Podoendin. A new cell surface protein of the podocyte and endothelium.

A new cell surface protein, podoendin, has been identified in Sprague-Dawley rats, and isolated using monoclonal antibody (mAb) G4. The distribution of podoendin is restricted to the surface of glomerular podocytes, urinary surface of the parietal epithelium of Bowman's capsule, and the luminal surface of endothelial cells. The antibody does not crossreact with podocytes or endothelia of human or mice. In newborn rats, the appearance of podoendin on glomerular epithelium is attendant on podocyte differentiation during glomerulogenesis of metanephrogenic vesicles. It disappears when podocytes retract and efface foot processes in tissue culture. Thus, podoendin appears to be a cell differentiation-dependent surface protein of podocytes. Podoendin is a protein of 62 kD mobility on 5% polyacrylamide gel electrophoresis. It stains intensely with Coomassie blue, but gives negative reactions to carbohydrate (periodic acid/Schiff reaction) and polyanions (alcian blue, colloidal iron, and carbocyanine). It is distinct from the major sialoglycoprotein of podocyte fuzzy coat, podocalyxin (11). Podoendin isolated and purified from endothelium of lungs appears to be identical with that from podocytes and endothelium of kidneys. Injection of mAb G4 into left ventricle of rats resulted in intense decoration of the endothelium and podocyte surface within 30 min. The decoration persisted throughout the 3-d period of observation. This was not accompanied by complement (C3) fixation. Preliminary results showed that the rats developed moderate proteinuria (100 mg/ml protein in urine), which was associated with the presence of hyaline droplets in renal tubules, on the third day. The proteinuria was not accompanied by effacement of podocyte pedicels. There were no morphologic alterations indicating glomerular or vascular injury in the kidneys.

Characterization of an apically derived epithelial membrane glycoprotein from bovine milk, which is expressed in capillary endothelia in diverse tissues.

A glycoprotein (PAS IV) of apparent Mr 76,000 was purified from bovine milk-fat-globule membrane and partially characterized. PAS IV contained mannose, galactose, and sialic acid as principal sugars (approximately 5.3% total carbohydrate [wt/wt]) and existed in milk in at least four isoelectric variants. The glycoprotein appeared to be an integral membrane protein by several criteria. PAS IV was recovered in the detergent phase of Triton X-114 extracts of milk-fat-globule membrane at room temperature. When bound to membrane, PAS IV was resistant to digestion by a number of proteinases, although after solubilization with non-ionic detergents, the protein was readily degraded. Amino acid analysis of the purified protein revealed a high percentage of amino acids with nonpolar residues. The location of PAS IV was determined in bovine tissues by using immunofluorescence techniques. In mammary tissue, PAS IV was located on both the apical surfaces of secretory epithelial cells and endothelial cells of capillaries. This glycoprotein was also detected in endothelial cells of heart, liver, spleen, pancreas, salivary gland, and small intestine. In addition to mammary epithelial cells, PAS IV was also located in certain other epithelial cells, most notably the bronchiolar epithelial cells of lung. The potential usefulness of this protein as a specific marker of capillary endothelial cells in certain tissues is discussed.

Differentiated microdomains on the luminal surface of the capillary endothelium. II. Partial characterization of their anionic sites.

To investigate the chemical nature of the cationic ferritin (CF)-binding sites of the differentiated microdomains of the capillary endothelium, the vasculature of the mouse pancreas and intestinal mucosa was perfused in situ with neuraminidase, hyaluronidase, chondroitinase ABC, heparinase, and three proteases: trypsin, papain, and pronase. Proteases of broad specificity removed all anionic sites, suggesting that the latter are contributed by acid glycoproteins or proteoglycans. Neuraminidase, hyaluronidase, and chondroitinase ABC reduced the density of CF-binding sites on the plasmalemma proper, but had no effect on either coated pits or fenestral diaphragms. Heparinase removed CF-binding sites from fenestral diaphragms and had no effect on coated pits. Taken together, these results indicate that the anionic sites of the fenestral diaphragms are contributed primarily by heparan sulfate and/or heparin, whereas those of the plasmalemma proper are of mixed chemical nature. The membranes and diaphragms of plasmalemmal vesicles and transendothelial channels do not bind CF in control specimens; this condition is not affected by the enzymic treatments mentioned above.

