Glomerular CD34 expression in short- and long-term diabetes.

Aging and diabetes are associated with exacerbated expression of adhesion molecules. Given their importance in endothelial dysfunction and their possible involvement in the alteration of glomerular permeability occurring in diabetes, we have evaluated expression of the sialomucin-type adhesion molecule CD34 in renal glomerular cells of normal and diabetic animals at two different ages by colloidal gold immunocytochemistry and immunoblotting. CD34 labeling was mostly assigned to the plasma membranes of glomerular endothelium and mesangial processes.

Early glycation products of endothelial plasma membrane proteins in experimental diabetes.

The participation of glucose and two intermediates of glucose metabolism: glucose-6-phosphate (G6P) and glyceraldehyde-3-phosphate (Gald3P) to the formation of early glycation products was comparatively evaluated in the endothelial plasma membrane of streptozotocin-induced diabetic rats. Antibodies risen to a carrier protein reductively glycated by each of the sugars mentioned above were used to probe by immunoblotting the proteins of the lung microvascular endothelium plasmalemma purified from normal and diabetic rats. The amount of glycated endothelial plasma membrane proteins was below the limit of detection in normoglycemic animals but increased dramatically in diabetic animals for glucose and G6P.

Correlated endothelial caveolin overexpression and increased transcytosis in experimental diabetes.

We investigated the mechanism by which diabetes renders the capillary endothelium more permeable to macromolecules in the lungs of short-term diabetic rats. We used quantitative immunocytochemistry (ICC) to comparatively assess the permeability of alveolar capillaries to serum albumin in diabetic and normoglycemic animals. The effect of diabetes on the population of endothelial caveolae was evaluated by morphometry and by ICC and immunochemical quantification of the amount of caveolin in the whole cell or associated with the purified endothelial plasma membrane.

Circulating bile salt-dependent lipase originates from the pancreas via intestinal transcytosis.

Background & Aims: Bile salt-dependent lipase (BSDL) has been detected in human blood, where it is assumed to play a substantial role in atherosclerosis. The origin of this circulating enzyme is unknown. The aim of this study was to show that blood BSDL originates from pancreatic exocrine secretions via a transcytotic motion across the intestinal epithelium.

Is diabetic hypercoagulability an acquired annexinopathy? Glycation of annexin II as a putative mechanism for impaired fibrinolysis in diabetic patients.

Diabetics die mainly from thrombotic complications and there is clear evidence that diabetes is a hypercoagulable state. Epidemiological and prospective intervention data link hyperglycemia to vascular complications and glycation of proteins is one favored molecular basis to explain this fact. Cell surface receptors may support fibrinolytic surveillance in both intravascular and extravascular locations by stimulating plasmin generation and by protecting plasmin from its inhibitors.

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).

Diversity in unity: the biochemical composition of the endothelial cell surface varies between the vascular beds.

The vascular endothelium represents a population of squamous epithelial cells characterized by a particular histological localization (intima of blood vessels) and by several physiological functions such as the transport of substances between blood and tissues, the modulation of the vascular tone, the control of blood coagulation and that of the leukocyte extravasation. In spite of all these elements in common and of an identical embryonic origin, endothelial cells show definite morphological and physiological variations that divide them into types and subtypes, each specifically associated to various categories of organs. Even within the vasculature of the same organ, there are clear segmental (arterial/capillary/venous) differentiations of the endothelial cells.

Vascular immunotargeting to endothelial surface in a specific macrodomain in alveolar capillaries.

A novel 85 kD glycoprotein (gp85) is a marker of the avesicular zone, a thin part of pulmonary endothelial cells separating alveolar and vascular compartments and lacking vesicles. This report presents the first evaluation whether mAb 30B3, a monoclonal antibody to gp85, can be used for targeting of drugs to the surface of lung endothelium. 125I-mAb 30B3 accumulated in isolated perfused lungs (IPL) (22.

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.

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.

Isolation, cloning, and localization of rat PV-1, a novel endothelial caveolar protein.

By using an immunoisolation procedure (Stan, R.-V., W.

A novel, 85 kDa endothelial antigen differentiates plasma membrane macrodomains in lung alveolar capillaries.

A monoclonal antibody raised against purified rat lung endothelial plasma membranes was found to recognize an apparently novel, 85 kD, integral endothelial plasma membrane glycoprotein. By immunofluorescence and electron microscope immunocytochemistry, this endothelial antigen was detected at the luminal and tissue fronts of all rat endothelia, except those of discontinuous type (liver and spleen sinusoids). In lung alveolar capillaries the antigen appeared to be uniquely associated with the very attenuated endothelial processes forming the blood-air barrier, and virtually absent on the surface of the rest of the cell, where the nucleus and the organelles are located.

P-glycoprotein is strongly expressed in the luminal membranes of the endothelium of blood vessels in the brain.

