthermore, these data imply that anti-VEGF therapies may prove therapeutically effective for hypertensive retinopathy and/or ameliorating the deleterious effects of coexistent hypertension on VEGF-associated disorders such as diabetic retinopathy.

Stereo nonmydriatic digital-video color retinal imaging compared with Early Treatment Diabetic Retinopathy Study seven standard field 35-mm stereo color photos for determining level of diabetic retinopathy.

OBJECTIVE: To evaluate the ability to determine clinical levels of diabetic retinopathy, timing of next appropriate retinal evaluation, and necessity of referral to ophthalmology specialists using stereoscopic nonmydriatic digital-video color retinal images as compared with Early Treatment Diabetic Retinopathy Study (ETDRS) seven standard field 35-mm stereoscopic color fundus photographs. DESIGN: Prospective, clinic-based, comparative instrument validation study. PARTICIPANTS: Fifty-four patients (108 eyes) with type 1 or type 2 diabetes mellitus selected after chart review from a single center to include the full spectrum of diabetic retinopathy. METHODS: Nonsimultaneous 45 degrees -field stereoscopic digital-video color images (JVN images) were obtained from three fields with the Joslin Vision Network (JVN) system before pupil dilation. Following pupil dilation, ETDRS seven standard field 35-mm stereoscopic color 30 degrees fundus photographs (ETDRS photos) were obtained. Joslin Vision Network images and ETDRS photos were graded on a lesion-by-lesion basis by two independent, masked readers to assess ETDRS clinical level of diabetic retinopathy. An independent ophthalmology retina specialist adjudicated interreader disagreements in a masked fashion. MAIN OUTCOME MEASURES: Determination of ETDRS clinical level of diabetic retinopathy, timing of next ophthalmic evaluation of diabetic retinopathy, and need for prompt referral to ophthalmology specialist. RESULTS: There was substantial agreement (kappa = 0.65) between the clinical level of diabetic retinopathy assessed from the undilated JVN images and the dilated ETDRS photos. Agreement was excellent (kappa = 0.87) for suggested referral to ophthalmology specialists for eye examinations. Comparison of individual lesions between the JVN images and the ETDRS photos and for interreader comparisons were comparable to the prior ETDRS study. CONCLUSIONS: Undilated digital-video images using the JVN system were comparable photographs for the determination of diabetic retinopathy level. The results validate the agreement between nonmydriatic JVN images and dilated ETDRS photographs and suggest that this digital technique may be an effective telemedicine tool for remotely determining the level of diabetic retinopathy, suggesting timing of next retinal evaluation and identifying the need for prompt referral to ophthalmology specialists. Thus, the JVN system would be an appropriate tool for facilitating increased access of diabetic patients into recommended eye evaluations, but should not be construed as a paradigm that would replace the need for comprehensive eye examinations.

von Willebrand factor and retinal circulation in early-stage retinopathy of type 1 diabetes.

OBJECTIVE: Although retinopathy is a common microvascular complication of type 1 diabetes, the mechanism for this complication is still unknown. Changes in retinal circulation have been noted before the development of overt retinal pathology. Because von Willebrand factor (vWF) is a marker for endothelial dysfunction and mediates platelet adhesion, we determined if there was an association between vWF and retinal circulation in the early stages of diabetic retinopathy. RESEARCH DESIGN AND METHODS: Twenty subjects (aged 32.4 +/- 7.8 years) with type 1 diabetes and minimal or no retinopathy were studied. The mean duration of diabetes was 4.7 +/- 2.6 years. Data were collected at baseline and after 4 months of 1,800 IU vitamin E therapy or placebo. Retinal circulation was evaluated by video fluorescein angiography. Plasma vWF antigen levels were measured by enzyme-linked immunosorbent assay and fibrinogen by the Clauss method. RESULTS: Retinal blood flow was negatively correlated with vWF levels (r = -0.44, P = 0.008), whereas retinal circulation time was positively correlated with vWF levels (r = 0.33, P = 0.048). Fibrinogen levels were not significantly associated with either retinal index. However, fibrinogen levels were positively associated with HbA1c levels (r = 0.34, P = 0.01), indicating an association between poor glycemic control and higher fibrinogen levels. CONCLUSIONS: Increased vWF was associated with a prolonged retinal circulation time and reduced retinal blood flow in early-stage retinopathy of type 1 diabetes. Reduced blood flow associated with increased vWF levels may promote stasis in the retinal circulation and lead to local hypoxemia. These changes might contribute to the microvascular complications of diabetes. Whether the vWF levels predict retinal complications deserves further investigation.

