Health-related behaviours in a remote Indigenous population with Type 2 diabetes: a Central Australian primary care survey in the Telehealth Eye and Associated Medical Services Network [TEAMSnet] project.

AIM: There is a wealth of data concerning the health behaviours of Indigenous Australians, but the health behaviours of Indigenous Australians with diabetes are not systematically documented. At the clinical level, understanding a person's health behaviours can help identify and address barriers to diabetes care and promote good clinical outcomes. METHODS: We used a novel survey tool to systematically collect health behaviour data on Smoking, Nutrition, Alcohol consumption, Physical activity and Emotional well-being (SNAPE) from Indigenous Australians with Type 2 diabetes in a remote primary care setting in Alice Springs. RESULTS: At least one of the five surveys in the SNAPE tool was completed by 210 participants: 30% male, mean age 52.6 years (range 22.9 - 87.4). Fifty per cent of men and 23% of women were current smokers (P < 0.001). None of the participants reported an adequate intake of vegetables. Only 9.6% reported an adequate fruit intake. Some 49% of men and 32% of women consumed alcohol in the past year (P = 0.022), and 46% of drinkers were considered high-risk or likely-dependent drinkers. On average, participants walked 10 min or more at a time 6.0 days a week and spent 4.8 h sitting on a weekday. Mean adapted Patient Health Questionnaire 9 score was 4.61, with 34% of participants having mild depressive symptoms and 11% having moderate-severe depressive symptoms. CONCLUSIONS: Our SNAPE survey tool results present a high-risk, disadvantaged Indigenous population with Type 2 diabetes. More resources will be needed to sustainably implement interventions with the goal of improving health behaviours and subsequent long-term health.

Evolving telehealth reimbursement in Australia.

Video-based consultation is the only telehealth service reimbursed by the Medicare Benefits Schedule in Australia, but the uptake of telehealth is still low and inconsistent. There is a clear need for the development of appropriate medical evidence to support implementation of telehealth services. With the ubiquitous use of mobile phones, mobile health becomes important in facilitating health services and impacting clinical outcomes anywhere.

Angiotensin AT(1) receptor antagonism normalizes retinal blood flow and acetylcholine-induced vasodilatation in normotensive diabetic rats.

AIMS/HYPOTHESIS: The renin angiotensin system is emerging as a potential therapeutic target for diabetic retinopathy. This study examines the effects of angiotensin-converting-enzyme inhibition by captopril and angiotensin AT(1) receptor antagonism using candesartan-cilexetil on retinal blood flow and acetylcholine-stimulated vasodilatation in normotensive diabetic rats. METHODS: Non-diabetic or streptozotocin-induced diabetic rats were treated for 2 weeks with captopril (100 mg/kg/day) or candesartan cilexetil (2 mg/kg/day). Retinal haemodynamics were measured using video fluorescein angiography. Effects of exogenous acetylcholine on retinal haemodynamics were examined following intravitreal injection. Total retinal diacylglycerol was labelled using diacylglycerol kinase, separated by thin-layer chromatography, and quantified using autoradiography. RESULTS: Diabetic rats had prolonged retinal mean circulation time and decreased retinal blood flow compared with non-diabetic rats. Treatment of diabetic rats with either captopril or candesartan blocked the development of these blood flow abnormalities. Intravitreal injection of acetylcholine (10(-5) mol/l) in non-diabetic rats increased retinal blood flow by 53.9+/-22.0% relative to baseline whereas this response to acetylcholine was blunted in diabetic rats (4.4+/-19.6%, p<0.001). Candesartan treatment of diabetic rats restored the acetylcholine-stimulated retinal blood flow response to 60.0+/-18.7% compared with a 56.2+20.1% response in candesartan-treated non-diabetic rats. Total retinal diacylglycerol levels were increased in diabetic rats (3.75+/-0.98 nmol/mg, p<0.05) compared with non-diabetic rats (2.13+/-0.25 nmol/mg) and candesartan-treatment of diabetic rats normalized diacylglycerol levels (2.10+/-0.25 nmol/mg, p<0.05). CONCLUSION/INTERPRETATION: This report provides evidence that angiotensin-converting enzyme inhibition and AT(1) receptor antagonism ameliorates retinal haemodynamic dysfunctions in normotensive diabetic rats.