Luminal membranes of the vascular endothelium were isolated from brain, heart and lungs by modification of their density. The presence of P-glycoprotein (P-gp) was detected by Western blotting in luminal membranes from the endothelium of the three tissues. Strong enrichment in brain capillary luminal membranes, compared with brain capillaries (17-fold) and whole membranes (400-500-fold), indicates that P-gp is mainly located on the luminal side of the brain endothelium.

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.

Immunoisolation and partial characterization of endothelial plasmalemmal vesicles (caveolae).

Plasmalemmal vesicles (PVs) or caveolae are plasma membrane invaginations and associated vesicles of regular size and shape found in most mammalian cell types. They are particularly numerous in the continuous endothelium of certain microvascular beds (e.g.

Endocytotic functions of ameloblasts and odontoblasts: immunocytochemical and tracer studies on the uptake of plasma proteins.

Background: Biochemical, (immuno)cytochemical, and radioautographic data accumulated over several years have lead to the view that ameloblasts carry out both secretory and degradative functions throughout amelogenesis. Whereas it has been assumed that maturation stage ameloblasts endocytose aged enamel proteins from the enamel layer, the origin of the newly formed ones detected in the endosomal/lysosomal compartment of ameloblasts from all stages remains to be elucidated. One possible source is from secretory products released ectopically along basolateral surfaces.

Immunocytochemical investigation of the in vivo endocytosis by renal tubular epithelial cells.

The internalization and degradation of glomerular filtered serum proteins by the proximal tubular epithelium has been extensively studied by microperfusion methods. By using a cationic probe that easily traverses the glomerular wall into the urinary space, we have performed a morpho-cytochemical and quantitative study of the in vivo endocytotic activity of the proximal tubular epithelial cell. Bovine serum albumin (BSA) was tagged with dinitrophenol (DNP) and cationized to pI over 8.

Glomerular handling of circulating glycated albumin in the normal mouse kidney.

In the present study, we have evaluated the glomerular handling of circulating glycated albumin in the normal mouse kidney by quantitative immunocytochemistry. Bovine serum albumin (BSA) was glycated in vitro and dinitrophenylated. Glycated and nonglycated probes were introduced into the circulation of anesthetized mice and traced by postembedding immunogold cytochemistry after 10 and 30 min of circulation.

Hapten-tagged plasma proteins as immunocytochemical probes for the study of vascular permeability.

Bovine serum albumin and transferrin were covalently coupled with fluorescein isothiocyanate and digoxigenin, respectively, and intravenously co-injected in equal amounts in mouse. The derivation of the two proteins induces minor alterations of their physicochemical properties as well as of their physiological functions. The two tracers were revealed within vascular and extravascular compartments of diaphragm by quantitative postembedding immunocytochemistry, using antibodies against each of the haptens in conjunction with the protein AG-gold complexes.

Immunocytochemical study of glomerular permeability to anionic, neutral and cationic albumins.

The renal handling of albumin of various isoelectric points (pI) was studied in mice by high resolution quantitative immunocytochemistry. Bovine serum albumin (BSA) was tagged with dinitrophenol (DNP) and cationized to pI 6.5 to 7.

Transendothelial transport of serum albumin: a quantitative immunocytochemical study.

The steady-state distribution of endogenous albumin in mouse diaphragm was determined by quantitative postembedding protein A-gold immunocytochemistry using a specific anti-mouse albumin antibody. Labeling density was recorded over vascular lumen, endothelium, junctions, and subendothelial space. At equilibrium, the volume density of interstitial albumin was 18% of that in circulation.

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.

Protein AG-gold complex: an alternative probe in immunocytochemistry.

The purpose of this study was to evaluate the use of protein AG tagged with colloidal gold as a reliable immunocytochemical reagent. Protein AG is a recombinant of 47.3 KD molecular weight and pI = 4.

Immunolabeling efficiency of protein A-gold complexes.

A systematic study of the adsorption of protein A on colloidal gold particles varying in size from 5-16 nm was performed at different protein concentrations. The number of protein A molecules bound per colloidal particle was evaluated and the Scatchard analysis of the adsorption parameters was applied for each size of the colloid. The binding of protein A to the colloidal gold surface exhibited the same affinity pattern for all of the particle sizes.

Fatty acids binding to albumin increases its uptake and transcytosis by the lung capillary endothelium.

To determine whether uptake and transcytosis of albumin (A) in continuous capillary endothelia are modified when this protein carries fatty acids, the transport of albumin-oleic acid and albumin-palmitic acid complexes was compared with that of defatted albumin. The probes, either radioiodinated or tagged with 5-nm gold particles (Au), or both, were perfused in situ or injected in vivo; after 3 or 30 min lung fragments were radioassayed or examined by electron microscopy. Both in situ and in vivo, the uptake of fatty acid-carrying albumin (A-FA) was consistently 2 to 3 times higher than that of defatted A.