A major endothelial plasmalemmal sialoglycoprotein, gp60, is immunologically related to glycophorin.

Glycophorins, the major sialoglycoproteins of red blood cells in many species, are generally considered to be specific to erythroid cells. Using polyclonal antibodies directed against mouse glycophorin (alpha gp), we have identified a glycoprotein antigenically related to glycophorin on the surface of bovine and rat cultured endothelial cells. Immunoblotting with alpha gp identified a single 60-kDa polypeptide on transfers of SDS/polyacrylamide gels of solubilized confluent endothelial monolayers. In addition, a 60-kDa polypeptide was immunoprecipitated by alpha gp from lysates of 125I-labeled intact endothelial cells. Controls with preimmune serum were negative. This antibody interaction was inhibited by murine erythrocyte ghosts and purified glycophorins. Our past work identified several endothelial surface sialoglycoproteins including a 60-kDa glycoprotein (gp60) that (i) interacts with albumin, (ii) binds Limax flavus, Ricinus communis, and Triticum vulgare agglutinins but not other lectins, (iii) is sequentially precipitated from 125I-labeled cell lysates by using R. communis agglutinin followed by T. vulgare agglutinin, and (iv) is sensitive to sialidase digestion. Immunoblotting of such precipitates with alpha gp demonstrates that lectins recognize the same glycoprotein, namely gp60. These results indicate that gp60, a major endothelial surface sialoglycoprotein, shares antigenic epitope(s) with glycophorin.

The tight junction protein ZO-1 is concentrated along slit diaphragms of the glomerular epithelium.

The foot processes of glomerular epithelial cells of the mammalian kidney are firmly attached to one another by shallow intercellular junctions or slit diaphragms of unknown composition. We have investigated the molecular nature of these junctions using an antibody that recognizes ZO-1, a protein that is specific for the tight junction or zonula occludens. By immunoblotting the affinity purified anti-ZO-1 IgG recognizes a single 225-kD band in kidney cortex and in slit diaphragm-enriched fractions as in other tissues. When ZO-1 was localized by immunofluorescence in kidney tissue of adult rats, the protein was detected in epithelia of all segments of the nephron, but the glomerular epithelium was much more intensely stained than any other epithelium. Among tubule epithelia the signal for ZO-1 correlated with the known fibril content and physiologic tightness of the junctions, i.e., it was highest in distal and collecting tubules and lowest in the proximal tubule. By immunoelectron microscopy ZO-1 was found to be concentrated on the cytoplasmic surface of the tight junctional membrane. Within the glomerulus ZO-1 was localized predominantly in the epithelial foot processes where it was concentrated precisely at the points of insertion of the slit diaphragms into the lateral cell membrane. Its distribution appeared to be continuous along the continuous slit membrane junction. When ZO-1 was localized in differentiating glomeruli in the newborn rat kidney, it was present early in development when the apical junctional complexes between presumptive podocytes are composed of typical tight and adhering junctions. It remained associated with these junctions during the time they migrate down the lateral cell surface, disappear and are replaced by slit diaphragms. The distribution of ZO-1 and the close developmental relationship between the two junctions suggest that the slit diaphragm is a variant of the tight junction that shares with it at least one structural protein and the functional property of defining distinctive plasmalemmal domains. The glomerular epithelium is unique among renal epithelia in that ZO-1 is present, but the intercellular spaces are wide open and no fibrils are seen by freeze fracture. The presence of ZO-1 along slit membranes indicates that expression of ZO-1 alone does not lead to tight junction assembly.

Localization of anticoagulantly active heparan sulfate proteoglycans in vascular endothelium: antithrombin binding on cultured endothelial cells and perfused rat aorta.

We have studied the interaction of 125I-antithrombin (125I-AT) with microvascular endothelial cells (RFPEC) to localize the cellular site of anticoagulantly active heparan sulfate proteoglycans (HSPG). The radiolabeled protease inhibitor bound specifically to the above HSPG with a Kd of approximately 50 nM. Confluent monolayer RFPEC cultures exhibited a linear increase in the amount of AT bound per cell for up to 16 d, whereas suspension RFPEC cultures possessed a constant number of protease inhibitor binding sites per cell for up to 5 d. These results suggest that monolayer RFPEC cultures secrete anticoagulantly active HSPG, which then accumulate in the extracellular matrix. This hypothesis was confirmed by quantitative light and EM level autoradiography which demonstrated that the AT binding sites are predominantly located in the extracellular matrix with only small quantities of protease inhibitor complexed to the cell surface. We have also pinpointed the in vivo position of anticoagulantly active HSPG within the blood vessel wall. Rat aortas were perfused, in situ, with 125I-AT, and bound labeled protease inhibitor was localized by light and EM autoradiography. The anticoagulantly active HSPG were concentrated immediately beneath the aortic and vasa vasorum endothelium with only a very small extent of labeling noted on the luminal surface of the endothelial cells. Based upon the above data, we propose a model whereby luminal and abluminal anticoagulantly active HSPG regulate coagulation mechanism activity.