Preferential distribution of anionic sites on the basement membrane and the abluminal aspect of the endothelium in fenestrated capillaries.

Cationized ferritin (CF) was injected interstitially to study the distribution of anionic sites on the basement membrane and abluminal aspect of the endothelium in the fenestrated capillaries of the mouse pancreas and intestinal mucosa. Extensive, but uneven removal of the basement membrane was obtained by collagenase perfusion of the vasculature before CF labeling. In the absence of collagenase treatment, CF label was essentially restricted to the lamina rara externa of the basement membrane and occurred in clusters distributed in a relatively ordered planar lattice. After collagenase digestion, labeling of the lamina rara interna and of the abluminal aspect of the endothelium became possible. In the lamina rara interna, the CF label occurred in clusters with a distribution comparable to that found in the lamina rara externa. On the abluminal aspect of the endothelium, the plasmalemma proper was extensively, though variably, labeled. Coated pits were heavily labeled, whereas the membranes and stomatal diaphragms of plasmalemmal vesicles and transendothelial channels remained free of CF decoration. In contradistinction with the heavy labeling of their luminal aspects, the abluminal surface of the fenestral diaphragms were free of any CF decoration. Pronase treatment removed all anionic sites detectable by CF binding. The findings establish the existence of differentiated microdomains on the abluminal aspect of the endothelial plasmalemma and suggest that the capillary wall selects permeant macromolecules according to charge, in addition to size.

Thrombomodulin is found on endothelium of arteries, veins, capillaries, and lymphatics, and on syncytiotrophoblast of human placenta.

We have used antibodies to human thrombomodulin isolated from placenta to investigate the distribution of this cofactor for protein C activation in human tissues. Thrombomodulin was found on endothelial cells of arteries, veins, capillaries, and lymphatics by immunocytochemical staining using an avidin-biotin peroxidase method. Thrombomodulin was not detected on sinusoidal lining cells of liver or on postcapillary high-endothelial venules of lymph node, although the latter contained another endothelial antigen, von Willebrand factor. Other cells noted to contain thrombomodulin antigen are those of the syncytiotrophoblast in placenta. The thrombomodulin in syncytiotrophoblast was primarily on the plasma membrane surface that forms the maternal blood sinus. Syncytiotrophoblast also stained with antibodies to von Willebrand factor, which implies that these cells have multiple endothelial functions. Thrombomodulin antigen was found in all organs studied, with the notable exception of brain.

Dextrans and glycogens as particulate tracers for studying capillary permeability.

Commercially available glycogens and dextrans can be used as biological particulate tracers in work on capillary permeability. These polysaccharides are well tolerated in intravenous injection and induce no vascular leakage when applied topically (cremaster test) in mice and in Wistar-Furth rats. The particles stain adequately with lead after aldehyde-OsO(4) fixation in phosphate buffer and provide a relatively wide set of probes ( approximately 45 A-300 A) for work on the large and small pore systems.

Ultrastructural immunoperoxidase demonstration of autologous albumin in the alveolar capillary membrane and in the alveolar lining material in normal rats.

The location of autologous serum albumin within the alveolar-capillary membrane was studied in the rat under physiological conditions using antialbumin antibodies labeled with peroxidase. Albumin was detected in the lung interstitium, and in numerous pinocytic vesicles within endothelial cells and type I alveolar epithelial cells. The immunoreaction was also positive at the level of plasmalemmal membranes of both cell types and in the alveolar lining material.

Sites of lipoprotein lipase activity in adipose tissue perfused with chylomicrons. Electron microscope cytochemical study.

