Ultrastructural alterations in capillaries of the diabetic hypertensive rat retina: protective effects of ACE inhibition

Aims/hypothesis: The ACE inhibitor cilazapril was administered to diabetic hypertensive rats to evaluate its ability to influence the development of retinal capillary alterations. Methods: Normotensive (strain: Wistar Kyoto) and genetically hypertensive (strain: spontaneously hypertensive) rats were rendered diabetic by intravenous injections of streptozotocin. Half of the diabetic animals received cilazapril with their daily food. At 20 weeks of diabetes, endothelial cells, pericytes and extracellular matrix were assessed by ultrastructural morphometry. Each experimental group consisted of seven animals. Results: Cilazapril normalised systolic arterial pressure in diabetic hypertensive rats (137±2mmHg compared with 188±16mmHg in non-medicated diabetic hypertensive rats, p<0.001). The number of endothelial intercellular junctions was reduced in untreated diabetic hypertensive rats (0.15±0.05, p<0.02, vs 0.47±0.20 in non-diabetic normotensive rats). In diabetic hypertensive animals treated with cilazapril, this loss was attenuated (0.32±0.16, p<0.05). The significant thickening of the basement membrane observed in the diabetic normotensive (132.8±19.4nm) and diabetic hypertensive (150.3±20.2nm) groups was decreased by cilazapril in the diabetic hypertensive group (116.7±11.0nm, p<0.01), but was unaffected in the normotensive (131.9±17.3nm) group. No protective effect of the drug was observed in either group on pericytes. Conclusions/interpretation: Long-term administration of an effective antihypertensive therapy normalises endothelial alterations and basement membrane thickness in diabetic hypertensive conditions, and thus may account for the well-known improvement of the blood-retinal barrier observed during antihypertensive treatmen

Isolation of plasma membrane domains from murine T lymphocytes.

Murine T-lymphoma cells have been homogenized in dense sucrose solution and centrifuged under isopycnic conditions for membrane components. Floating fractions banding between 10% and 22.5% sucrose ("light" membranes) and between 22.5% and 35% sucrose ("heavy" membranes) were shown to consist of smooth membrane vesicles. The amounts of cholesterol and phospholipids recovered after chloroform/methanol extraction were similar in both fractions, but heavy membranes contained two to three times more protein than light membranes. The most striking difference between the two membrane fractions was revealed by their labeled surface glycoprotein patterns on polyacrylamide gels, suggesting that (i) the smooth membrane vesicles originated from the plasma membrane and (ii) two distinct segments of the plasma membrane can be recovered in fractions characterized by specific surface glycoproteins. Light membranes were enriched in Thy-1 antigen, whereas Ly-5 antigen and a 170,000-dalton surface glycoprotein were recovered almost exclusively from heavy membranes, as were metabolically labeled protein spots comigrating with the H-2k/d antigen in two-dimensional electrophoresis. The patterns of the unlabeled proteins in light and heavy membranes appeared similar, except for polypeptides of 180,000 and 85,000 daltons that were found preferentially in heavy membranes. These results support the concept of plasma membrane domains by showing that two distinct populations of plasma membrane vesicles can be isolated and that these populations contain different sets of cell surface glycoproteins

The rationale of retinal endovascular fibrinolysis in the treatment of retinal vein occlusion : from experimental data to clinical application.

PURPOSE: : We describe a retinal endovascular fibrinolysis technique to directly reperfuse experimentally occluded retinal veins using a simple micropipette. METHODS: : Retinal vein occlusion was photochemically induced in 12 eyes of 12 minipigs: after intravenous injection of 10% fluorescein (1-mL bolus), the targeted retinal vein segment was exposed to thrombin (50 units) and to Argon laser (100-200 mW) through a pars plana approach. A beveled micropipette with a 30-μm-diameter sharp edge was used for micropuncture of the occluded vein and endovascular microinjection of tissue plasminogen activator (50 μg/mL) in 11 eyes. In one control eye, balanced salt solution was injected. The lesion site was examined histologically. RESULTS: : Retinal vein occlusion was achieved in all cases. Endovascular microinjection of tissue plasminogen activator or balanced salt solution led to reperfusion of the occluded retinal vein in all cases. Indicative of successful reperfusion were the following: continuous endovascular flow, unaffected collateral circulation, no optic disk ischemia, and no venous wall bleeding. However, balanced salt solution injection was accompanied by thrombus formation at the punctured site, whereas no thrombus was observed with tissue plasminogen activator injection. CONCLUSION: : Retinal endovascular fibrinolysis constitutes an efficient method of micropuncture and reperfusion of an experimentally occluded retinal vein. Thrombus formation at the punctured site can be prevented by injection of tissue plasminogen activator

Effect of disruption of actin filaments by Clostridium botulinum C2 toxin on insulin secretion in HIT-T15 cells and pancreatic islets.

