Biomedical subjects

N Sorgente

Publications and source records attributed to N Sorgente.

At least 37 records · Page 2Linked to original sources

PubMed1988

Human retinal pigment epithelial cell cultures: phenotypic modulation by vitreous and macrophages.

In proliferative vitreoretinopathy (PVR), retinal pigment epithelial (RPE) cells migrate into the vitreous, where they may acquire a fibroblast-like morphology. Such cells may eventually form contractile periretinal membranes, resulting in traction retinal detachment. Among the environmental influences that could cause this change in RPE phenotype, exposure to vitreous and to macrophages is most obvious, as macrophages are invariably found in epiretinal membranes and precede membrane formation in experimental traction retinal detachment. We initiated studies to define modulation of cultured RPE cell morphology by exposure to vitreous or to macrophage-conditioned media. Vitreous, serum, and albumin alone had no effect on the epithelial appearance of RPE cells in vitro. However, macrophage-conditioned media and vitreous-serum or vitreous-albumin mixtures induced a reversible fibroblast-like appearance in these cells. These findings show that macrophages produce a morphoplastic substance for RPE cells, and suggest that vitreous also contains a factor(s) that affects RPE cell shape, and that requires mediation by serum components.

Actins↗

PubMed1988

Breakdown of Bruch's membrane after subretinal injection of vitreous. Role of cellular processes.

It was recently shown that the injection of autologous vitreous beneath the retina of rabbits leads to retinal degeneration, subretinal cellular proliferation and neovascularization. The current study, using electron microscopy, was designed to determine the cellular processes involved in the breakdown of Bruch's membrane in this model. Bruch's membrane was not mechanically damaged by the injection and appeared intact for the first 1 to 2 days after injection. Subsequently, numerous breaks in Bruch's membrane were found associated with invasion of macrophages and fibroblasts; in addition, budding and penetration of retinal pigment epithelial (RPE) and choroidal endothelial cells into Bruch's membrane were noted. Although it was not proven that these cells, per se, were responsible for the breaks, that these cells actively penetrate Bruch's membrane is a reasonable hypothesis.

Animals↗

PubMed1988

Experimental changes resembling the pathology of drusen in Bruch's membrane in the rabbit.

Drusen-like changes in Bruch's membrane following subretinal injection of vitreous in the rabbit were studied by electron microscopy. A sequence of changes is seen that closely panellels those observed during drusen formation in primates. The initial event is the budding of retinal pigment epithelial (RPE) cells into Bruch's membrane; the buds, which contain cytoplasm and plasma membrane, are connected to the cytoplasm of the parent RPE cell. Most buds are surrounded by basement membrane, but some penetrate RPE basement membrane into Bruch's membrane. Later, RPE buds completely separate from the parent RPE cell and show degeneration and disintegration. Finally, drusen-like material, including vesicular, granular, tubular and linear structures, is dispersed from the bud remnant into Bruch's membrane. The study described herein thus supports the hypothesis that drusen are the result of budding from the RPE.

Animals↗

PubMed1988

Liquefaction of rabbit vitreous by ferrous ions.

Intravitreal injection of 0.7 mumol of ferrous chloride in 0.1 ml into the rabbit eye resulted in liquefaction of the vitreous gel and condensation of vitreous collagen fibrils within two weeks; injection of 0.1 mumol did not cause any obvious vitreous degeneration, although retina damage was noted in the posterior pole. Macrophages migrated at the vitreoretinal interface and local posterior vitreous separation was observed after the injection of ferrous solution. This suggests that the least amount of ferrous ions necessary to cause liquefaction of the rabbit vitreous is in the range of 16.8 to 39.2 micrograms of elemental iron, a concentration of 0.3 to 0.7 mM in the vitreous. Since 0.1 ml of blood contains approximately 50 micrograms of iron, it is possible, at least theoretically, that the iron released by hemoglobin following vitreous hemorrhage could induce liquefaction of the vitreous.