Lipoprotein lipase activity was studied in rat parametrial adipose tissue perfused with chylomicrons and in gelatin blocks containing postheparin plasma and chylomicrons. The tissues and blocks were fixed in glutaraldehyde and incubated in 0.035 M CaCl(2)-0.1 M Tris medium (pH 8.3) at 38 degrees C. The doubly labeled chylomicron triglycerides (glycerol-(3)H and palmitate-(14)C) in the tissues and blocks were hydrolyzed during incubation to free fatty acids (FFA) and the FFA remained in the specimens; hydrolysis was inhibited by 0.004 M diethyl paranitrophenyl phosphate (E-600). Incubated blocks and tissue were treated with 0.05 M Pb(NO(3))(2), postfixed in OsO(4), dehydrated with acetone, embedded in Epon, and examined by electron microscopy. The incubated blocks contained electronlucent areas and granular and laminar precipitates at sites of hydrolysis. Similar precipitates were found in incubated tissue, within vacuoles and microvesicles of capillary endothelium, and in the subendothelial space (between the endothelium and pericytes), but not in the capillary lumen or in or near fat cells. The cytochemical reaction was greatly reduced, in blocks and tissues incubated with E-600. It is concluded that plasma glycerides are hydrolyzed by lipoprotein lipase in capillary endothelial cells and in the subendothelial space of adipose tissue and that glycerides across the endothelial cells within a membrane-bounded system.

The cellular site of action of angiotensin.

The site of action and the distribution of angiotensin II have been studied in the mouse. A comparison of the ratios of angiotensin-(14)C and inulin-(3)H at the time of the pressor effect reveals an extracellular pattern of distribution. Morphological studies were made using angiotensin coupled to exogenous enzymes which can be demonstrated histochemically. Coupling of angiotensin to horseradish peroxidase or cytochrome c, with glutaraldehyde or difluorodinitrodiphenylsulfone (FNPS) as the coupling agent, does not alter the pattern of its vasopressor response or that of its inactivation; nor are differences present between angiotensin and the angiotensin-enzyme complexes in the stimulation of in vitro tissue preparations. Dissociation of the complexes was shown not to occur in vitro, but the possibility of a serum factor splitting the complexes immediately after intravenous injection cannot be excluded. Since these complexes are localized on the endothelium and not on the smooth muscle at the time of maximum hypertension, the endothelium is proposed as the site of action for angiotensin.

Immunofluorescent localization of antihemophilic factor antigen and fibrinogen in human renal diseases.

Tissue localization of antihemophilic factor (AHF, factor VIII) antigen and fibrinogen by immunofluorescent microscopy was determined in 146 specimens of normal and diseased kidneys. AHF antigen was present in the endothelial cells of glomeruli, peritubular capillaries, arteries, and veins of normal kidneys; a distribution similar to that in other tissues. In scleroderma and malignant hypertension, deposition of AHF antigen and fibrinogen was limited to the markedly thickened endothelial layers of arteries. More extensive intense deposition of both AHF antigen and fibrinogen in glomeruli and in arterial walls were present in hyperacute renal homograft rejection, hemolyticuremic syndrome, postpartum renal failure, and in some cases of acute homograft rejection. In contrast, deposition of fibrinogen was observed in glomerular epithelial cresents in severe proliferative glomerulonephritis, but AHF deposition was not present in these lesions. Glomerular deposition of fibrinogen without increased AHF standing was also detected in renal tissue from patients with anaphylactoid purpura nephritis and in recurrent macroscopic hematuria with focal glomerulonephritis. Increased staining of peritubular capillaries with anti-AHF was seen in diseased kidneys irrespective of etiology. Immunofluorescent localization of AHF, a participant in the intrinsic coagulation pathway, offers a new way by which to analyze the mechanisms responsible for fibrinogen deposition in disease.

Identification of persistent defects in insulin receptor structure and function capillary endothelial cells from diabetic rats.

