Marketta Hormia

Factor VIII-related antigen: a pericellular matrix component of cultured human endothelial cells

We studied the extracellular localization of factor VIII-related antigen (VIIIR: Ag) in cultures of human endothelial cells. The cells deposited both VIIIR: Ag and fibronectin already during their initial adhesion phase and in immunofluorescence microscopy of spread cells extracellular VIIIR: Ag was localized to fibrils coaligning with pericellular fibronectin. When human fibroblasts, which do not synthesize VIIIR: Ag, were cultured in endothelial cell post-culture medium, a fibrillar matrix localization of VIIIR: Ag was seen, comparable to that of endothelial cell cultures. A fibrillar VIIIR: Ag-specific staining was also seen in cell-free pericellular matrices of endothelial cells, produced by deoxycholate treatment. In immunoelectron microscopy, VIIIR: Ag was seen in fibrillar extracellular material between and underneath the cells and in cell-free matrices of of endothelial cells as well. In immunofluorescence microscopy of cell-free matrices, VIIIR: Ag codistributed with both fibronectin and type III procollagen. Digestion of the matrices with purified bacterial collagenase abolished the type III procollagen-specific fluorescence, whereas the fibrillar VIIIR: Ag-specific staining, codistributing with fibronectin, remained unaffected. In electrophoresis of isolated, metabolically labelled endothelial cell matrices, major polypeptides with Mr 220-240; 180; 160 and 45 kD and some minor polypeptides were resolved. In addition, immunoblotting revealed fibronectin, VIIIR: Ag and type III procollagen as components of cell-free matrices of endothelial cells. Direct overlay of iodinated cellular fibronectin on electrophoretically separated polypeptides of cultured endothelial cells, transferred to nitrocellulose, suggested that fibronectin binds directly to VIIIR: Ag. Our results indicate that VIIIR: Ag produced by human endothelial cells is a component of the pericellular matrix and is not bound to collagen but may directly associate with fibronectin.

Immunolocalization of Integrin α6β4 in Mouse Junctional Epithelium Suggests an Anchoring Function to both the Internal and the External Basal Lamina

The localization of the integrin α 6β4, a transmembrane adhesion molecule associated with hemidesmosomes, was studied in mouse junctional epithelium (JE) by the use of monoclonal antibodies in indirect immunofluorescence microscopy. The results showed that the integrin a6 subunit was expressed throughout the JE and was localized to the cell membranes, including the aspects facing the internal and external basal laminae. The β4 subunit had a more restricted distribution. It was expressed only in cells facing the internal and the external basal laminae and had a basally polarized distribution. In other parts of gingival epithelium, both integrin subunits were mainly expressed at the basal aspects of basal epithelial cells. The basement membrane components, type IV collagen and laminin, could be detected only in the external basal lamina and in other basement membranes of gingival epithelium. The results indicate that the a6β4 integrin, expressed in mouse JE, has a role in mediating the attachment of the cells to the basement membranes facing the connective tissue and the tooth.

Identification of UEA I-binding surface glycoproteins of cultured human endothelial cells

Ulex europaeus agglutinin (UEAI) binds mainly to endothelial cells in human tissues. In cultured human umbilical vein endothelial cells TRITC-UEAI gave an even surface staining but no binding to pericellular material. After permeabilization of the cells UEAI decorated the Golgi apparatus as a juxtanuclear structure. Electrophoresis of Triton X-100 lysates of 35S-methionine labeled cells bound to lectin agarose beads showed that a similar set of polypeptides was recognized by UEA-I and WGA while distinctly different polypeptides were bound to LcA-agarose. Surface labelling revealed major glycoproteins with Mr 220 kD, 160 kD, 140 kD, 120 kD, 80 kD and 50 kD, most of which could be extracted with Triton X-100. However, only the 140 kD gp, 120 kD gp and 80 kD gp showed binding to UEA 140 kD gp, 120 kD gp and 80 kD gp showed binding to UEA I-lectin. The results show that among a distinct set of surface glycoproteins in cultured human endothelial cells only a few have alpha-l-fucosyl moieties capable of binding to UEAI lectin.