Animals↗

PubMed1987

Separation of luminal and abluminal membrane enriched domains from cultured bovine aortic endothelial cells: monoclonal antibodies specific for endothelial cell plasma membranes.

Two kinds of membrane (luminal and abluminal membrane domains) fractions have been isolated from bovine aortic endothelial cells by fractionation of whole cell homogenate on discontinuous sucrose density gradients. The luminal membrane domain was enriched 12-16-fold for angiotensin-converting enzyme activity and 8-10-fold in alkaline phosphatase activity. The abluminal membrane domain displayed an enrichment of 8-fold in (Na+ + K+)-ATPase activity. Both of the membrane domains were minimally contaminated with mitochondria, microsomes and Golgi bodies, as assessed by their corresponding marker enzyme activities. 125I-labeling of endothelial cell monolayers by the Enzymo-Bead lactoperoxidase-catalyzed iodination procedure, followed by isolation of membranes, revealed that the radioactivity was predominantly associated with membranes enriched in angiotensin-converting enzyme activity, corresponding to the luminal membrane domain. However, when cells were radioiodinated in suspension culture, radioactivity was found equally associated in both the luminal and abluminal membrane fractions. Electron microscopy of freeze-fractured and sectioned material showed both luminal and abluminal membrane domains to be in the form of vesicles varying in size from 100 to 400 nm in diameter. To characterize the separation of endothelial cell membrane domains, we have attempted to prepare monoclonal antibodies specific for endothelial cells. Several clones were obtained, producing antibodies which bound to endothelial cells of arterial, venous and capillary origin. Two antibodies of these clones, XIVC6 and XVD2, were studied in more detail. In the ELISA assay, these antibodies reacted with bovine vascular endothelial cells, but not with human umbilical cord endothelial cells, nor with bovine corneal endothelial cells, smooth muscle cells or fibroblasts. Both of these antibodies are directed against an antigen of approximately 130 kDa, under reducing and non-reducing conditions, as assayed by the immunoprecipitation method. Western blot analysis of luminal and abluminal membrane fractions revealed that only MAb XVD2 reacted with an antigen, indicating that the antibody XIVC6 is directed against an epitope which is denatured by SDS. Moreover, MAb XVD2 preferentially reacted with the luminal membrane compared to the abluminal membrane domain of the endothelial cell. These monoclonal antibodies do not react with platelet membrane proteins, indicating that this 130 kDa membrane antigen is not common to both endothelial cells and platelets.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1987

Increased adherence of oxidant-treated human and bovine erythrocytes to cultured endothelial cells.

Bovine erythrocytes, which normally lack phosphatidyl choline in their membranes, when treated with either H2O2 or diamide (1-3 mM), showed a partial appearance of phosphatidyl ethanolamine (PE 40%) and phosphatidyl serine (PS, 30-33%) in the external leaflet of the bilayer and a concomitant increased (four- to five-fold) propensity to adhere to cultured bovine aortic endothelial cells. Similar treatment of normal human erythrocytes caused an alteration in the organization of the phospholipid bilayer and also resulted in their increased adherence to endothelial cells derived either from human umbilical vein or bovine aorta. Treatment of RBCs with H2O2 at low concentration (0.5 mM) resulted in cross-linking of spectrin without significant changes in the orientation of aminophospholipids but the RBCs exhibited 15-20% increase in adherence to endothelial cells. Pretreatment of either human or bovine erythrocytes with antioxidants such as vitamin E (2 mM) prevented both oxidant-induced reorganization of phospholipids in the bilayer and enhancement of adherence to endothelial cells. Introduction of either phosphatidyl serine or phosphatidyl ethanolamine but not phosphatidyl choline into erythrocyte membranes increased their adherence to endothelial cells threefold. Oxidant-treated RBCs exhibited enhanced binding and fluorescence of Merocyanine 540 dye (MC-540), which is sensitive to the packing of lipids in the lipid bilayer. On flow cytometric analysis, 78% of H2O2 (0.5 mM)-treated erythrocytes compared to 30% of untreated RBCs exhibited MC-540 binding and fluorescence, indicating differences in the lipid packing in the outer leaflet of the bilayer. Oxidant-treated erythrocytes adhere preferentially to endothelial cells rather than to bovine aortic smooth muscle cells and skin fibroblasts. It is suggested that the alterations in the erythrocyte membrane surface due to spectrin cross-linking and the organization of the phospholipids concomitant with less ordered packing in the external leaflet of the bilayer, either induced by oxidative manipulation in normal RBC or in pathological erythrocytes, play a role in erythrocyte-endothelial cell interaction.