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.

Leukocyte-mediated endothelial cell injury and death in the diabetic retina.

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.

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.

Endothelin-3 regulation of retinal hemodynamics in nondiabetic and diabetic rats.

PURPOSE: To investigate the mechanisms of action of endothelin (ET)-3 on the regulation of retinal hemodynamics in diabetic and nondiabetic rats. METHODS: Retinal blood flow changes were measured using video fluorescein angiography. Measurements were made before and after intravitreal injections of different ET-3 concentrations in nondiabetic rats and rats with streptozotocin (STZ)-induced diabetes. The effect of ET-3 on retinal blood flow was also investigated in nondiabetic rats after pretreatment with N:(G)-monomethyl-L-arginine (L-NMMA), a nitric oxide synthase (NOS) inhibitor; BQ-788, an ET receptor B (ETB) antagonist; and BQ-123, an ET receptor A (ETA) antagonist. Control animals were injected intravitreally with vehicle alone. RESULTS: In nondiabetic rats, ET-3 induced a dose-dependent rapid increase in retinal blood flow 2 minutes after intravitreal injection (maximal at 10(-)(8) M, P < 0. 01) followed 15 and 30 minutes after ET-3 injection by dose-dependent decreases in retinal blood flow (maximal effect at 10(-)(6) M, P < 0.05). The ET-3-stimulated retinal blood flow increase was inhibited by 10(-)(4) M BQ-788 (P < 0.01) and 10(-)(3) M L-NMMA (P < 0.05). The ET-3-stimulated decrease in retinal blood flow at later times (15 minutes) was inhibited (P < 0.03) by 10(-4) M BQ-123. In diabetic rats, baseline retinal blood flows were decreased compared with nondiabetic rats (P < 0.01), showed dose-dependent increases 2 minutes after ET-3 injection (P < 0.03), and at later times remained significantly increased (P < 0.05) in contrast to flows in nondiabetic rats. CONCLUSIONS: The ET-3-induced initial rapid retinal blood flow increase in nondiabetic rats is mediated by the ET-3/ETB and NOS action. The subsequent retinal blood flow decrease is mediated by ET-3/ETA action. Diabetic rats showed comparable ET-3-induced retinal blood flow increases indicating normal ET-3/ETB action. However, at later times, retinal blood flow remained increased, suggesting an abnormal ET-3/ETA action.

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.

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.

Can protein kinase C inhibition and vitamin E prevent the development of diabetic vascular complications?

Hyperglycemia causes vascular complications of diabetes possible by the activation of protein kinase C (PKC). We have provided substantial evidence that activation of PKC can lead to a whole host of vascular dysfunction in diabetes. The activation of PKC induced by hyperglycemia appears to be due to an increase in diacylglycerol (DAG) levels, a physiological activator of PKC. Studies involving cultural cells, animal models of diabetes and patients have shown that inhibition of PKC by specific PKC inhibitor was able to reverse many of the vascular dysfunctions in the retina, kidney and cardiovascular systems induced by either hyperglycemia or diabetes. In addition high doses of vitamin E were shown to decrease the level of DAG and PKC induced by diabetes or hyperglycemia. Thus animal and clinical studies have shown that high doses of vitamin E treatment can apparently reverse some of the changes in the retinal and renal vessels.

High-dose vitamin E supplementation normalizes retinal blood flow and creatinine clearance in patients with type 1 diabetes.

