Robert O. Kelley

TIMP-2 reduces proteolytic opening of blood-brain barrier by type IV collagenase

Intracerebral hemorrhage occurs in tumors, stroke and head trauma. Proteolysis of the extracellular matrix around cerebral capillaries by naturally occurring mammalian 72-kDa type IV collagenase may initiate this pathologic event. To investigate this hypothesis adult rats underwent intracerebral injection of type IV collagenase purified from human melanoma cells. Histologically, at 4 h there was perivascular cellular infiltration with hemorrhage, and by 24 h there was infarction with necrosis, edema and hemorrhage. Ultrastructurally, the basal lamina of endothelial cells was disrupted at 2 h. Brain uptake of [14C]dextran and [3H]sucrose increased after intracerebral injection of type IV collagenase compared to controls (P less than 0.0001). Tissue inhibitor of metalloproteinase-2 (TIMP-2) reduced the tracer uptake (P less than 0.02). Metalloproteinase inhibitors reduce extracellular matrix proteolysis and protect the blood-brain barrier.

Ligand-mediated osmium binding: Its application in coating biological specimens for scanning electron microscopy

The osmium-bridging properties of thiocarbohydrazide have been used to bind additional osmium to a variety of soft tissue surfaces before critical-point drying. The procedure gives satisfactory protection to specimens under a scanning electron beam at accelerating voltages between 5 and 25 kV and provides the microscopist with a simple, efficient, and economical alternative to evaporative coating with other heavy metals prior to analysis in the scanning electron microscope.

Bacterial collagenase disrupts extracellular matrix and opens blood-brain barrier in rat ☆

Bacterial collagenase causes hemorrhagic necrosis of brain. We studied the enzymes effect on blood-brain barrier (BBB) permeability and extracellular matrix (ECM) structure by radiolabeled tracers and electron microscopy. Adult rats had intracerebral injection of bacterial collagenase. Brain uptake from blood of [14C]sucrose was measured in 24 rats 0.5 h to 14 days after injection. 12 rats had ultrastructural studies 1 h after collagenase injection. Brain uptake of [14C]sucrose is maximally increased at 0.5 h, remaining significantly increased for 7 days. Ultrastructurally, some vessels had widening of basal lamina while others had severe disruption of basal lamina with stretching of endothelial cells. We conclude that bacterial collagenase disrupts ECM and opens BBB.

Loss of organized fibronectin matrix from the surface of aging diploid fibroblasts (IMR-90)

Indirect immunofluorescence was used to investigate the production of a fibrillar fibronectin matrix by human diploid fibroblasts (IMR-90) as cells progress through their in vitro lifespan. Early and mid-passage cultures displayed a prominent fibrillar reticulum over the cell layer which formed within 24 hours of seeding. Even sparsely seeded early-passage cells exhibited fibrils of fibronectin on external surfaces. In contrast, fibrillar fibronectin was reduced or absent on surfaces of late-passage cells. However, the larger, non-proliferating, late-passage cells were producing fibronectin, 1h determined by radioimmunoassay of the medium.

An ultrastructural analysis of cell and matrix differentiation during early limb development in Xenopus laevis

Abstract Early development of the hind limb of Xenopus (stages 44–48) has been analyzed at the level of ultrastructure with emphasis on differentiation of extracellular matrix components and intercellular contacts. By stages 44–45, mesenchyme is separated from prospective bud epithelium by numerous adepidermal granules in a subepithelial compartment (the lamina lucida), a continuous basal lamina and several layers of collagen (the basement lamella). Tricomplex stabilization of amphoteric phospholipid demonstrates that each adepidermal granule consists of several membranelike layers (electron-lucent band 25–30 A; electron-dense band 20–40 A), which are usually parallel to the basal surface of adjacent epithelial cells. Collagen fibrils are interconnected by filaments (35 A in diameter) which stain with ruthenium red. Epithelial cells possess junctional complexes at their superficial borders, numerous desmosomes at apposing cell membranes and hemidesmosomes at their basal surface. Mesenchymal cells predominantly exhibit close contacts (100–150 A separation) with few focal tight junctions at various areas of their surface. By stages 47–48, adepidermal granules are absent beneath bud epithelium and layers of collagen in the basement lamella lose filamentous cross-linking elements. Filopodia of mesenchymal cells penetrate the disorganized matrix and abut the basal lamina. Hemidesmosomes disappear at the basal surface of the epidermis and mesenchymal cells immediately subjacent to epithelium exhibit focal tight junctions and gap junctions at their lateral borders. These structural changes may be instrumental in the epitheliomesenchymal interactions of early limb development. Degradation of oriented collagenous lamellae permits direct association of mesenchymal cell surfaces (filopodia) with surface-associated products of epithelial cells (organized into the basal lamina). Development of structural pathways for intercellular ion and metabolite transport in mesenchyme may coordinate events specific to limb morphogenesis.

Development of the aging cell surface: variation in the distribution of intramembrane particles with progressive age of human diploid fibroblasts.