Animals↗

PubMed1987

Posterior vitreous separation and retinal detachment induced by macrophages.

Macrophages, which migrate into the vitreous in conditions such as vitreous hemorrhage and penetrating ocular injury, may contribute to the development of intravitreous cellular proliferation and posterior vitreous separation. To investigate this possibility, activated macrophages were harvested from the peritoneal cavity and injected into the vitreous of rabbits. As early as 8 days after macrophage injection, posterior vitreous separation and glial epiretinal membrane formation began to occur. Two weeks after injection, vitreous strands that approached the optic disc and medullary rays were evident; fibroblasts proliferated over the disc or rays and induced retinal detachment. These findings support the hypothesis that macrophages in the vitreous may, in part, mediate cellular proliferation and posterior vitreous separation.

Animals↗

PubMed1987

Differential effects of soluble and immobilized fibronectins on aortic endothelial cell proliferation and attachment.

We studied the effects of soluble and immobilized forms of plasma fibronectin on bovine aortic endothelial cell (AEC) proliferation and attachment. Soluble fibronectin stimulated AEC growth at 10 micrograms/ml, but at higher concentrations of soluble fibronectin AEC growth was progressively inhibited. The growth rates of arterial smooth muscle cells (ASMC) and dermal fibroblasts (DF) were not altered by soluble fibronectin concentrations of 10 to 100 micrograms/ml. Plasma fibronectin, immobilized by attachment to culture dish surfaces, had no significant effects on the proliferation of any of the cell types examined. The attachment rates of AEC were decreased in the presence of 50 micrograms/ml soluble fibronectin. Immobilized fibronectin increased the rate of AEC attachment, but had no significant effects on ASMC or DF attachment; however, 12 h after plating there was nearly 100% attachment in all groups, whether or not fibronectin was present in the system. That soluble and immobilized fibronectins elicit disparate cellular responses is consistent with published reports of different cell surface receptors for different forms of the protein; in this manner, cells enmeshed in an interstitial matrix containing immobilized fibronectin could still respond to soluble fibronectin in the extracellular milieu.

Animals↗

PubMed1987

Morphologic observations on experimental subretinal neovascularization in the monkey.

Subretinal neovascularization is a poorly understood and potentially disastrous feature of many eye diseases. We used light and electron microscopy to study the sequence of events that lead to the formation of new vessels after laser photocoagulation of the retina and choroid of primates. In this animal model there is a rapid development of new blood vessels; one day after photocoagulation, endothelial cell degeneration and thrombus formation were observed in the capillaries, venules and arterioles of the choroid around the center of the lesion. Re-endothelialization began in some thrombosed choroidal vessels, with migration of the activated endothelial cells within the old basement membrane. Two days after photocoagulation, re-endothelialization was observed in almost all thrombosed choroidal vessels, and the initial stage of the endothelial cell budding was observed in the pre-existing choroidal vessels; this was especially prominent in venules with pericytes. Three days after photocoagulation, not only the endothelial cells in pre-existing vessels but also those in re-endothelialized vessels showed budding and lumen formation. The lumen of vessels was formed by the budding of adjacent endothelial cells that were coupled by transient intercellular junctions. Mitotic figures were frequently found in the endothelial cells distal to the growing tip. Five to eight days after photocoagulation, many new vessels extended into the subretinal space.