OBJECTIVE: To determine the effectiveness of vitamin E treatment in normalizing retinal blood flow and renal function in patients with <10 years of type 1 diabetes. RESEARCH DESIGN AND METHODS: An 8-month randomized double-masked placebo-controlled crossover trial evaluated 36 type 1 diabetic and 9 nondiabetic subjects. Subjects were randomly assigned to either 1,800 IU vitamin E/day or placebo for 4 months and followed, after treatment crossover, for a further 4 months. Retinal blood flow was measured using video fluorescein angiography, and renal function was assessed using normalized creatinine clearance from timed urine collections. RESULTS: After vitamin E treatment, serum levels of vitamin E were significantly elevated (P<0.01) in both type 1 diabetic and control patients. Hemoglobin A1c was not affected by vitamin E treatment. Diabetic patient baseline retinal blood flow (29.1+/-7.5 pixel2/s) was significantly (P = 0.030) decreased compared with that of nondiabetic subjects (35.2+/-7.2 pixel2/s). After vitamin E treatment, diabetic patient retinal blood flow (34.5+/-7.8 pixel2/s) was significantly increased (P<0.001) and was comparable with that of nondiabetic subjects. Additionally, vitamin E treatment significantly (P = 0.039) normalized elevated baseline creatinine clearance in diabetic patients. CONCLUSIONS: Oral vitamin E treatment appears to be effective in normalizing retinal hemodynamic abnormalities and improving renal function in type 1 diabetic patients of short disease duration without inducing a significant change in glycemic control. This suggests that vitamin E supplementation may provide an additional benefit in reducing the risks for developing diabetic retinopathy or nephropathy.

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.

Joslin Vision Network Validation Study: pilot image stabilization phase.

PURPOSE: This preliminary study is designed to evaluate the quality of initial images obtained using the Joslin Vision Network (JVN) image capture and retrieval system. METHODS: Digitized images of 18 patients (36 eyes) were obtained using Topcon Non-mydriatic Fundus Camera and various imaging protocols as described for each series of images. Level of retinopathy ranged from no diabetic retinopathy to proliferative diabetic retinopathy. Images were reviewed and evaluated in comparison to medical record notes and Early Treatment Diabetic Retinopathy Study (ETDRS) seven-standard field stereoscopic 35-mm retinal fundus photographs--when available--to determine the ability of JVN digitized images to allow appropriate diagnosis and clinical management. JVN images were also evaluated for presence of photographic artifacts and stereoscopic presentation. The various displays and relative value of each JVN display were also evaluated. RESULTS: Using combinations of JVN image displays, JVN images were shown to match diagnosed level of diabetic retinopathy as determined from record notes and/or ETDRS seven-standard field stereoscopic 35-mm retinal fundus photography. Viewing images with various combinations of JVN image displays delineates possible sources of artifacts in JVN images. Areas to guide future image capture and image reading are identified. CONCLUSIONS: The JVN image capture system and reading of images by certified readers should allow appropriate photodiagnostic information to support the JVN, the diabetes mellitus disease management telemedicine initiative of the Joslin Diabetes Center. A rigid, full-scale, masked study to compare the Joslin Vision Network to seven-standard field stereoscopic 35-mm retinal fundus photography should be pursued to validate the image-gathering and image-reading strategy to support the Joslin Vision Network.

Reversibility of retinal flow abnormalities is disease-duration dependent in diabetic rats.

Decreased retinal blood flow has been measured in streptozotocin (STZ)-induced diabetes of 1 week's duration, and primary insulin intervention was effective in maintaining normal retinal blood flow in diabetic rats. Retinal blood-flow abnormalities precede clinical diabetic retinopathy in both diabetic animals and patients. An important characteristic of diabetic retinopathy is the difficulty of reversibility once it has been established. Because altered retinal hemodynamics is a possible marker of early diabetic retinopathy, we investigated in this study whether retinal blood-flow changes in rats can be normalized by secondary insulin intervention following short and chronic periods of untreated STZ-induced diabetes. Subcutaneous insulin pumps were placed into diabetic rats for 1 week after 1 week of diabetes (2-week group) and after 3 weeks of diabetes (4-week group). Retinal circulatory parameters were determined using image analysis of video fluorescein angiogram recordings. For the 2-week group, retinal blood flow was significantly (P < 0.05) reduced in the untreated diabetic rats compared with nondiabetic and insulin-treated diabetic rats (80.6+/-29.2, 131.9+/-50.1, and 151.3+/-54.0 pixels2/s respectively). Retinal blood flow was also significantly (P < 0.05) reduced in the 4-week untreated diabetic rats compared with nondiabetic rats (95.7+/-22.2 vs. 125.7+/-29.5 pixels2/s). In contrast to the shorter-duration group, insulin treatment for 1 week after 3 weeks of diabetes did not totally normalize retinal blood flow (117.5+/-32.4 pixels2/s). These results suggest that vascular abnormalities could become more resistant to normalization following short-term (1 week) insulin treatment after longer periods of untreated diabetes.