Vascular endothelial growth factor induces expression of connective tissue growth factor via KDR, Flt1, and phosphatidylinositol 3-kinase-akt-dependent pathways in retinal vascular cells.

Fibroblastic proliferation accompanies many angiogenesis-related retinal and systemic diseases. Since connective tissue growth factor (CTGF) is a potent mitogen for fibrosis, extracellular matrix production, and angiogenesis, we have studied the effects and mechanism by which vascular endothelial growth factor (VEGF) regulates CTGF gene expression in retinal capillary cells. In our study, VEGF increased CTGF mRNA levels in a time- and concentration-dependent manner in bovine retinal endothelial cells and pericytes, without the need of new protein synthesis and without altering mRNA stability. VEGF activated the tyrosine receptor phosphorylation of KDR and Flt1 and increased the binding of phosphatidylinositol 3-kinase (PI3-kinase) p85 subunit to KDR and Flt1, both of which could mediate CTGF gene induction. VEGF-induced CTGF expression was mediated primarily by PI3-kinase activation, whereas PKC and ERK pathways made only minimal contributions. Furthermore, overexpression of constitutive active Akt was sufficient to induce CTGF gene expression, and inhibition of Akt activation by overexpressing dominant negative mutant of Akt abolished the VEGF-induced CTGF expression. These data suggest that VEGF can increase CTGF gene expression in bovine retinal capillary cells via KDR or Flt receptors and the activation of PI3-kinase-Akt pathway independently of PKC or Ras-ERK pathway, possibly inducing the fibrosis observed in retinal neovascular diseases.

Role of vascular endothelial growth factor in diabetic vascular complications.

BACKGROUND: Much of the morbidity and mortality associated with diabetes mellitus predominantly reflects its deleterious effects on microcirculation and macrocirculation. During the past few years, rapid advancement has been made in our understanding of the mechanisms and molecules involved in the pathogenesis of diabetic microvasculopathy. This is particularly true with regard to retinal vascular disease and the role of the angiogenesis- and vasopermeability-inducing molecule, vascular endothelial growth factor (VEGF). METHODS: Biochemical studies in many relevant cell types have been performed. Effects of VEGF action and inhibition have been evaluated in animals. Interventions that block the biochemical pathways initiated by VEGF have been tested both in culture and in animals. Human clinical trials have begun. RESULTS: VEGF induces vascular endothelial cell proliferation, migration and vasopermeability in many cells and tissues. In vivo, VEGF has been identified as a primary initiator of proliferative diabetic retinopathy, and as a potential mediator of nonproliferative retinopathy. In addition, VEGF has been implicated in the development of neuropathy and nephropathy in the patient with diabetes. In patients with diabetes and coronary artery or peripheral vascular disease, VEGF may induce development of cardiac and limb vascular collateralization, respectively. Many biochemical processes mediating these actions have now been elucidated. CONCLUSIONS: VEGF appears to play a central role in mediating diabetic vasculopathy in many organs. Improved understanding of the molecular mechanisms underlying these processes has permitted development of novel therapeutic interventions, several of which are now in human clinical trials. These scientific advances and various implications for the future care of vasculopathy associated with diabetes will be discussed.

Retinal expression, regulation, and functional bioactivity of prostacyclin-stimulating factor.