Nonmuscle and smooth muscle myosin isoforms in bovine endothelial cells.

A panel of monoclonal antibodies, specific for human platelet (NM-A9, NM-F6, and NM-G2) and for bovine smooth muscle (SM-E7) myosin heavy chains (MHC), were used to study the composition and the distribution of myosin isoforms in bovine endothelial cells (EC), in vivo and in vitro. Using indirect and double immunofluorescence techniques, we have found that in the intact aortic endothelium there is expression of nonmuscle MHC (NM-MHC), exclusively. By contrast, hepatic sinusoidal endothelium as well as cultured bovine aortic EC (BAEC) in the subconfluent phase of growth show coexistence of NM- and smooth muscle MHC (SM-MHC) isoforms. SM myosin immunoreactivity disappears when cultured BAEC become confluent. In this phase of cell growth, NM-MHC isoforms are localized differently within the cells, i.e., in the cytoplasm around the nucleus or in the cortical, submembranous region of EC cytoplasm. A third type of intracellular distribution of NM-MHC immunoreactivity was evident in the cell periphery of binucleated, confluent BAEC. These data indicate that (1) several myosin isoforms are differently distributed in bovine endothelia; and (2) SM myosin expression and the specific subcellular localization of NM myosin isoforms within EC might be regulated by cell-cell interactions.

Sulfated glucuronyl paragloboside in rat brain microvessels.

In patients with neuropathy associated with paraproteinemia, there are monoclonal immunoglobulin M antibodies reacting with myelin-associated glycoprotein and sulfated glucuronyl glycolipids. There are indications that the monoclonal antibodies may be responsible for these neuropathies. However, the mechanism by which the antibodies gain access to the nervous tissue, which is separated by the blood-brain barrier or blood-nerve barrier, is still unknown. In this study, we examined the presence of the sulfated glucuronyl glycolipid antigens on brain endothelial cells. Microvessels were isolated from adult Lewis rat brain cortex. Sulfated glucuronyl paragloboside (SGPG) was detected in the acidic lipid fraction by a TLC immunostaining method. Immunofluorescence studies showed positive staining on the surface of microvessels. In addition, SGPG could be detected in the cultured endothelial cells of human umbilical vein. These findings suggest that the endothelial cells contain antigenic sites for interaction with the autoantibodies. This type of interaction may result in damages to the endothelial cell function and may be responsible for changes in the blood-brain barrier permeability and the ensuing penetration of large molecules, such as immunoglobulins, into the endoneurial space.

Specialized localization of P-glycoprotein recognized by MRK 16 monoclonal antibody in endothelial cells of the brain and the spinal cord.

This communication describes the cellular and ultrastructural localization in the central nervous system of P-glycoprotein (P-GP) recognized by a murine monoclonal antibody, MRK 16. At the ultrastructural level P-GP was strictly confined to the luminal surface of the endothelial cells which comprise the capillary vessels of the brain and the spinal cord. No P-GP was found in the endothelial cells of other organs. Our findings may be useful as a means to define the blood-brain barrier, and they imply that the blood-brain barrier is anatomically characterized by the presence of intercellular tight junctions between continuous nonfenestrated endothelial cells.

Involvement of endothelin in the regulation of human vascular tonus. Potent vasoconstrictor effect and existence in endothelial cells.