Insulin actions and receptors were studied in capillary endothelial cells cultured from diabetic BB rats and their nondiabetic colony mates. The endothelial cells from diabetic rats of 2 mo duration had persistent biological and biochemical defects in culture. Compared with normal rats, endothelial cells from diabetic rats grew 44% more slowly. Binding studies of insulin and insulin-like growth factor I (IGF-I) showed that cells from diabetic rats had 50% decrease of insulin receptor binding (nondiabetic: 4.6 +/- 0.7; diabetic: 2.6 +/- 0.4% per milligram protein, P less than 0.01), which was caused by a 50% decrease in the number of binding sites per milligram protein, whereas IGF-I binding was not changed. Insulin stimulation of 2-deoxy-glucose uptake and alpha-aminoisobutyric acid uptake were also severely impaired with a 80-90% decrease in maximal stimulation, in parallel with a 62% decrease in insulin-stimulated autophosphorylation (P less than 0.05). 125I-insulin cross-linking revealed an 140-kD alpha subunit of the insulin receptor similar to that in cells from nondiabetic rats, although bands at greater than 200 kD were also detected. The molecular weight of the insulin receptor beta subunit (by SDS-PAGE) was smaller in cells from diabetic than from normal rats (88-90 vs. 95 kD). Neuraminadase treatment of the partially purified insulin receptors decreased the molecular weight of the insulin receptors from nondiabetic rats to a greater degree than its diabetic counterpart. In contrast, Northern blot analysis of insulin receptor mRNAs using human cDNA probes revealed two species of 9.4 and 7.2 kb with no difference in mRNA abundance between cells from diabetic and nondiabetic rats. We conclude that the exposure of capillary endothelial cells to a diabetic milieu in vivo can cause specific and persistent changes in the insulin receptor and insulin action.

Evidence for the chylomicron origin of lipids accumulating in diabetic eruptive xanthomas: a correlative lipid biochemical, histochemical, and electron microscopic study.

Plasma lipoprotein alterations in nine insulin-dependent diabetics with hyperlipemia have been related to the lipid accumulating in eruptive xanthomas evolving in these patients. Histochemical and electron microscopic examination of xanthomas have been correlated with the lipid analyses in order to obtain additional evidence regarding the lipoprotein origin of lipids accumulating in the lesions. Both analytical and morphologic evidence suggested that circulating chylomicrons significantly contribute to the xanthoma lipids. All the patients had large quantities of circulating triglyceriderich chylomicrons which carried approximately 70% of the triglyceride found in the plasma. The fatty acid pattern of chylomicron and xanthoma triglycerides were similar. Triglyceride constituted the major lipid found in the xanthomas when they were sampled during their eruption. These findings, take in conjunction with histochemical and electron microscopic evidence of chylomicron particles in the dermal capillary walls, support the theory that blood lipoproteins, and particularly chylomicrons, permeated the vascular walls and the triglycerides carried by these lipoproteins apparently accumulated in tissue macrophages and perithelial cells which evolved into foam cells. Initiation of appropriate therapy resulted in clearance of the chylomicronemia and a concomitant resolution of the xanthomas as reflected by a decrease in total xanthoma lipid. Sequential studies of resolving xanthomas in five patients revealed that xanthoma triglyceride was mobilized more rapidly than cholesterol, resulting in a redistribution of the xanthoma lipids, so that the resolving lesions were cholesterol rich. Consistent with this change in lipid composition, correlative electron microscopy revealed loss of amorphous material from many of the foam cell vacuoles.

31P nuclear magnetic resonance spectroscopy studies of tumor energy metabolism and its relationship to intracapillary oxyhemoglobin saturation status and tumor hypoxia.

Relationships between tumor bioenergetic status on the one hand and intracapillary oxyhemoglobin (HbO2) saturation status and fraction of radiobiologically hypoxic cells on the other were studied using two murine sarcoma lines (KHT, RIF-1) and two human ovarian carcinoma xenograft lines (MLS, OWI). Tumor energy metabolism was studied in vivo by 31P nuclear magnetic resonance (NMR) spectroscopy and the resonance area ratio (PCr + NTP beta)/Pi was used as parameter for bioenergetic status. Intracapillary HbO2 saturation status reflects the oxygen supply conditions in tumors and was measured in vitro using a cryospectrophotometric method. The KHT, RIF-1, and MLS lines showed decreasing bioenergetic status, i.e., decreasing PCr and NTP beta resonances and an increasing Pi resonance, with increasing tumor volume, whereas the OWI line showed no changes in these resonances during tumor growth. The volume-dependence of the HbO2 saturation status differed similarly among the tumor lines; HbO2 saturation status decreased with increasing tumor volume for the KHT, RIF-1, and MLS lines and was independent of tumor volume for the OWI line. Moreover, linear correlations were found between bioenergetic status and HbO2 saturation status for individual tumors of the KHT, RIF-1, and MLS lines. These observations together indicated a direct relationship between 31P-NMR spectral parameters and tumor oxygen supply conditions. However, this relationship was not identical for the different tumor lines, suggesting that it was influenced by intrinsic properties of the tumor cells such as rate of respiration and ability to survive under hypoxia. Similarly, there was no correlation between bioenergetic status and fraction of radiobiologically hypoxic cells across the four tumor lines. This indicates that 31P-NMR spectroscopy data have to be supplemented with other data, e.g., rate of oxygen consumption, cell survival time under hypoxic stress, and/or fraction of metabolically active, nonclonogenic hypoxic cells, to be useful in quantitative determination of tumor hypoxia and hence prediction of tumor radioresistance caused by hypoxia.

