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.

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.

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.

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.

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.

Diabetic retinopathy.

INTRODUCTION: To provide a review of the current standard of care in diabetic eye management. METHODS: A non-systematic evidence-based review utilising available data and consensus statements on the subject matter. RESULTS: Diabetes mellitus affects some 9.0% of Singaporeans, and more than 60% of patients with diabetes in this population remained undiagnosed. Diabetic retinopathy is an important complication among diabetic patients and adverse visual outcome associated with this condition can be reduced by more than 95% by taking measures including good glycaemic control, timely and appropriate laser therapy, and vitrectomy surgery when indicated. An important aspect of management is the accurate disease classification which is essential for prognostication, appropriate follow-up schedule, and timing of therapeutic intervention purposes. CONCLUSION: Diabetic retinopathy will remain a significant problem if the current trend in diabetes among Singapore residents prevails. As the severe visual impairment associated with diabetic retinopathy can be largely prevented with appropriate and timely intervention, the major challenge to the health care providers today is the identification and education of patients with diabetes, and the enrollment of these patients in a life-long comprehensive ophthalmic management programme in order to minimise visual morbidity.

Vascular endothelial growth factor and diabetes: the agonist versus antagonist paradox.

Much of the morbidity and mortality associated with diabetes is primarily attributable to sequelae of microvascular and macrovascular disease. Over the past decade, dramatic progress has been achieved in elucidating the fundamental processes underlying the pathogenesis of these complications. Angiogenic factors in particular now appear to play a pivotal role in the development of microvascular complications as well as the response to macrovascular disease. Hyperglycemia, other growth factors, advanced glycation end products, oxidative stress, and ischemia can increase growth factor expression. In some microvascular tissues, the result is pathologic neovascularization and increased vascular permeability. These responses account for much of the visual loss associated with diabetic retinopathy and may, in addition, serve a significant role in nephropathy and neuropathy. In contrast, recent data suggest that vascular collateralization resulting from ischemia-induced growth factor release in tissues compromised by macrovascular disease may be important in reducing clinical symptoms and tissue damage. This angiogenic response, which may be beneficial in coronary artery and peripheral limb disease, appears to be reduced in patients with diabetes. Thus, two apparently diametrically opposed therapeutic paradigms are arising for the treatment of vascular complications in diabetes. Indeed, growth factor antagonists have been used successfully in diabetes-related animal models to block angiogenic and permeability complications in the retina and kidney. Conversely, growth factor agonists have been successfully used to stimulate collateral vessel formation and reduce ischemic symptoms from macrovascular disease in the coronary arteries and peripheral limbs. Both of these approaches are currently being evaluated in clinical trials for their respective indications. Thus, as these divergent therapeutic modalities begin to enter the clinical arena, this apparent paradox necessitates careful consideration of the potential risks, benefits, and interactions of the opposing regimens. Using vascular endothelial growth factor as a classic example of growth factor involvement, we discuss the current preclinical and clinical data supporting these approaches and the implications arising from the probable coexistence of these two therapeutic modalities.

Prevention of leukostasis and vascular leakage in streptozotocin-induced diabetic retinopathy via intercellular adhesion molecule-1 inhibition.

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.

Basic fibroblast growth factor induces expression of VEGF receptor KDR through a protein kinase C and p44/p42 mitogen-activated protein kinase-dependent pathway.

Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are angiogenic molecules whose combined mitogenic activity is potently synergistic. However, the molecular mechanism underlying this synergy is incompletely understood. We examined whether VEGF and bFGF affect expression of each other or alter expression of the VEGF receptor KDR in retinal capillary endothelial cells. In addition, we investigated the intracellular signaling mechanisms involved in this response. VEGF-induced [3H]thymidine uptake was tightly correlated with KDR mRNA and protein concentrations, suggesting that increased KDR expression might account for VEGF's synergistic activity in the presence of bFGF. bFGF (10 ng/ml) induced KDR mRNA expression within 4 h and attained a 4.0-fold increase after 24 h. KDR protein expression was increased 7.5-fold after 48 h. VEGF (= 50 ng/ml) did not alter bFGF, VEGF, or KDR mRNA expression under serum-deprived conditions. In contrast, VEGF increased KDR mRNA expression 87% under growth conditions and 2.9-fold under serum-deprived conditions in the presence of bFGF. The protein kinase C (PKC) agonist phorbol myristate acetate (PMA) induced KDR mRNA expression 5.1-fold at 100 nmol/l. bFGF increased p44/p42 mitogen-activated protein kinase (MAPK) phosphorylation within 5 min, reaching a maximum within 15 min and remaining significantly elevated for >6 h. bFGF-induced MAPK phosphorylation and KDR mRNA expression were almost completely inhibited by 5 micromol/l GFX, a non-isoform-selective PKC inhibitor. MAPK inhibitor PD98059 reduced KDR mRNA expression 72% at concentrations that inhibited bFGF-induced MAPK phosphorylation 100%, suggesting that pathways in addition to MAPK might also be involved. Inhibitors of the beta isoform of PKC (LY333531), protein kinase A (PKA) (H89), and phosphotidylinositol (PI) 3 kinase (wortmannin) had no significant effect. These data suggest that bFGF stimulates KDR expression through a PKC and p44/p42 MAPK-dependent pathway not primarily involving the beta isoform of PKC, PKA, or PI-3 kinase. Since bFGF induces VEGF expression and since increased KDR expression potentiates VEGF action, resulting in additional KDR expression and marked mitogenic activity, these data provide a novel mechanistic explanation for the angiogenic synergy between VEGF and bFGF.

Hypoxia induces vascular endothelial growth factor gene and protein expression in cultured rat islet cells.

The formation of new microvasculature by capillary sprouting at the site of islet transplantation is crucial for the long-term survival and function of the graft. Vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen with potent angiogenic and vascular permeability-inducing properties, may be a key factor in modulating the revascularization of islets after transplantation. In this study, we examined the gene expression of VEGF mRNA in three tumor cell lines and in isolated whole and dispersed rat islets in vitro by Northern blot hybridization in normoxic (5% CO2, 95% humidified air) and hypoxic (1% O2, 5% CO2, 94% N2) culture conditions. Increased expression of VEGF mRNA was observed in beta-TC3, RAW 264.7, and IC-21 tumor cell lines when subjected to hypoxia. With isolated whole islets in normoxic culture, a threefold increase in VEGF mRNA (P < 0.001) was seen at 48 h as compared with freshly isolated islets. This response was similar to the 3.8-fold increase observed with islets subjected to hypoxia. Dispersed rat islet cell clusters cultured on Matrigel for 24 h under hypoxic conditions showed a 3.4-fold increase (P < 0.01) in VEGF mRNA compared with those cultured in normoxia. This correlated with increased VEGF secretion as determined by enzyme-linked immunosorbent assay. Immunohistochemical studies revealed the presence of increased expression of VEGF protein near the center of islets after 24 h of normoxic culture. Islet cell clusters on Matrigel showed intense cellular localization of VEGF in both beta-cells and non-beta-cells. These findings suggest that rat islet cells, when subjected to hypoxia during the first few days after transplantation, may act as a major source of VEGF, thereby initiating revascularization and maintaining the vascular permeability of the grafted islets.

Hypoxia upregulates glucose transport activity through an adenosine-mediated increase of GLUT1 expression in retinal capillary endothelial cells.

