concanavalin-a and Diabetic-Retinopathy

To study if the endogenous renin-angiotensin system affects diabetic retinal leukostasis, rats with streptozotocin-induced diabetes were treated with an ACE inhibitor (ramipril), an angiotensin II AT(1) receptor antagonist (losartan) and the Ca channel blocker, (nifedipine). In the diabetic rats, these drug treatments reduced systolic blood pressure by approximately 16 mmHg but did not change blood glucose. After 2 weeks, the rats were examined for retinal leukostasis in vivo with a scanning laser ophthalmoscope (SLO). Retinal leukostasis, which was defined as no movement of arrested leukocytes over 2 min, was markedly higher in diabetic rats than normal controls (P<0.01). Leukostasis was significantly decreased by ramipril and losartan (P<0.01 vs. untreated diabetic rats) but was still higher than normal. Retinal leukostasis after nifedipine treatment was not significantly different than in untreated diabetic rats. The same trend was observed when leukostasis was analyzed on retinal flat mounts with concanavalin A and CD45 immunofluorescence; ramipril and losartan treatment, however, decreased leukostasis to values no different than controls. Retinal leukostasis was lowered by nifedipine (P<0.05, untreated diabetes vs. nifedipine-treated) but was still higher than in normal, ramipril-, or losartan-treated rats. Assays of gene expression of retinal intercellular adhesion molecule (ICAM-1) by semi-quantitative RT-PCR indicated that ICAM-1 mRNA was increased in diabetic rats but was decreased markedly by treatment with losartan or ramipril, and modestly by nifedipine. In summary, suppressing the activity of the endogenous renin-angiotensin system markedly decreases, perhaps even normalizes, the retinal leukostasis that accompanies type I diabetes in rats. These effects seem to be partly independent of blood pressure and to be associated with a decrease in ICAM-1 gene expression. Angiotensin II may, thus, mediate retinal leukostasis in early diabetes.

Topics: Angiotensin II; Angiotensin II Type 1 Receptor Blockers; Angiotensin-Converting Enzyme Inhibitors; Animals; Blood Glucose; Blood Pressure; Calcium Channel Blockers; Concanavalin A; Diabetes Mellitus, Experimental; Diabetic Retinopathy; Intercellular Adhesion Molecule-1; Leukocyte Common Antigens; Leukostasis; Losartan; Male; Nifedipine; Protein Tyrosine Phosphatase, Non-Receptor Type 1; Ramipril; Rats; Rats, Long-Evans; Retina; Retinal Diseases

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

Topics: Animals; Blood-Retinal Barrier; Blotting, Western; Capillary Permeability; Concanavalin A; Diabetes Mellitus, Experimental; Diabetic Retinopathy; Endothelial Growth Factors; Enzyme-Linked Immunosorbent Assay; Fluorescein-5-isothiocyanate; Immunohistochemistry; Injections; Intercellular Adhesion Molecule-1; Intercellular Signaling Peptides and Proteins; Leukocyte Common Antigens; Leukostasis; Lymphokines; Oligonucleotides; Protein Isoforms; Rats; Rats, Long-Evans; Retinal Vessels; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors; Vitreous Body

Diabetic retinopathy increases the permeability of the blood-retinal barrier, but the specific vessels that become permeable have not been identified. Both transcellular and paracellular pathways of vascular solute flux have been proposed. This study was conducted to test the hypothesis that paracellular flux contributes to increased retinal vascular permeability after VEGF treatment or diabetes, and to map the types of vessels that became permeable.. Regions of paracellular flux were identified by perfusion with fluorescent concanavalin A (ConA). Rats were injected intravitreally with VEGF or made diabetic with streptozotocin (STZ). After specified times, the rats were perfused with fixative followed by ConA, which binds to the basement membrane but not the luminal surface of endothelial cells. With this approach, ConA labels only blood vessels with paracellular permeability. Retinas were also labeled by immunofluorescence for the tight junction proteins occludin and claudin-5 and examined by confocal microscopy.. ConA labeling increased in the superficial arterioles and postcapillary venules, 2 weeks after the onset of diabetes. After 1 month, ConA labeling dramatically increased and extended to the capillaries of the outer plexiform layer. There was an inverse relationship between occludin immunoreactivity and ConA binding, but no change in claudin-5 immunoreactivity was detected. Injection of VEGF gave similar results.. Diabetes and VEGF increase paracellular vascular permeability in the retina, associated with redistribution of occludin. This permeability begins in the superficial arterioles and postcapillary venules and progresses to the capillary bed.

Topics: Animals; Blood-Retinal Barrier; Capillary Permeability; Claudin-5; Concanavalin A; Diabetes Mellitus, Experimental; Diabetic Retinopathy; Fluorescein-5-isothiocyanate; Immunohistochemistry; Injections; Male; Membrane Proteins; Microscopy, Confocal; Microscopy, Fluorescence; Occludin; Rats; Rats, Sprague-Dawley; Retinal Vessels; Vascular Endothelial Growth Factor A; Vitreous Body

Immune responses to normal retinal proteins, including S-antigen, have been demonstrated in patients with a variety of retinal disorders, as well as in those who have received panretinal laser photocoagulation. T-cell lymphocytes (T cells) have been implicated in the pathogenesis of several ocular inflammatory diseases of possible autoimmune etiology. We used synthetic peptides that correspond to the amino acid sequence of S-antigen in lymphocyte proliferation assays to identify specific sites in the molecule recognized by human T cells. Ten patients with type II diabetes were studied before and after initial panretinal laser photocoagulation for proliferative diabetic retinopathy. T-cell responses, expressed as a stimulation index, to S-antigen and peptides were negative in all patients before treatment. Three weeks after panretinal laser photocoagulation, eight of 10 assays were positive (stimulation index greater than 2; P less than .01) when lymphocytes were stimulated with peptide BSA(273-292); six of nine were positive (P less than .01) with peptide BSA(303-332); and six of six were positive (P less than .001) with peptide BSA(343-362). Our study identifies several specific sites in S-antigen that elicit human immune responses. The implications of these findings with regard to the pathogenesis and treatment of autoimmune uveitis are discussed.

Topics: Amino Acid Sequence; Antigens; Arrestin; Binding Sites; Concanavalin A; Diabetic Retinopathy; Eye Proteins; Female; Humans; Light Coagulation; Lymphocyte Activation; Male; Middle Aged; Molecular Sequence Data; Peptides; T-Lymphocytes

The concentration and degree of glycosylation of serum ferritin was evaluated in type I male diabetic patients at different levels of glycaemic control. Serum ferritin did not appear to be affected by hyperglycaemia, but some patients undergoing photocoagulation had abnormally high levels of serum ferritin. The glycosylated, (concanavalin A binding), proportion of serum ferritin was essentially the same in the control and diabetic groups. The finding that hyperglycaemia does not affect the degree of enzymatic glycosylation of this serum protein is discussed.

Topics: Adolescent; Adult; Carbohydrates; Concanavalin A; Diabetes Mellitus, Type 1; Diabetic Retinopathy; Ferritins; Glycated Hemoglobin; Humans; Male; Middle Aged; Phototherapy