Endothelin, a recently discovered endothelium-derived peptide, has been reported to produce potent vasoconstriction in various vessels of experimental animals. To study the involvement of endothelin in the regulation of vascular tonus in humans, isolated human mesenteric arteries were investigated by both pharmacological and immunohistochemical methods. The vasoconstrictor action of endothelin-1 was examined on ring segments of human mesenteric arteries. Endothelin-1 induced a slowly developing and sustained contraction, with an EC50 value (half-maximal effective concentration) of 2.9 x 10(-9) M, two orders of magnitude smaller than that of norepinephrine (EC50 of 3.9 x 10(-7) M), indicating that the vasoconstrictor action of endothelin-1 is about 100 times more potent than that of norepinephrine. The contractile effect of endothelin-1 was affected neither by adrenergic, cholinergic, histaminergic, nor serotonergic antagonists, nor by inhibitors of arachidonic acid metabolism. The vasoconstrictor response to endothelin-1 was effectively antagonized by nicardipine, a dihydropyridine Ca2+ channel blocker. Endothelin-1 profoundly augmented contractile response to Ca2+ in partially depolarized tissues. Immunohistochemical studies revealed for the first time that endothelin-like immunoreactivity was localized in endothelial cells of human mesenteric artery. The results of the present study indicate that endothelin-1 is one of the most potent vasoconstrictors in the human mesenteric artery and that it induces vasoconstriction via an ultimately accelerating Ca2+ influx through voltage-dependent Ca2+ channels. Since endothelin-1 can be located in human endothelial cells, it may play an important physiological or pathophysiological role.

Plasma immunoreactive endothelin in essential hypertension.

PURPOSE: Endothelin plays a role in the regulation of vascular tonus. Therefore, it has been hypothesized that increased production or release of endothelin or both may contribute to the pathogenesis of hypertension. To assess any changes in the plasma endothelin concentration in essential hypertension, plasma immunoreactive endothelin concentrations were measured in patients with essential hypertension. PATIENTS AND METHODS: We measured plasma immunoreactive endothelin concentrations in 42 subjects with essential hypertension, 12 subjects with borderline hypertension, and 25 normotensive control subjects. RESULTS: The concentrations were higher in hypertensive patients than in borderline hypertensive patients and normotensive subjects (both p less than 0.05), although values in normotensives and hypertensives overlapped. Reverse-phase high-performance liquid chromatography (HPLC) and radioimmuno-assay showed two components of plasma endothelin, one corresponding to synthetic endothelin-1 (1-21) and the other corresponding to synthetic big endothelin (human, 1-38). The HPLC profile of plasma endothelin of hypertensive patients was the same as that of normotensive subjects. Hypertensives with reduced glomerular filtration rates or increased serum creatinine levels had higher plasma endothelin concentrations than hypertensive patients as a whole (p less than 0.05). Mean blood pressure and serum creatinine levels were correlated to plasma endothelin in the hypertensives. Correlation was negative between glomerular filtration rate and the endothelin level in the hypertensives. CONCLUSION: Plasma endothelin was elevated in many hypertensive patients with severe hypertension or renal involvement. Its major components were endothelin-1 and big endothelin.

Tissue distribution of HRF20, a novel factor preventing the membrane attack of homologous complement, and its predominant expression on endothelial cells in vivo.

A 20,000 molecular weight (MW) homologous restriction factor (HRF20), detected by 1F5 monoclonal antibody (mAb), is present on blood cell surfaces and inhibits the terminal stage of the formation of membrane attack complexes by homologous complement activation. The tissue distribution of HRF20 was studied by immunohistochemical analysis using 1F5. HRF20 was predominantly expressed on endothelial cells of systemic arteries, veins and capillaries, as well as on the surface of cultured human umbilical vein endothelial cells. HRF20 was also detected, to a lesser extent, on the Schwann sheath of peripheral nerve fibres, ependymal cells and certain epithelial cells such as acinar cells of the salivary gland, bronchial epithelium, renal tubules and squamous epithelium. The distribution pattern of HRF20 differed somewhat from that of decay-accelerating factor (DAF), which is another membrane inhibitor of homologous complement activation.

Desmopressin is a potent vasorelaxant of aorta and pulmonary artery isolated from rabbit and rat.

Hypotension related to the intraoperative use of desmopressin acetate to improve platelet function following cardiopulmonary bypass has recently been reported. To investigate the direct vascular actions of this drug as a potential mechanism of its induced hypotension, cumulative, dose-dependent (3.7 X 10(-10) to 1.2 X 10(-7) M) effects of desmopressin were studied in isolated phenylephrine precontracted rings of rat and rabbit thoracic aorta and rabbit pulmonary artery. Desmopressin was a potent vasodilator of all vessel types studied with significant (P less than 0.05) vasodilation beginning at 7.5 X 10(-9) M. Vascular relaxation of all vessels was greater when the vascular endothelium was intact (P less than 0.05). Indomethacin potentiated (P less than 0.05) vascular relaxation in rat and rabbit aortic rings and partially inhibited (P less than 0.05) relaxation in rabbit pulmonary artery rings. Selective antagonists of vasopressin V1 (d(CH2)5-Tyr(Me)AVP, 1 X 10(-6) M) and V2 (d(CH2)5[D-Ile2,Ala-NH2(9)] AVP, 1 X 10(-6) M) receptors and of histamine H1 (diphenhydramine, 1 X 10(-5) M) and H2 (cimetidine 1 X 10(-5) M) receptors had no effect on desmopressin-induced relaxation of rat aortic rings. Chlorobutanol, the diluent in which desmopressin is supplied, was devoid of vascular effects. To study the effects of desmopressin on vascular cyclic GMP and cyclic AMP concentrations, a cultured bovine aortic smooth muscle--rat vascular smooth muscle coculture model was employed. Desmopressin (1 X 10(-7) and 1 X 10(-8) M) did not significantly alter control values of either cyclic nucleotide.(ABSTRACT TRUNCATED AT 250 WORDS)