Prostacyclin-stimulating factor (PSF) acts on vascular endothelial cells to stimulate the synthesis of the vasodilatory molecule prostacyclin (PGI2). We have examined the expression, regulation, and hemodynamic bioactivity of PSF both in whole retina and in cultured cells derived from this tissue. PSF was expressed in all retinal cell types examined in vitro, but immunohistochemical analysis revealed PSF mainly associated with retinal vessels. PSF expression was constitutive in retinal pericytes (RPCs) but could be modulated in bovine retinal capillary endothelial cells (RECs) by cell confluency, hypoxia, serum starvation, high glucose concentrations, or inversely by soluble factors present in early vs. late retinopathy, such as TGF-beta, VEGF, or bFGF. In addition, RPC-conditioned media dramatically increased REC PGI2 production, a response inhibited by blocking PSF with a specific antisense oligodeoxynucleotide (ODN). In vivo, PGI2 increased retinal blood flow (RBF) in control and diabetic animals. Furthermore, the early drop in RBF during the initial weeks after inducing diabetes in rats, as well as the later increase in RBF, both correlated with levels of retinal PSF. RBF also responded to treatment with RPC-conditioned media, and this effect could be partially blocked using the antisense PSF ODN. We conclude that PSF expressed by ocular cells can induce PGI2, retinal vascular dilation, and increased retinal blood flow, and that alterations in retinal PSF expression may explain the biphasic changes in RBF observed in diabetes.

Mechanisms of hepatocyte growth factor-induced retinal endothelial cell migration and growth.

PURPOSE: Hepatocyte growth factor (HGF), also called scatter factor, stimulates growth and motility in nonocular endothelial cells and smooth muscle cells through its receptor c-Met. Recent reports suggest that HGF is increased in the serum and vitreous of patients with proliferative diabetic retinopathy and that smooth muscle cells and retinal pigment epithelial cells secrete HGF in the eye. However, little is known about HGF's action in the retina. In this study, the activity, expression, and signaling pathways of HGF were investigated in bovine retinal microvascular endothelial cells (BRECs). METHODS: Mitogenic and motogeneic effects of HGF on BRECs were examined using cell counts, thymidine uptake, and migration assays. MAP kinase (MAPK) phosphorylation was examined by Western blot analysis. Protein kinase C (PKC), MAPK, and PI3 kinase involvement were evaluated using selective inhibitors and activity assays. Expression of HGF and c-Met was evaluated by reverse transcription-polymerase chain reaction. RESULTS: HGF and c-Met were both expressed in BRECs. HGF stimulated BREC growth in a time- and dose-dependent manner, observed at HGF concentrations of 5 ng/ml or more and maximal (410%) at 100 ng/ml (P < 0.001). HGF increased BREC migration in a dose-dependent manner with a maximal 3.4-fold increase at 50 ng/ml after 5 hours. HGF induced time- and dose-dependent MAPK phosphorylation, initially evident at 5 minutes (P < 0.001) or 5 ng/ml (P < 0.050) and maximal after 15 minutes (>80-fold, P < 0.001) or 50 ng/ml (>20-fold, P < 0.001), respectively. MAPK phosphorylation was maintained for more than 2 hours. This response was inhibited 31% by 0.1 microm wortmannin and 76% by 30 microm LY294002, another PI3 kinase inhibitor. The non-isoform-selective PKC inhibitor GFX inhibited HGF-induced MAPK phosphorylation by only 15% at 5 microm. Combined PKC and PI3 kinase inhibition was additive (P < 0.05). Cell migration was inhibited 30% by wortmannin (P < 0.01) and 32% by GFX (P < 0.05), and again the effect was additive (P < 0.001). HGF-induced BREC growth was suppressed by PI3 kinase, PKC, or MAPK inhibition (all P < 0.01). HGF (50 ng/ml) stimulated PI3 kinase activity 347% (P < 0.001) and PKC activity 37% (P < 0.05). HGF-induced MAPK phosphorylation and mitogenesis were not inhibited by vascular endothelial growth factor (VEGF)-neutralizing antibody. CONCLUSIONS: HGF and its receptor are expressed in BREC, and HGF stimulates both BREC growth and migration at concentrations observed in the human eye with diabetic retinopathy. HGF signaling appears to involve activation of both PKC and PI3 kinase, inducing MAPK phosphorylation that is critical for migration and growth. However, VEGF does not appear to mediate these initial HGF effects. These results indicate that HGF could have a significant role in mediating retinal endothelial cell proliferation and migration in diabetic retinopathy, and they begin to elucidate the signal transduction pathway by which this action may occur.

Vascular endothelial growth factor (VEGF)-induced retinal vascular permeability is mediated by intercellular adhesion molecule-1 (ICAM-1).

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.

Integrin-mediated neutrophil adhesion and retinal leukostasis in diabetes.

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.