concanavalin-a and Leukostasis

Retinal hemorrhages occur in a variety of sight-threatening conditions including ocular trauma, high altitude retinopathy, and chronic diseases such as diabetic and hypertensive retinopathies. The goal of this study is to investigate the effects of blood in the vitreous on retinal vascular function in rats.. Intravitreal injections of autologous blood, plasma kallikrein (PK), bradykinin, and collagenase were performed in Sprague-Dawley and Long-Evans rats. Retinal vascular permeability was measured using vitreous fluorophotometry and Evans blue dye permeation. Leukostasis was measured by fluorescein isothiocyanate-coupled concanavalin A lectin and acridine orange labeling. Retinal hemorrhage was examined on retinal flatmounts. Primary cultures of bovine retinal pericytes were cultured in the presence of 25 nM PK for 24 hours. The pericyte-conditioned medium was collected and the collagen proteome was analyzed by tandem mass spectrometry.. Intravitreal injection of autologous blood induced retinal vascular permeability and retinal leukostasis, and these responses were ameliorated by PK inhibition. Intravitreal injections of exogenous PK induced retinal vascular permeability, leukostasis, and retinal hemorrhage. Proteomic analyses showed that PK increased collagen degradation in pericyte-conditioned medium and purified type IV collagen. Intravitreal injection of collagenase mimicked PK's effect on retinal hemorrhage.. Intraocular hemorrhage increases retinal vascular permeability and leukostasis, and these responses are mediated, in part, via PK. Intravitreal injections of either PK or collagenase, but not bradykinin, induce retinal hemorrhage in rats. PK exerts collagenase-like activity that may contribute to blood-retinal barrier dysfunction.

Topics: Animals; Blood; Blood-Retinal Barrier; Bradykinin; Capillary Permeability; Cattle; Cells, Cultured; Collagenases; Concanavalin A; Evans Blue; Fluorescein-5-isothiocyanate; Fluorophotometry; Intravitreal Injections; Leukostasis; Male; Pericytes; Plasma Kallikrein; Rats; Rats, Long-Evans; Rats, Sprague-Dawley; Retinal Diseases; Retinal Hemorrhage; Retinal Vessels; Tandem Mass Spectrometry; Vitreous Body

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