Elevation of intracellular glucose within retinal vascular cells is believed to be an important causal factor in the development of diabetic retinopathy. The intracellular glucose concentration is regulated by both the rate of glucose metabolism and glucose transport. Because retinal hypoxia often precedes proliferative diabetic retinopathy, we have studied the regulation of the glucose transport system by hypoxia in cultured bovine retinal endothelial cells (BRECs). Because retinal ischemia is known to increase intracellular adenosine levels, which subsequently regulate hypoxia-inducible genes, such as vascular endothelial growth factor and erythropoietin, the role of adenosine and its receptor-mediated pathways has also been evaluated. Hypoxia (0.5% O2, 5% CO2, and 94.5% N2) stimulated GLUT1 mRNA expression in BRECs in a time-dependent manner with an 8.9 +/- 1.5-fold (P < 0.01) increase observed after 12 h. GLUT1 mRNA expression returned to baseline (1.4 +/- 0.3-fold of control) within 12 h after reinstitution of normoxia. N6-Cyclopentyl adenosine (adenosine A1 receptor agonist, Kd = 1 nmol/l) did not affect GLUT1 mRNA expression at concentrations up to 1 micromol/l, while 2-p-(2-carboxyethyl)-phenethyl-amino-5'-N-ethylcarboxamidoadenosine and 5'-(N-ethylcalboxamido)-adenosine (adenosine A2 receptor [A2R] agonists, Kd = 15 and 16 nmol/l, respectively) increased mRNA levels at concentrations as low as 10 nmol/l. Maximal stimulation was 2.3 +/- 0.2- and 2.1 +/- 0.2-fold, respectively (P < 0.01). The adenosine A2a receptor antagonist 8-(3-chlorostyryl)caffeine (CSC) (Kd = 100 nmol/l for A2R) inhibited hypoxia-stimulated GLUT1 mRNA expression by 40 +/- 8% at 100 nmo/l. Hypoxia upregulated GLUT1 protein expression by 3.0 +/- 0.3-fold after 12 h (P < 0.01), but this response was attenuated by CSC (P < 0.05). Hypoxia increased glucose transport activity by 2.1 +/- 0.3-fold (P < 0.001) after 12 h, a response inhibited 65% by CSC (P < 0.01). A protein kinase A (PKA) inhibitor (H89, 20 micromol/l) suppressed hypoxia-induced GLUT1 mRNA expression by 42 +/- 9% (P < 0.01). These data suggest that hypoxia in BRECs upregulates glucose transport activity through an increase of GLUT1 expression that is partially mediated by adenosine, A2R, and the cAMP-PKA pathway.

A specific increased expression of insulin receptor substrate 2 in pancreatic beta-cell lines is involved in mediating serum-stimulated beta-cell growth.

Certain nutrients and growth factors can stimulate pancreatic beta-cell growth. However, the appropriate mitogenic signaling pathways in beta-cells have been relatively undefined. In this study, differential gene expression in NEDH rat insulinoma was compared with NEDH rat primary islet beta-cells. Differential mRNA display analysis revealed an elevated expression in insulinoma of VL30 transposons, S24 ribosomal protein, and cytochrome-C oxidaseVIIc that is typical for cells undergoing mitosis. A gene candidate approach revealed that mRNA levels of the oncogenes c-fos and c-jun were equivalently expressed in insulinoma and islet cells, as was the mRNA for the mitogenic signal transduction molecule insulin receptor substrate (IRS)-1. However, in contrast to that of IRS-1, IRS-2 gene expression was 60- to 70-fold higher in the insulinoma tissue compared with islets, which was reflected at the protein as well as the mRNA level. The specific elevated IRS-2 expression was a consistent observation across all rodent pancreatic beta-cell lines. To investigate whether IRS-2 was functional, serum-stimulated beta-cell proliferation was examined in isolated insulinoma cells. After a 48-h period of serum withdrawal, 24 h of serum refeeding rendered an 8- to 10-fold increase in [3H]thymidine incorporation into insulinoma cells. This serum-stimulated DNA synthesis was prevented by inhibitors of tyrosine protein kinase and phosphatidylinositol (PI) 3-kinase activities, as well as the activation of mitogen-activated protein (MAP) kinase and p70S6K. Examination of IRS-mediated signal transduction pathways indicated that after 10-15 min of serum refeeding, there was increased tyrosine phosphorylation of IRS-2 and pp60, and PI 3-kinase recruitment to IRS-2. Serum also increased the association of growth factor-bound protein 2/murine sons of sevenless 1 protein to a PI 3-kinase/IRS-2 protein complex. Moreover, serum also activated MAP-kinase (erk-1 and erk-2 isoforms) and 70 kD S6 kinase. Thus IRS-mediated signal transduction pathways are functional in pancreatic beta-cells. It is conceivable that IRS-2 expression in beta-cells contributes to maintaining the islet beta-cell population, complementary to observations in the IRS-2 knockout mouse in which beta-cell mass is markedly reduced.