To examine their role in insulin secretion, actin filaments (AFs) were disrupted by Clostridium botulinum C2 toxin that ADP-ribosylates G-actin. Ribosylation also prevents polymerization of G-actin to F-actin and inhibits AF assembly by capping the fast-growing end of F-actin. Pretreatment of HIT-T15 cells with the toxin inhibited stimulated insulin secretion in a time- and dose-dependent manner. The toxin did not affect cellular insulin content or nonstimulated secretion. In static incubation, toxin treatment caused 45-50% inhibition of secretion induced by nutrients alone (10 mM glucose + 5 mM glutamine + 5 mM leucine) or combined with bombesin (phospholipase C-activator) and 20% reduction of that potentiated by forskolin (stimulator of adenylyl cyclase). In perifusion, the stimulated secretion during the first phase was marginally diminished, whereas the second phase was inhibited by approximately 80%. Pretreatment of HIT cells with wartmannin, a myosin light chain kinase inhibitor, caused a similar pattern of inhibition of the biphasic insulin release as C2 toxin. Nutrient metabolism and bombesin-evoked rise in cytosolic free Ca2+ were not affected by C2 toxin, indicating that nutrient recognition and the coupling between receptor activation and second messenger generation was not changed. In the toxin-treated cells, the AF web beneath the plasma membrane and the diffuse cytoplasmic F-actin fibers disappeared, as shown both by staining with an antibody against G- and F-actin and by staining F-actin with fluorescent phallacidin. C2 toxin dose-dependently reduced cellular F-actin content. Stimulation of insulin secretion was not associated with changes in F-actin content and organization. Treatment of cells with cytochalasin E and B, which shorten AFs, inhibited the stimulated insulin release by 30-50% although differing in their effects on F-actin content. In contrast to HIT-T15 cells, insulin secretion was potentiated in isolated rat islets after disruption of microfilaments with C2 toxin, most notably during the first phase. This effect was, however, diminished, and the second phase became slightly inhibited when the islets were degranulated. These results indicate an important role for AFs in insulin secretion. In the poorly granulated HIT-T15 cells actin-myosin interactions may participate in the recruitment of secretory granules to the releasable pool. In native islet beta-cells the predominant function of AFs appears to be the limitation of the access of granules to the plasma membrane

Effect of disruption of actin filaments by Clostridium botulinum C2 toxin on insulin secretion in HIT-T15 cells and pancreatic islets.

To examine their role in insulin secretion, actin filaments (AFs) were disrupted by Clostridium botulinum C2 toxin that ADP-ribosylates G-actin. Ribosylation also prevents polymerization of G-actin to F-actin and inhibits AF assembly by capping the fast-growing end of F-actin. Pretreatment of HIT-T15 cells with the toxin inhibited stimulated insulin secretion in a time- and dose-dependent manner. The toxin did not affect cellular insulin content or nonstimulated secretion. In static incubation, toxin treatment caused 45-50% inhibition of secretion induced by nutrients alone (10 mM glucose + 5 mM glutamine + 5 mM leucine) or combined with bombesin (phospholipase C-activator) and 20% reduction of that potentiated by forskolin (stimulator of adenylyl cyclase). In perifusion, the stimulated secretion during the first phase was marginally diminished, whereas the second phase was inhibited by approximately 80%. Pretreatment of HIT cells with wartmannin, a myosin light chain kinase inhibitor, caused a similar pattern of inhibition of the biphasic insulin release as C2 toxin. Nutrient metabolism and bombesin-evoked rise in cytosolic free Ca2+ were not affected by C2 toxin, indicating that nutrient recognition and the coupling between receptor activation and second messenger generation was not changed. In the toxin-treated cells, the AF web beneath the plasma membrane and the diffuse cytoplasmic F-actin fibers disappeared, as shown both by staining with an antibody against G- and F-actin and by staining F-actin with fluorescent phallacidin. C2 toxin dose-dependently reduced cellular F-actin content. Stimulation of insulin secretion was not associated with changes in F-actin content and organization. Treatment of cells with cytochalasin E and B, which shorten AFs, inhibited the stimulated insulin release by 30-50% although differing in their effects on F-actin content. In contrast to HIT-T15 cells, insulin secretion was potentiated in isolated rat islets after disruption of microfilaments with C2 toxin, most notably during the first phase. This effect was, however, diminished, and the second phase became slightly inhibited when the islets were degranulated. These results indicate an important role for AFs in insulin secretion. In the poorly granulated HIT-T15 cells actin-myosin interactions may participate in the recruitment of secretory granules to the releasable pool. In native islet beta-cells the predominant function of AFs appears to be the limitation of the access of granules to the plasma membrane