Morpho-functional studies of the blood-brain barrier in streptozotocin-induced diabetic rats.

AIMS/HYPOTHESIS: We undertook the characterization of the capillary bed of the rat frontal cortex and their permeability properties in short-term and long-term diabetic rats. METHODS: Diabetes was induced by strepozotocin injection. Rats were maintained hyperglycaemic without insulin treatment during 4 to 5 months (short-term) and 8 to 13 months (long-term). Rats from an additional short-term hyperglycaemic group received an injection of exogenous dinitrophenylated albumin 15 min before being killed. Tissues were processed for electron microscopy and quantitative immunocytochemistry. Endogenous and dinitrophenylated exogenous albumin were revealed with high resolution over the capillary wall using specific antibodies and the protein A-gold complex. Morphometrical analyses were carried out. RESULTS: Albumin is transported across endothelial cells by plasmalemmal vesicles or caveolae and larger vacuolar structures. This transport increased in diabetic rats by an increment in the number of vesicles. Albumin distribution across the capillary basement membrane showed that the restrictive properties of the basement membrane present in normoglycaemic rats are altered in the diabetic condition, as was its thickness. Similar alterations of the basement membrane structure and function were encountered in old normoglycaemic rats but to a lesser extent. CONCLUSION/INTERPRETATION: The results indicate that diabetes seems to accelerate the ageing process of the vascular wall and that the central nervous system capillary bed is also a target for diabetic microangiopathy.

Actin and annexins I and II are among the main endothelial plasmalemma-associated proteins forming early glucose adducts in experimental diabetes.

An immunochemical and biochemical study was performed to reveal which of the endothelial plasma membrane proteins become glycated during the early phases of diabetes. The blood front of the lung microvascular endothelial plasmalemma was purified by the cationic colloidal silica method from normal and diabetic (streptozotocin-induced) rats and comparatively analyzed by two-dimensional electrophoresis. No major qualitative differences in the general spectrum of endothelial plasmalemmal proteins were recorded between normoglycemic and hyperglycemic animals. By probing with anti-glucitollysine antibodies, we found that at 1 month after the onset of diabetes, several endothelial membrane polypeptides contained glucose covalently linked to their lysyl residues. Ten days of insulin treatment restored euglycemia in the diabetic animals and completely abolished the membrane nonenzymatic glycosylation. All the glycated polypeptides of the endothelial plasma membrane belong to the peripheral type and are associated with its cytoplasmic face (cell cortex). They were solubilized by buffers of high pH and were not detected in the lung cytosolic fraction (100,000 g). By microsequencing, the major proteins labeled by the anti-glucitollysine have been identified as being actin, annexin I, annexin II, the p34 subunit of the Arp2/3 complex, and the Ras suppressor protein-1. Conversely, the intrinsic endothelial membrane proteins do not seem to be affected by hyperglycemia. This defines the internal face of the endothelial plasma membrane, particularly the cortical cytoskeleton, as a preferential target for nonenzymatic glycosylation in diabetes, with possible consequences on the fluidity of the endothelial plasmalemma and impairment of the endothelial mechanotransducing ability.

Alterations of the rat mesentery vasculature in experimental diabetes.

The alteration induced by diabetes on vascular permeability to serum albumin was investigated in the mesentery of streptozotocin-induced hyperglycemic rats. Double-tagged ((125)I and dinitrophenol-haptenated) heterologous albumin was intravenously administered in normal and hyperglycemic animals, and the extravasation of the tracer was evaluated by radioactivity measurements and by morphometry at the ultrastructural level using quantitative protein A-colloidal gold immunocytochemistry. The results demonstrate that diabetes induces a significant increase in the permeability of the mesentery vessels to albumin. This increase is due to a more efficient transport of macromolecules by endothelial plasmalemmal vesicles and not to leakier interendothelial junctions. Passage across the endothelial basement membranes did not appear to be restricted in either the control or diabetic condition. However, in diabetes, the mesothelial basement membrane appeared to become modified and to restrain the passage of albumin toward the peritoneal cavity. After 3 months of diabetes, the rats presented a net increase in the average diameter of the blood vessels localized in the mesentery arcada (macrovascular hyperplasy) and a notable angiogenesis, manifested at the level of the microvasculature in the mesenteric windows.

Antibodies specific to the plasma membrane of rat lung microvascular endothelium.

The highly purified, luminal domain of rat lung endothelial plasma membranes was used as an immunogen to obtain monoclonal antibodies to the endothelial cell surface. The procedure was highly efficient, yielding antibodies which recognize three seemingly novel endothelial integral membrane glycoproteins of molecular weights of 170, 114, and 95 kDa, respectively. By immunofluorescence, two of these antigens (170 and 95 kDa) appeared to be uniquely expressed by the lung microvascular endothelium. The third one, the 114-kDa polypeptide, was detected in the continuous endothelium of the lung, but also in the fenestrated endothelia of pancreas, intestinal villi, and kidney peritubular capillaries. Partition in Triton X-100-soluble and -insoluble plasmalemmal components suggests that two of these novel endothelial antigens (170 and 114 kDa) are specific for the plasma membrane proper only, while that of 95 kDa is present both in the caveolae and on the rest of the cell surface.

Distribution of endogenous apoprotein B-containing lipoproteins in normal and injured aortas of normocholesterolemic rabbits.

The distribution of endogenous apolipoprotein B (apo B) was studied in both normal and balloon catheter-injured aortas of standard fed rabbits. Using light and electron microscopy, the distribution within entire aortic walls and individual tissue compartments was investigated by immunocytochemistry using an antibody raised against rabbit apo B. The concentration of apo B across the vessel wall dropped sharply from the luminal front towards the media of the normal aortas. The strong superficial reaction was mainly due to a heavy, yet specific, labelling of endothelial cells. Significant concentrations of apo B were also detected within the innermost regions of the extracellular space. The characteristics associated with the labelling of the intimal layer suggested an intense uptake and transcellular transport of apo B by endothelial cells. In contradistinction, normal smooth muscle cells did not appear to be labelled. In the previously injured aortas, the same features of strong superficial apo B labelling were present in the areas covered by regenerated endothelial cells, but not in those persistently deendothelialized. The smooth muscle cells of these regions appeared to show a low uptake of apo B. The increased concentrations of apo B in deeper interstitial areas of injured aortas, indicated the contribution of the extracellular matrix to apo B accumulation. This was especially prominent in the advanced lesions, selectively developed within neointima covered by regenerated endothelium. A rather uniform labelling pattern accompanying small lipid particle deposits, suggested a direct extracellular accumulation of circulating lipoproteins. Intensely labelled foam cells and irregularly distributed apo B within areas of cellular necrosis were detected as well. Injury-mediated responses of the cellular and extracellular aortic components can trigger the development of lipoprotein accumulations characteristic of atherosclerosis within aortas of normocholesterolemic animals.