Prevention of diabetes-induced abnormal retinal blood flow by treatment with d-alpha-tocopherol.

Hyperglycemia in diabetes mellitus has been shown to activate diacylglycerol (DAG)-protein kinase C (PKC) pathway in the vascular tissues, possibly altering vascular function. We have characterized the effects of vitamin E (d-alpha-tocopherol) on activation of PKC and DAG levels in retinal tissues of diabetic rats, and correlated its effects to retinal hemodynamics using video-based fluorescein angiography (VFA). Comparing streptozotocin-induced diabetic rats to controls, membranous PKC specific activities were increased by 71% (p < 0.05). Western blot analysis showed that the membranous PKC beta II isoform was significantly increased by 133 +/- 45% (p < 0.05). Intraperitoneal injection of d-alpha-tocopherol (40 mg/kg) every other day prevented the increases in membranous PKC specific activity and PKC beta II protein shown by immunoblots. Similar to PKC activities, total DAG levels were increased in the retina and were normalized by d-alpha-tocopherol treatment. Physiologically, abnormalities of retinal blood hemodynamics, as measured using VFA, which previously have been reported to be associated with increases of DAG and PKC levels in the diabetic rats, were prevented by d-alpha-tocopherol treatment in diabetic rats. The direct effect of d-alpha-tocopherol on total DAG and [3H]-palmitate incorporation into DAG were also examined using cultured bovine retinal endothelial cells (REC). Exposure of REC to 22 mM glucose for three days increased total DAG and [3H]-palmitate labeled DAG levels by 35 +/- 8% and 50 +/- 8%, respectively (p < 0.05). The presence of d-alpha-tocopherol (50 micrograms/ml) prevented the increase of both total DAG and [3H]-palmitate labeled DAG levels in cells exposed to 22 mM glucose. These findings suggested that the mechanism of the d-alpha-tocopherol's effect appears to be mediated by the normalization of the hyperglycemia-induced activation of the DAG-PKC pathway which leads to the normalization of abnormal retinal blood flow seen in diabetes mellitus.

Vascular endothelial growth factor and severity of nonproliferative diabetic retinopathy mediate retinal hemodynamics in vivo: a potential role for vascular endothelial growth factor in the progression of nonproliferative diabetic retinopathy.

PURPOSE: To determine the effect of vascular endothelial growth factor and retinopathy level on retinal hemodynamics in nondiabetic and diabetic rats and to evaluate retinal hemodynamics in nondiabetic and diabetic patients. METHODS: Forty-eight diabetic and 22 nondiabetic patients had their diabetic retinopathy levels determined from fundus photographs according to Early Treatment Diabetic Retinopathy Study (ETDRS). Fluorescein angiograms were recorded from the left eye by video fluorescein angiography. Retinal blood flow was calculated from the digitized angiograms. Human recombinant vascular endothelial growth factor or vehicle alone was injected intravitreally into 13 nondiabetic and 11 diabetic rats. RESULTS: Retinal blood flow decreased 33% in patients with ETDRS retinopathy level 10 compared with control patients (P = .001) and increased sequentially in more advanced stages of retinopathy, with a strong correlation between retinal blood flow and retinopathy level (r2 = 0.434, P = .001). In the diabetic rats, retinal blood flow was decreased 35.6% (P = .01). Vascular endothelial growth factor maximally increased retinal blood flow by 36.1% in nondiabetic rats after 25 minutes (P = .001) and by 73.7% in diabetic rats after only 5 minutes (P = .01) and caused a greater response in diabetic than in nondiabetic rats. CONCLUSIONS: Retinal blood flow increases with advancing nonproliferative diabetic retinopathy in humans, and diabetes accentuates the vascular endothelial growth factor-induced increase in retinal blood flow and venous dilation in rats. Vascular endothelial growth factor may contribute to the changes in retinal hemodynamics and morphology observed in early diabetic retinopathy.

Vascular endothelial growth factor-induced retinal permeability is mediated by protein kinase C in vivo and suppressed by an orally effective beta-isoform-selective inhibitor.