Presence of glycosaminoglycans in retinal capillary basement membrane.

Retinal microvessels were isolated from bovine eyes and the basement membranes were purified either directly or after incubation with [35S]sulfate and [14C]glucosamine. The basement membranes, which were purified by osmotic lysis and sequential treatment with detergents, had the general compositional features associated with basement membrane collagens, including high levels of hydroxyproline and hydroxylysine and the presence of 3-hydroxyproline and cystine. After pronase digestion, cellulose acetate electrophoresis of glycosaminoglycans from retinal microvessel basement membrane revealed material comigrating with heparan sulfate that was insensitive to digestion with Streptomyces hyaluronidase ad chondroitinase ABC. Retinal microvessels also incorporated [35S]- and [14C]glucosamine into glycosaminoglycans that were isolated following pronase digestion of the retinal microvessel basement membrane purified from these incubations. The findings provide the first demonstration that glycosaminoglycans are integral components of the retinal microvascular basement membrane and suggest that heparan sulfate is the major glycosaminoglycan species in this basement membrane.

Skeletal muscle basement membrane in maturity-onset diabetes in the young.

We have studied skeletal muscle capillary basement membrane width (CBMW) and intensity of skeletal muscle extracellular basement membrane staining for albumin and IgG in eight families with maturity-onset diabetes in the young (MODY). Ninety-two MODY patients were identified. Sixty-three of these patients, 33 relatives with nondiagnostic oral glucose tolerance studies, and 61 normoglycemic relatives were studied for glucose and insulin. Twenty-six of these MODY patients, 20 normoglycemic relatives, and 16 unrelated normal controls had skeletal muscle capillary morphologic studies. The muscle capillary basement membrane was significantly increased in MODY patients younger than 40 yr when compared with unrelated normal subjects and relatives of the same age (P less than 0.001). However, in these families, the CBMW of MODY patients showed no significant thickening with age (slope = 0.45, P less than 0.14), as expected and seen in the normal subjects and in the normoglycemic relatives of the patients (slope = 1.21, P less than 0.001). The slope derived from the linear regression of age and CBMW in MODY patients (0.45 +/- 0.29) was significantly less (P less than 0.05) than that of the nondiabetic subjects (1.21 +/- 0.19). The mean intensity of skeletal muscle extracellular basement membrane staining for albumin was higher in MODY patients (1.1 +/- 0.15) than in unrelated normal subjects (0.7 +/- 0.1, P less than 0.025) and normal MODY family members (0.6 +/- 0.08, P less than 0.01). The unexpected absence of basement membrane thickening with age in the MODY patients may explain the paucity of vascular complications that have been reported by some.(ABSTRACT TRUNCATED AT 250 WORDS)

Light and electron microscopic localization of D-galactosyl residues in capillary endothelial cells of the canine cerebral cortex.

Ricinus communis agglutinin I (RCA-I), a lectin that binds to D-galactosyl residues, intensely stained capillaries in cryostat sections of canine cerebral cortex when evaluated by the avidin-biotin-peroxidase complex method. Of seven lectins tested, only RCA-I gave strong staining of vessels and capillaries with little staining of other cortical cells. Ultrastructural studies using ferritin-, biotin-, and peroxidase-labeled RCA-I indicated that this lectin was bound to the luminal membrane of the cerebral capillary endothelial cell and that lectin receptors were distributed continuously along this membrane. Plasmalemma invaginations that bound RCA-I were also present in endothelial cells. Primary cultures of cerebral capillary endothelial cells grown on plastic or gelatin-coated glass substrates demonstrated staining of the cell membrane and perinuclear structures which appeared to be the Golgi complex and secondary lysosomes. These staining characteristics were retained when the cells were subcultured and were confirmed by ultrastructural studies. In contrast, light microscopy showed that fibronectin was more widely distributed in the cytoplasm, a finding consistent with its occurrence in the endoplasmic reticulum. This work provides support for the concept that lectins may be useful endothelial cell markers in both intact tissue and cell culture.