A human endothelial cell membrane protein that binds Staphylococcus aureus in vitro.

We have investigated S. aureus adherence to human endothelial cells utilizing an in vitro model. Staphylococcus binding to confluent endothelial cell monolayers was saturable in both dose and time response studies suggesting that the binding interaction was specific. We have developed a technique, based on the pH dependent affinity of iminobiotin for streptavidin, for the isolation of an endothelial cell membrane component that binds S. aureus, in vitro. A 50-kD membrane component was isolated and purified using this approach. This component was trypsin sensitive, periodate insensitive, and did not label with [3H]glucosamine. [35S]Methionine and [125I]iodine labeling confirmed that the protein was synthesized by and expressed on the endothelial cell surface. Functional binding studies demonstrated that staphylococci, but not endothelial cells, bound to the protein when immobilized on microtiter wells. Preincubation of staphylococci with the purified protein significantly (P less than 0.001) reduced staphylococcal binding to cultured endothelial cells. The capacity of S. aureus to colonize and invade endovascular surfaces may in part be a consequence of staphylococcal interaction with this endothelial cell membrane protein.

Glycosaminoglycans of bovine aorta endothelial cells: identification and localization by use of a platelet factor 4-fluorescein probe.

We utilized platelet factor 4 (PF4) conjugated to fluorescein to stain the proteoglycans of permeabilized fixed bovine aorta endothelial cells in monolayer culture. Treatment of the monolayers with chondroitin ABC lyase and/or a preparation from Flavobacterium heparinum was used to remove chondroitin sulfate and/or heparan sulfate before staining, with resultant separate identification and partial localization of these glycosaminoglycans. When PF4-fluorescein was utilized with untreated control monolayers, fairly uniform reticular, perinuclear, and cell surface fluorescence was seen. After treatment with chondroitin ABC lyase, fluorescence was retained only on the cell surface. In contrast, treatment with the F. heparinum preparation resulted in the loss of all cell surface fluorescence. Use of both glycosaminoglycan lyases together resulted in loss of essentially all the fluorescence. The cell surface heparan sulfate observed by fluorescence after removal of cell surface chondroitin sulfate appeared to be unevenly distributed, with a heavier accumulation at one pole of each cell. This technique offers a specific method for identification and partial localization of cell surface heparan sulfate.

Phospholipid molecular species in human umbilical artery and vein endothelial cells.

Molecular species of several phospholipid classes and subclasses were quantitatively determined in human umbilical artery and vein endothelial cells. Both types of endothelial cells were similar in phospholipid class composition, whereas they were markedly different in phospholipid subclass and molecular species composition. The amounts of two ether subclasses in phosphatidylcholine and phosphatidylethanolamine were higher in artery endothelial cells than those in vein endothelial cells. The relative content of alkylacyl subclass in phosphatidylcholine, a precursor of platelet-activating factor, was about three times higher in artery endothelial cells than in vein endothelial cells. In artery endothelial cells, arachidonic acid was in highest amounts in alkenylacyl phosphatidylethanolamine, followed by diacyl phosphatidylcholine, diacyl phosphatidylethanolamine, and phosphatidylinositol. In the vein endothelial cells, arachidonic acid was highest in phosphatidylinositol, followed by diacyl phosphatidylethanolamine, diacyl phosphatidylcholine, and alkenylacyl phosphatidylethanolamine. Artery endothelial cells had higher amounts of molecular species containing arachidonic acid than vein endothelial cells in all phospholipid classes and subclasses. These differences are thought to reflect the functional differences of artery and vein endothelial cells.

Immunoelectrophoretic analysis of membrane glycoproteins in cultured human endothelial cells.