Increased vascular permeability and excessive neovascularization are the hallmarks of endothelial dysfunction, which can lead to diabetic macular edema and proliferative diabetic retinopathy in the eye. Vascular endothelial growth factor (VEGF) is an important mediator of ocular neovascularization and a known vasopermeability factor in nonocular tissues. In these studies, we demonstrate that intravitreal injection of VEGF rapidly activates protein kinase C (PKC) in the retina at concentrations observed clinically, inducing membrane translocation of PKC isoforms alpha, betaII, and delta and >threefold increases in retinal vasopermeability in vivo. The effect of VEGF on retinal vascular permeability appears to be mediated predominantly by the beta-isoform of PKC with >95% inhibition of VEGF-induced permeability by intravitreal or oral administration of a PKC beta-isoform-selective inhibitor that did not inhibit histamine-mediated effects. These studies represent the first direct demonstration that VEGF can increase intraocular vascular permeability through activation of PKC in vivo and suggest that oral pharmacological therapies involving PKC beta-isoform-selective inhibitors may prove efficacious for the treatment of VEGF-associated ocular disorders such as diabetic retinopathy.

Specific retinal diacylglycerol and protein kinase C beta isoform modulation mimics abnormal retinal hemodynamics in diabetic rats.

PURPOSE: Elevation of diacylglycerol (DAG) and protein kinase C (PKC) levels in diabetic vascular tissue is associated with abnormalities of retinal and renal hemodynamics. The object of this study was to determine whether direct elevation of retinal DAG levels, in the absence of diabetes or hyperglycemia, can mimic the hemodynamic abnormalities normally observed in diabetic rats. Retinal DAG levels were elevated using an inhibitor of DAG kinase that converts DAG to phosphatidic acid. The effectiveness of a specific PKC-beta isoform inhibitor introduced directly into the retinas of diabetic rats in reversing diabetes-related abnormal retinal hemodynamics was also investigated. METHODS: For retinal blood flow studies, diacylglycerol kinase (DGK) inhibitor R59949, at various concentrations, was injected into the vitreous of nondiabetic Sprague-Dawley rats (n = 33), and a PKC-beta isoform-selective inhibitor LY333531 was injected into the vitreous of rats with streptozotocin (STZ)-induced diabetes of 2 weeks' duration (n = 21). Retinal hemodynamic changes were quantitated using video-based fluorescein angiography. Total DAG levels were assayed from five nondiabetic rat retinas after DGK inhibition and retinal PKC activities were assayed from six diabetic rat retinas after PKC-beta inhibition. RESULTS: DGK inhibitor R59949 injected into the vitreous dose dependently increased the mean circulation time (MCT) and decreased retinal blood flow (EC50 = 10(-8) M). After 30 minutes, 10(-5) M R59949 induced a 1.7-fold increase in total retinal DAG levels, compared with the levels in vehicle-injected eyes, an increase in MCT from 0.87 +/- 0.05 seconds to 1.44 +/- 0.12 seconds (P < 0.01) and a decrease in retinal blood flow from 105.3 +/- 6.5 pixel2/second to 64.1 +/- 5 pixel2/second (P < 0.01). The effect of R59949 was sustained for 60 minutes after injection. These retinal hemodynamic parameters after DGK inhibition were comparable to those measured at baseline in rats with STZ-induced diabetes of 2 weeks' duration (MCT = 1.38 +/- 0.20 seconds; retinal blood flow = 68 +/- 11.2 pixel2/second). Intravitreal injection of the PKC-beta inhibitor (LY333531) at 10(-5) M in diabetic rats decreased by a factor of 1.6 the diabetes-related increased PKC activation, decreased the prolonged MCT (0.98 +/- 0.13 seconds; P < 0.01) and increased retinal blood flow (93.4 +/- 14.2 pixel2/second; P < 0.01). The measured retinal circulatory parameters after PKC inhibition in the retina were comparable to those measured at baseline in the nondiabetic rats. CONCLUSIONS: These results provide direct evidence that DAG elevation and subsequent PKC-beta isoform activation are the primary biochemical sequelae responsible for the development of the abnormal retinal hemodynamics observed in diabetic rats.