Sven-Erik Bursell

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.

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.

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.

Extracellular carbonic anhydrase mediates hemorrhagic retinal and cerebral vascular permeability through prekallikrein activation

Excessive retinal vascular permeability contributes to the pathogenesis of proliferative diabetic retinopathy and diabetic macular edema, leading causes of vision loss in working-age adults. Using mass spectroscopy–based proteomics, we detected 117 proteins in human vitreous and elevated levels of extracellular carbonic anhydrase-I (CA-I) in vitreous from individuals with diabetic retinopathy, suggesting that retinal hemorrhage and erythrocyte lysis contribute to the diabetic vitreous proteome. Intravitreous injection of CA-I in rats increased retinal vessel leakage and caused intraretinal edema. CA-I–induced alkalinization of vitreous increased kallikrein activity and its generation of factor XIIa, revealing a new pathway for contact system activation. CA-I–induced retinal edema was decreased by complement 1 inhibitor, neutralizing antibody to prekallikrein and bradykinin receptor antagonism. Subdural infusion of CA-I in rats induced cerebral vascular permeability, suggesting that extracellular CA-I could have broad relevance to neurovascular edema. Inhibition of extracellular CA-I and kallikrein-mediated innate inflammation could provide new therapeutic opportunities for the treatment of hemorrhage-induced retinal and cerebral edema.

Suppression of diabetic retinopathy with angiopoietin-1.

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

Normalization of Diacylglycerol-Protein Kinase C Activation by Vitamin E in Aorta of Diabetic Rats and Cultured Rat Smooth Muscle Cells Exposed to Elevated Glucose Levels

Hyperglycemia and diabetes have been shown to increase diacylglycerol (DAG) level and activate protein kinase C (PKC) activity in the vascular tissues, possibly altering vascular function. We have characterized the effects of D-α-tocopherol (vitamin E) on PKC activities and DAG levels in rat aortic smooth muscle cells (ASMCs) cultured with elevated glucose levels as well as in the vascular tissues obtained from control and diabetic rats. In ASMCs, the specific PKC activity from the membraneous fraction and total DAG level were increased by 31 ± 4% (P < 0.05) and 50 ± 7% (P < 0.05), respectively, when the glucose levels were changed from 5.5 to 22 mmol/1. The addition of D-α-tocopherol and another lipophilic antioxidant, probucol, prevented the glucose-stimulated increases in DAG level and PKC activity. By immunoblotting studies, D-α-tocopherol treatment was able to reduce the enhancement of PKC (ill isoform in the membraneous fraction isolated from ASMCs. Comparing streptozotocin-induced diabetic rats with their nondiabetic controls, both membraneous-specific PKC activities and total cellular DAG levels were increased in aorta by 162% (P < 0.05) and 60% (P < 0.05), respectively. Intraperitoneal injection of D-α-tocopherol (40 mg/kg) every other day prevented the increases in membraneous-specific PKC activities and total DAG levels in parallel with a significant increase of D-α-tocopherol contents in the aorta and plasma. These findings have demonstrated that D-α-tocopherol can prevent the activation of PKC activities in the vascular cells and tissues induced by hyperglycemia by lowering DAG levels, possibly via its antioxidant effect.

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.

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.

Evaluating Retinal Circulation Using Video Fluorescein Angiography in Control and Diabetic Rats

Video fluorescein angiography has been used to evaluate retinal circulatory parameters in diabetic and non-diabetic Sprague-Dawley rats. Video fluorescein angiograms were recorded from the retina using a modified retinal fundus camera following a 5 ul bolus injection of sodium fluorescein dye into the jugular vein. Retinal circulatory parameters were measured using computer assisted image analysis. These analyses were performed on 25 diabetic rats with 1 week duration of diabetes and 26 matched, non-diabetic, rats. There was a significant (p = .0001) increase in retinal Mean Circulation Time (MCT) in the diabetic group (1.83 +/- 0.40 s) compared to the control group (1.09 +/- 0.27 s). There were no significant differences in arterial or venous diameters comparing diabetic and control groups. In a separate paired experiment, measurements were made from the same animals both before and after one week duration of diabetes. A paired t-test analysis demonstrated significantly increased MCT times in the 6 diabetic animals (p = .001) while there was no significant differences detected in the 4 corresponding control animals. These results indicate that significant increases in retinal circulation times can be measured as early as 1 week after streptozotocin induced diabetes in this animal model.

Retinal Blood Flow in Diabetes

Objective: This is a review of work focused on characterizing retinal blood flow in diabetes. The review describes results on validation of the methodology for retinal blood flow measurements, the mechanisms of action of various factors that contribute to abnormalities in retinal blood flow in diabetic rodent models, and the translation of these results to clinical studies demonstrating the effectiveness of different therapeutic agents in normalizing retinal blood flow abnormalities in patients with diabetes.