Human endothelial cells isolated from umbilical cords were solubilized in Triton X-100 and examined by crossed immunoelectrophoresis using rabbit antiserum against endothelial cells. Endogenous labelling of the endothelial cell proteins with 14C-mannose followed by crossed immunoelectrophoresis and autoradiography revealed about 10 immunoprecipitates. Four of these endothelial cell glycoproteins were labelled by lactoperoxidase catalyzed iodination and thus were surface located. Three of the surface located glycoproteins showed reduced electrophoretic mobility after incubation of the endothelial cells with neuraminidase and were therefore sialoglycoproteins. Amphiphilicity of endothelial cell glycoproteins was studied by crossed hydrophobic interaction immunoelectrophoresis with phenyl-Sepharose in the intermediate gel. Amphiphilic proteins also show increasing electrophoretic migration velocity with decreasing concentration of Triton X-100 in the first dimension gels. Five of the endothelial cell glycoproteins were shown to be amphiphilic using these two techniques. Two monoclonal antibodies against the platelet glycoprotein complex IIb-IIIa and glycoprotein IIIa, respectively, reacted with the same precipitate of endothelial cells. When a polyclonal antibody against the platelet glycoprotein complex IIb-IIIa was incorporated into the intermediate gel the position of two endothelial cell precipitates were lowered. One of these was a sialoglycoprotein.

Endothelium-derived constricting factor(s): the last novelty--endothelin.

The vascular endothelium is not merely a passive physical barrier between the blood and the tissue surrounding the blood vessel, but may actively participate in key processes of metabolic, secretory, and vasoregulatory character. In addition, the endothelium plays an important role in the control of platelet activation. Under certain conditions endothelial cells have been shown to produce powerful vasodilators, like endothelium-derived relaxing factor (EDRF) and prostacyclin (GPI2), and vasoconstrictors like endothelium-derived constricting factor (EDCF) and endothelin (ET) (Griffith et al., 1988; Vanhoutte & Katusic, 1988). In contrast to the extensive studies performed to characterize the actions and nature of EDRF, recently identified chemically as nitric oxide (Moncada et al., 1988), relatively little is known about EDCF(s). This paper reviews recent data on EDCF, with special emphasis on the newly discovered vasoconstrictor peptide, endothelin (ET).

A sandwich-type enzyme immunoassay to detect immunoreactive big-endothelin-1 in plasma.

A sensitive sandwich-type enzyme immunoassay (sandwich-EIA) has been established for human big endothelin-1 (big-ET-1). A monoclonal antibody AwETN40 which recognizes the N terminal portion of big-ET-1 was used as an immobilized antibody. The Fab' fragment of rabbit antibodies to the human big-ET-1 C terminal peptide (22-38) was used as the enzyme-labeled antibody after being coupled to horseradish peroxidase (HRP). The assay is sensitive enough to detect as little as 0.2 pg/well (5 x 10(-17) mol/well) of big-ET-1 without crossreactivity with ET-1. Immunoreactive big-ET-1 in plasma was determined after being extracted with Sep-pak C-18 cartridges. The average levels were 5.2 +/- 1.0 pg/ml for males and 5.7 +/- 1.6 pg/ml for females (the values were corrected for recovery efficiency). Big-ET-1 was found to be eliminated from the blood stream at a slower rate than ET-1. This may be an important reason for the higher plasma levels of immunoreactive big-ET-1 compared to the levels of immunoreactive ET-1.

PECAM-1 (CD31) cloning and relation to adhesion molecules of the immunoglobulin gene superfamily.

An antibody to a platelet integral membrane glycoprotein was found to cross-react with the previously identified CD31 myelomonocytic differentiation antigen and with hec7, an endothelial cell protein that is enriched at intercellular junctions. This antibody identified a complementary DNA clone from an endothelial cell library. The 130-kilodalton translated sequence contained six extracellular immunoglobulin (Ig)-like domains and was most similar to the cell adhesion molecule (CAM) subgroup of the Ig superfamily. This is the only known member of the CAM family on platelets. Its cell surface distribution suggests participation in cellular recognition events.

Factor VIII von Willebrand protein in haemolytic uraemic syndrome and systemic vasculitides.

von Willebrand protein (vWF) is reduced by dithiothreitol (DTT) and, as a result, is not detected by enzyme-linked immunosorbent assay (ELISA). Plasma samples from normal subjects, children with haemolytic uraemic syndrome (HUS), and adults with vasculitis and vWF prepared from endothelium were treated with DTT before vWF assay. vWF in HUS and vasculitis resembled the endothelial form in being resistant to reduction. DTT modification of the ELISA assay may be useful as a marker of disease severity in conditions associated with endothelial cell damage.