The Junctional Epithelium around Murine Teeth differs from Gingival Epithelium in its Basement Membrane Composition

It is not known whether epithelial differentiation patterns are reflected in the composition of gingival basement membranes (BMs). We have investigated the expression of laminin isoforms and associated BM components in the murine dento-epithelial junction by using immunofluorescence microscopy. Our results show that chains of laminins 5/6/7/10/11 are expressed in the BM of outer gingival epithelium. The external BM between junctional epithelium (JE) and connective tissue differs from gingival BM by lacking laminin-7 and -11 chains. The internal basal lamina (IBL) between JE and tooth contains only laminin-5. Collagen chains α1,2(IV) and nidogen-1 are present in other BMs except the IBL. The dento-epithelial junction thus has a unique BM composition, suggesting that epithelial cells are able to secrete two extracellular matrices in a polarized manner. The exclusive expression of the non-self-polymerizing laminin-5 indicates that the IBL is not a BM by definition, but rather a simple extracellular matrix lacking network structure.

Actomyosin organization in stationary and migrating sheets of cultured human endothelial cells

We used immunofluorescence microscopy to study the organization of actin, myosin and vinculin in confluent endothelial cells and in cells migrating into an experimental wound and interference reflection microscopy to assess the cell-substratum adhesion pattern in these cells. In confluent stationary endothelial cell monolayers actin showed a distinct cell-to-cell organization. Myosin, on the other hand, was diffusely distributed and was clearly absent from cell peripheries. Vinculin was confined as linear arrays to cell-cell contact areas. Interference reflection microscopy revealed areas of close and distant adhesion but no focal adhesion sites in these cultures. Twelve hours after experimental wounding a distinct zone of advancing cells was seen at the wound edge. These cells showed a spreadout morphology and, in contrast to stationary cells, had a stress fibre-type organization of both actin and myosin. Vinculin was in the migrating cells seen as plaques at the ventral cell surface. In interference reflection microscopy numerous focal adhesions were seen. The results indicate that the actomyosin system forms the structural basis for monolayer organization of endothelial cells and responds by reorganization upon cell migration.

Identification of cytoskeletal intermediate filaments of vascular endothelial cells as targets for autoantibodies in patient sera

Abstract Autoantibodies present in sera of patients suffering from various inflammatory conditions react with fibrillar structures, “microfibrils” (MF), which are abundant in developing fetal connective tissue. The purpose of this study was to further characterize the target antigens and to look for potential antigenic target structures in the adult organism using the indirect immunofluorescence (IFL) technique. Bright vascular endothelial staining was regularly observed in various tissues, e.g., kidneys, skin, and vessels accompanying nerves using anti-MF-positive patient area. A weak staining of the media was seen in arteries. Placental vessels were convenient in vitro targets in a screening assay for anti-MF antibodies. The cytoplasmic structures responsible for the endothelial cell fluorescence were identified using cultured umbilical vein endothelial cells as the IFL substrate. In these cells the fluorescence had the typical perinuclear distribution and fibrillar appearance of cytoskeletal intermediate filaments (IMF). We could further show that anti-IMF and anti-MF autoantibodies gave indistinguishable staining patterns. Some anti- MF IMF -positive sera could be neutralized by “insoluble” placental preparations obtained by procedures for preparation of IMF polypeptides. Immunization with a 50,000-dalton “insoluble” polypeptide produced antibodies with a reactivity similar to that of the autoantibodies. The IFL staining and absorption results were consistent with the ultrastructurally demonstrated large amounts of intermediate filaments in vascular endothelial cells. The results suggest that circulating anti-IMF autoantibodies might be involved in inflammatory reactions in cases of vascular endothelial injury.

Dolichos biflorus agglutinin (DBA) reacts selectively with mast cells in human connective tissues