Cell membranes of early and late passage human embryo fibroblasts were freeze-fractured and the distribution of intramembrane particles was compared using statistical methods. Aged cells do not exhibit dramatic differences in total numbers of intramembrane particles when compared to younger cells. However, significant alterations develop with age between the distribution of particles on P and E fracture faces. Younger cells exhibit more particles on their P faces than corresponding membrane halves from older cells. In contrast, the E fracture faces of aged cells have significantly more particles than do corresponding surfaces of younger cell membranes. The observations suggest that properties of membrane fluidity may change with increased cellular age.

Variation in cytoskeletal assembly during spreading of progressively subcultivated human embryo fibroblasts (IMR-90)

The cytoskeletons of early and late passage IMR-90 human diploid fibroblasts have been directly imaged in replicas of Triton X-100 extracted cells during spreading following reseeding. All cells from both young and sensescent cultures exhibit a cytoskeletal network of actin microfilaments, intermediate (10 nm) filaments, microtubules, and interconnecting thin filaments (6-8 nm in diameter) which do not interact with heavy meromyosin. Early passage cells assemble linear aggregates of actin filaments within 1 h of spreading. By 4 h of incubation, these bundles establish a structural bond with the cell membrane which results in resistance by the plasmalemma to detergent extraction at these sites. Furthermore, these membrane regions are associated with developing stress fibers of well-spread cells. In contrast, late passage cells exhibit slower spreading which correlates with a retarded assembly of actin bundles. In addition, by 8 h of spreading, cells of older cultures do not exhibit the regions of membrane-actin interaction which impart detergent resistance to the plasmalemma. We conclude that the ability to reassemble actin-actin and actin-membrane association during cell spreading is reduced with increased serial subcultivation of cells.

Development of the aging cell surface: Reduction of gap junction-mediated metabolic cooperation with progressive subcultivation of human embryo fibroblasts (IMR-90)

Abstract Gap junctions develop between IMR-90 fibroblasts in cultures which have been serially subcultivated until reduced proliferative rates are produced. However, distribution is sparse and the junctions exhibit relatively few component subunits. To investigate whether a concomitant reduction in the ability of senescent cells to maintain metabolic coupling occurs, autoradiography was used to determine the relative exchange of [ 3 H]uridine nucleotides between early and late passage cells. In addition, the rate of reappearance of gap junctions between dissociated cells of both sample groups was examined by freeze-fracturing and electron microscopy. Gap junctions form between young cells approx. 1 h after reassociation in culture. Structurally complete gap junctions are present within 2 h. In contrast, membranes of cells in senescent cultures do not exhibit particle arrays typical of forming junctions until one or more hours of incubation. Small but assembled junctions are observed after 4 or more hours of reassociation. Autoradiography reveals some 70% of young cells to be receiving label from young donors following a 4 h period of metabolic exchange, whereas only 30% of older cells receive label after comparable periods of incubation. Statistical analyses of reciprocal experiments using young donor/old recipients and old donor/young recipients suggest that recipient cells may have a slightly larger effect on the exchange reaction than donor cells. We conclude that contact-mediated metabolic cooperation is reduced with increased serial subcultivation of cells.

Cell surface oligosaccharide modulation during differentiation. I. Modulation of lectin binding.

IMR-90 human lung fibroblasts exhibit quantitative and qualitative changes in their cell surface oligosaccharide expression as a result of low-density growth, contact inhibition of growth and changes in population doubling level (PDL). Binding of Concanavalin-A (CON-A), wheat germ agglutinin (WGA) and Ricinus communis (RCA-120) was shown to decrease by 20-40% as the cells advanced through their lifespan (but significantly before morphological manifestations commonly associated with senescence, which occur at approximately PDL 45). The binding of Dolichos biflorus agglutinin (DBA) increased by 100% over the same time interval. These changes were detected by quantitation of FITC conjugated lectins on single cells and the use of a sensitive analytical biotin-avidin-enzyme amplification assay on whole populations of cells. These studies were performed on randomly growing cultures. These results suggest an important and dynamic role for the cell surface oligosaccharide in growth control and cellular senescence.

Development of the aging cell surface: Concanavalin a-mediated intercellular binding and the distribution of binding sites with progressive subcultivation of human embryo fibroblasts

External surfaces of early and late passage human embryo fibroblasts were reacted with concanavalin A to determine whether quantitative and qualitative variations in receptor sites develop with increased serial subcultivation. Comparative analyses of direct con A binding to cell surfaces; lectin-mediated cell-to-cell binding and agglutination; and ultrastructural distribution of con A receptor sites were made on the surfaces of both cell groups. Subtle variations were observed in the patterns of intercellular binding between early and late passage cells as assayed by both agglutination and the binding of cells in suspension to substrate-attached monolayers. However, no major differences in the total number of binding sites per cell were expressed on the external surfaces of either group. Hemocyanin-labeled binding sites tended to be more clustered on membranes of late passage cells in contrast to more homogeneous patterns of distribution in early passage specimens. These observations suggest that variations in binding patterns are not the result of changes in numbers of binding sites but may be the result of alterations in the concerted actions of numerous factors which include cell surface topography (e.g. villous projections) and the relative distribution of lectin binding sites on the cell periphery.