Animals↗

PubMed1987

Immunofluorescent studies of fibronectin and laminin in the human eye.

The topographic distribution of fibronectin and laminin in young and old human eyes was determined by indirect immunofluorescent techniques. These two glycoproteins may play a role in the attachment of the vitreous to the internal limiting membrane (ILM) and the internal limiting membrane to the Mueller cell processes. A double-laminated pattern of fluorescence for both glycoproteins was frequently found at the ILM of the posterior retina of aged eyes. This pattern of fluorescence, which was rarely seen in young eyes, may represent senescent changes in the ILM which could predispose the eye to posterior vitreous detachment.

Adolescent↗

PubMed1987

Alterations in the distribution of fibronectin and laminin in the diabetic human eye.

The distribution of fibronectin and laminin in diabetic human eyes was determined by indirect immunofluorescent techniques. The intense fluorescence suggests increased amounts of fibronectin and laminin in the diabetic internal limiting membrane (ILM). A double laminated pattern of fluorescence for both glycoproteins suggests structural abnormalities of the ILM of the posterior retina. Preretinal and subretinal proliferative tissues fluoresced strongly and diffusely with antifibronectin. This study indicates that in diabetic patients, the ILM, especially in the posterior retina, is biochemically and morphologically abnormal.

Aged↗

PubMed1986

Cellular migration, proliferation, and contraction. An in vitro approach to a clinical problem--proliferative vitreoretinopathy.

Presently used animal models of proliferative vitreoretinopathy reflect only cell proliferation and contraction. We used an in vitro model that measured cell migration, proliferation, and contraction. The following four drugs were assayed on this system: daunomycin, taxol, colchicine, and cytochalasin B. Daunomycin was the most effective drug against cell proliferation and cell migration but had no effect on cell contraction; taxol and colchicine affected all three parameters. Cytochalasin B was the least effective drug tested.

Alkaloids↗

PubMed1986

Aging changes in Bruch's membrane of monkeys: an electron microscopic study.

Bruch's membrane from monkeys of various ages was studied by electron microscopy. In monkeys under 15 years of age, Bruch's membrane rarely contained a small amount of polymorphous material that did not appear to increase with advancing age up to 15 years. However, the polymorphous material did increase over 20 years of age. The accumulation of vesicular, granular, tubular and linear polymorphous material in Bruch's membrane was though to be a result of evagination of a minimal portion of a retinal pigment epithelial (RPE) cell between adjacent basal infoldings, and its subsequent degeneration. The plasma membrane of the evagination seemed to be the primary source of the tubular or linear material, vesicles the main source of vesicular material, and cytosol and basement membranes to be the source of the granular material. The fibrous long-spacing collagen was associated with the basement membrane of the choriocapillaris and RPE cells. The granular deposits between the plasma infoldings and the basement membrane of RPE cells may originate from the basement membrane of the RPE, and could be the initial lesion of basal linear deposits.

Aging↗

PubMed1986

Endothelial cells in culture synthesize a potent bone cell active mitogen.

Bovine aortic endothelial cells (BAEC) are known to synthesize in vitro multiple factors which act on a variety of cells in culture. The fact that vascularization appears to be required for endochondral and intramembranous ossification promoted us to examine whether BAEC produce a bone cell active mitogen. Conditioned media collected from exponential and confluent BAEC cultures were concentrated by ultrafiltration and assayed for growth-stimulating activity on bone cell populations isolated from 1-day-old rat calvaria by sequential enzymatic digestions. As assessed by the incorporation of [3H]thymidine into DNA, it was found that BAEC synthesized a potent bone cell active mitogen. Bovine and rat fibroblasts, as well as rabbit articular chondrocytes, were not affected by the factor which was produced by exponential and confluent cultures, regardless of whether BAEC were cultured in the presence or absence of fetal bovine serum. Employing gel filtration chromatography, Bio-Gel P60, the mitogenic activity eluted as a major peak. It was flanked on either side by a minor one. Apparent mol wt were calculated to be 43,000, 38,000, and 30,000, respectively. Upon heat treatment, 56 C for 30 min, the mitogenic activity was fully retained. No effect of the endothelial derived-growth factor on the synthesis of collagen was found. Our data suggest that endothelial cells, by virtue of their ability to synthesize bone cell active mitogen(s), may represent an important element in the genesis of bone formation.