Demonstration of anionic sites on the luminal and abluminal fronts of endothelial cells with poly-L-lysine-gold complex.

An attempt was made to demonstrate the anionic sites on the endothelial cell (EC) surfaces of mouse brain micro-blood vessels (MBVs) after embedding of tissue samples in hydrophilic media: Lowicryl K4M, LR White, and Polyamph-10. As a cationic probe, poly-L-lysine-gold complex (PLG), prepared according to the procedure of Skutelsky and Roth (J Histochem Cytochem 34:693, 1986), was used. In ultra-thin sections of brain samples embedded in Lowicryl K4M and LR White, the anionic sites were demonstrated in the entire cross-section of the vessel wall. After embedding in Polyamph-10, however, the anionic sites could not be detected. Brain capillaries, representing blood-brain barrier type MBVs, showed polar distribution of anionic sites, evidenced by more intense labeling of luminal than of abluminal plasma membrane of the EC. Some differences in labeling of ECs and of basement membrane in arterioles and venules were also noted. The use of cationic gold and the ultra-thin sections of tissue samples embedded in hydrophilic media (Lowicryl K4M and LR White) seems to be a promising new method for detection of anionic constituents located on both luminal and abluminal surfaces of the EC, in the basement membrane, and in other components of the vessel wall.

Immunocytochemical studies of desmin and vimentin in pericapillary cells of chicken.

The composition of intermediate filaments in pericytes was examined by immunofluorescent and immunoelectron microscopic labeling of frozen sections of various chicken microvascular beds in situ. Pericytes in capillaries of cardiac muscle, exocrine pancreas, and kidney (peritubular capillary) were found to contain both desmin and vimentin. In some capillaries where pericytes do not exist, cells apposed to endothelial cells--the Ito cell in the hepatic sinusoid and the reticular cell in the splenic sinusoid--were shown to contain both of the intermediate filament proteins. In contrast, podocytes and mesangial cells around renal glomerular capillaries contained only vimentin. The presence of desmin supports the hypothesis that pericytes may have a contractile apparatus similar to that of vascular smooth muscle cells. Our results also revealed that even in microvascular beds where pericytes are not found, cells having both desmin and vimentin exist next to endothelial cells and may assume similar functions to pericytes.

Distribution of sialoglycoconjugates on the luminal surface of the endothelial cell in the fenestrated capillaries of the pancreas.

Sialic acid-bearing molecules on the luminal surface of the vascular endothelium in mouse and rat pancreatic capillaries were detected electron microscopically by using a procedure with ferritin hydrazide (FH), after preferential oxidation of sialyl residues with sodium periodate. The distribution of FH on the endothelial surface demonstrated the existence of microdomains with various densities of sialoglycoconjugates oxidizable by sodium periodate and accessible to the tracer. On the plasmalemma proper, FH binding sites were heterogeneously distributed. Their concentration on various microdomains decreased as follows: plasmalemma proper greater than coated pits greater than stomal diaphragms of plasmalemmal vesicles and transendothelial channels, and fenestral diaphragms. The membrane of plasmalemmal vesicles and transendothelial channels was not labeled by FH. Nonspecific binding of FH to the nonoxidized endothelial surface or that oxidized after neuraminidase treatment was relatively low.

Alpha-smooth muscle actin, a differentiation marker of smooth muscle cells, is present in microfilamentous bundles of pericytes.

alpha-Smooth muscle (alpha-sm) actin, an isoform typical of smooth muscle cells (SMC) and present in high amounts in vascular SMC, was demonstrated in the cytoplasm of pericytes of various rat and human organs by means of immunocytochemistry at the electron microscopic level. In SMC and pericytes, alpha-sm actin was localized in microfilament bundles, strengthening the assumption that it is the functional isoform in these cell types and supporting the assumption that pericytes exert contractile functions.