Dolichos biflorus agglutinin (DBA) binds to N-acetyl-D-galactosamine (GalNAc) residues in glycoconjugates and agglutinates erythrocytes carrying blood group antigen A. In cryostat sections of various tissues from blood group-specified humans, fluorochrome-coupled DBA bound preferentially to fusiform connective tissue cells and to certain epithelial cells. The connective tissue cells were identified as mast cells by their typical metachromasia in consecutive staining with toluidine blue. Double labeling with DBA and conjugated avidin revealed two distinct populations of mast cells. In several tissues the DBA-reactive cells likewise displayed uniform avidin reactivity. In intestinal mucosa, however, morphologically distinct DBA-binding mast cells were found, which were labeled with the avidin conjugates only in specially fixed paraffin sections. DBA did not bind to vascular endothelial cells, which could be identified by double staining with antibodies to factor VIII-related antigen. Labeling with Helix pomatia agglutinin (HPA), another blood group A-reactive lectin, resulted in distinct blood group-dependent fluorescence of the endothelia. Sophora japonica agglutinin (SJA), a blood group B-reactive lectin, labeled vascular endothelial cells in tissues from blood group A, AB, and B donors. HPA and SJA reacted with small mast cells in the gastrointestinal mucosa but failed to label large mast cells in any of the tissues. These results indicate that the blood group reactivity of lectins, as determined by erythroagglutination, is not necessarily consistent with their reactivity with blood group determinants in tissue sections. Moreover, DBA conjugates appear to be a reliable probe for detection of mast cells in various human connective tissues.

Vimentin filaments in cultured endothelial cells form butyrate-sensitive juxtanuclear masses after repeated subculture

Abstract In primary cultures of human umbilical vein endothelial cells vimentin-specific fluorescence was seen in spreading cells, as distinct juxtanuclear aggregates and in fully spread cells as typical perinuclear coils and cytoplasmic fibrils. After repeated subculture the cells increased in size and showed vimentin-specific staining as juxtanuclear cap-like aggregates also in fully spread cells. Treatment of late-passage cultures with sodium butyrate led to flattening of the cells and reappearance of fibrillar vimentin organization. Treatment of the late-passage cells with anti-mitotic drugs brought about the formation of solitary juxtanuclear masses of vimentin. In primary cultures, an occasional coalignmelt of microtubules and vimentin filaments was seen in double IIF, but in late-passage cells only after culture in the presence of sodium butyrate. Similarly centrioles could be shown to be close to the juxtanuclear vimentin aggregates. The results indicate that vimetin filaments in cultured umbilical vein endothelial cells have a spontaneous tendency to aggregate independently of the organization of microtubules.

Psophocarpus tetragonolobus Agglutinin Reveals N-Acetyl Galactosaminyl Residues Confined to Endothelial Cells and Some Epithelial Cells in Human Tissues'

We studied the binding of Psophocarpus tetragonolobus agglutinin (PTA) conjugates to human adult tissues. In all kidney specimens studied, PTA bound in a blood group-independent way to endothelia in glomerular and intertubular capillaries as well as in larger vessels. In addition, a heterogeneous binding to collecting duct cells was seen. In specimens of human smooth, cardiac, and skeletal muscle, cerebellum, lung, thyroid gland, liver, proliferative endometrium, and placenta, PTA bound only to endothelial of capillaries and larger vessels. In epidermis and gingiva, PTA conjugates additionally revealed reactivity with keratinocytes. Similarly, in salivary gland, urinary bladder, gastrointestinal tract, mammary gland, and renal pelvis, PTA reacted with some epithelial cell layers. The PTA conjugates gave an even cell surface membrane staining of cultured umbilical vein endothelial cells. Lectin-affinity binding of radioactively surface-labeled endothelial cells showed that PTA and Ulex europaeus I agglutinin (UEA-I) recognized related major cell surface glycoproteins. The results with PTA conjugates show that certain N-acetyl galactosaminyl residues are, in addition to some epithelial cells, confined to endothelial cells in human tissues.

Surface glycoproteins of cultured human umbilical vein endothelial cells

Radioactive surface-specific and metabolic labeling techniques were used to characterize the surface glycoprotein pattern of cultured human endothelial cells. Electrophoretic analysis of whole cells, surface labeled either by the galactose oxidase/sodium borotritide or the periodate/sodium borotritide method, revealed several major polypeptides in the Mr region of ca 40-220. During primary culture, the surface labeling pattern showed no changes related to cell density or to the establishment of confluence. A slightly different polypeptide profile was, however, seen when primary culture cells were labeled as an intact monolayer and not in suspension. On the other hand, in cells from later passages, when compared to their parental cells of early passages, there was a distinct intensification of polypeptides with Mr 155 and 90.