Animals↗

PubMed1986

Pathogenesis of drusen in the primate.

Two monkey eyes that showed clinical evidence of drusen were studied by light and electron microscopy. The drusen-like spots had several different morphological patterns: the appearance of typical drusen, budding retinal pigment epithelial (RPE) cells, and vacuolation of retinal pigment epithelial cells. Several stages of budding were seen. In some lesions, part of the RPE cell protruded into the sub-RPE space. The upper portion of the budding cell was connected to the cytoplasm of the parent RPE cell and was surrounded by basement membrane of the RPE cell. These budding cells had plasma membranes, cytoplasm that contained organelles, and a nucleus. Disconnected buds, separate from the parent RPE cell, were also seen; these showed degeneration. Finally, an accumulation of vesicular, granular, tubular and linear material was found in the nodular space beneath the RPE cell. It is suggested that this budding of RPE cells is the initial event in drusen-formation.

Animals↗

PubMed1985

Effects of intravitreal administration of steroids on experimental subretinal neovascularization in the subhuman primate.

To elucidate the role of inflammation in the occurrence of experimental subretinal neovascularization caused by high-intensity laser photocoagulation, we investigated the effects of vitreal infusion of steroids on laser lesions in a primate model. Dexamethasone, with or without triamcinolone, was infused continuously for two weeks through an indwelling cannula system. The animals were followed up clinically for up to eight weeks. The frequency of subretinal neovascularization in the steroid-treated animals was significantly lower than that in a control group of untreated animals. Although steroids have multiple effects, these results suggest that the inflammatory response, possibly macrophage infiltration, may plan an important role in the occurrence of subretinal neovascularization in our experimental model.

Animals↗

PubMed1985

Altered characteristics of B16 melanoma cells induced by chemically crosslinking fibronectin to cell surfaces.

The loss of fibronectin from tumor cell surfaces has been correlated with an increased incidence of metastases. To determine directly whether cell surface fibronectin influences the metastatic potential of solid tumors, we chemically crosslinked fibronectin to B16 murine melanoma cells using a photosensitive heterobifunctional crosslinking reagent, N-succinimidyl-4-azidophenyl-1,3 dithiopropionate (SADP). Cell attachment to plastic surfaces was increased in cells to which fibronectin was attached; cell growth over a 24-hr period was not significantly affected by the addition of fibronectin. When C57BL/6 mice were injected with fibronectin-crosslinked B16 cells, there was a 63% reduction in the number of pulmonary nodules compared to untreated controls. These results are consistent with the hypothesis that fibronectin enhances the recognition and removal of tumor cells from the circulation, possibly by cells of the reticuloendothelial system.

Animals↗

PubMed1985

Fibronectin of the chorioretinal interface in the monkey: immunohistochemical and immunoelectron microscopic studies.

The distribution of fibronectin in the chorioretinal interface of the monkey eye was studied by indirect immunofluorescent and immunoelectron microscopic techniques. Immunofluorescent staining revealed fibronectin in Bruch's membrane and the choriocapillaris. Immunoelectron microscopic techniques revealed fibronectin associated with basement membranes, collagen fibers and elastic fibers in Bruch's membrane; the stromal side of the basement membrane of the choriocapillaris also showed staining. This study thus demonstrates that fibronectin is an integral component of Bruch's membrane in the monkey eye.

Animals↗