Anchorage independent growth and plasminogen activator production by bovine endothelial cells.

Endothelial cells obtained from the aortae of 1- to 2-d-old calves were cloned at high efficiency using fibrin-coated dishes. Primary cultures as well as clones derived from them produced high fibrinolytic activity when grown on 125I-fibrin-coated dishes which was 90% dependent upon the presence of plasminogen. High plasminogen-dependent proteolytic activity was also demonstrated in endothelial cell lysates and in the culture medium of the cells. The production and secretion of the plasminogen activator(s) were found to increase during the log phase of cell growth and to reach a maximum level at confluence. These endothelial cells exhibited morphological phenotypes comparable to those of transformed cells when grown in the presence of acid-treated fetal calf, dog, or human serum. Furthermore, they demonstrated anchorage independent growth, and large colonies were formed in semisolid media. Spontaneous neoplastic transformation of these cells was excluded by karyotypic analysis, lack of tumorigenicity in athymic nude mice, and limited lifespan in culture. Cell clones isolated from colonies grown in agarose demonstrated the same growth characteristics and proteolytic activity as before plating in agarose. High fibrinolytic activity, morphological changes in the appropriate serum, and growth in semisolid media may therefore be indicative of the migratory and/or invasive capacity of both nontransformed endothelial cells as well as tumor cells.

Chemotaxis of aortic endothelial cells in response to fibronectin.

We have determined the chemotactic response of bovine aortic endothelial cells to fibronectin and to endothelial cell mitogens (endothelial cell growth supplement, tumor extract), using blind-well chemotaxis chambers. Fibronectin induced a chemotactic response in bovine aortic endothelial cells; at 100 micrograms/ml, chemotaxis increased by 440% above that observed in negative controls (p less than 0.001). The chemotactic response plateaued with time, paralleling the disappearance of the fibronectin concentration gradient in the chambers. As further evidence that chemotaxis was measured, we observed that cell migration did not occur when cells were incubated with fibronectin in the absence of a concentration gradient. Endothelial cell mitogens increased bovine aortic endothelial cell proliferation in our experiments but did not stimulate chemotaxis above background levels. In contrast, fibronectin inhibited cell proliferation by 23%. We present a hypothetical model of the role of fibronectin in evoking endothelial cell chemotaxis during tumor neovascularization.

Fibronectin-induced protein carboxy-O-methylation in aortic endothelial cells.

In a previous report (Bowersox, J.C. and Sorgente, N. (1982) Cancer Res. 42, 2547-2551) we demonstrated that the glycoprotein fibronectin is a chemoattractant for vascular endothelial cells. In probing the mechanisms by which fibronectin induces endothelial cell chemotaxis, we have discovered that the carboxy-O-methylation of cellular proteins is stimulated by fibronectin. By measuring the incorporation of L-[methyl-3H]methionine into alkali-labile, [3H]methyl ester linkages, we determined that fibronectin stimulated protein carboxy-O-methylation in aortic endothelial cells in a time- and concentration-dependent manner; the greatest stimulation occurred at 100 micrograms/ml fibronectin (approx. 35% above controls). When inhibitors of carboxymethylation were added, fibronectin-induced stimulation of protein methylation did not occur. Furthermore, inhibitors of methylation prevented the chemotaxis of endothelial cells in response to fibronectin. These data support our hypothesis that fibronectin mediates endothelial cell chemotaxis, such as that occurring during neovascularization. As carboxy-O-methylation of cell proteins is also effected by fibronectin, transmethylation reactions may be an important component of endothelial cell chemotaxis.

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)

Altered growth kinetics of dermal fibroblasts and arterial smooth muscle cells from spontaneously diabetic BB rats.

We cultured arterial smooth muscle cells and dermal fibroblasts from spontaneously diabetic BB rats and normoglycemic littermate controls. Although there were no detectable differences in the morphology of cells obtained from diabetic rats, significant differences existed in growth parameters of the diabetic smooth muscle cells. These cells grew more rapidly than smooth muscle cells from normal rats (population doubling times: normal = 42.6 +/- 3.2 h, diabetic = 31.8 +/- 3.7 h) and attained greater densities at confluence. The growth rates of the diabetic smooth muscle cells were dependent on the initial seeding density of cells, a characteristic not observed in cells from normal rats. Although growth rates of the diabetic smooth muscle cells were increased, their plating efficiencies were reduced. Dermal fibroblasts from diabetic rats grew at the same rates as fibroblasts from control animals and the plating efficiencies of the diabetic fibroblasts were increased rather than reduced. In these studies, we have shown that vascular-derived cells from diabetic rats have growth defects not detected in dermal fibroblasts from the same animals. This emphasizes the importance of using blood vessel cells to probe the pathophysiology of diabetic vascular disease. Furthermore, our results establish the validity of using spontaneously diabetic rats as a model system for examining inherent cell defects in insulin-dependent diabetes mellitus.

Demineralized bone matrix mediates differentiation of bone marrow stromal cells in vitro: effect of age of cell donor.

Bone maintenance requires a continuous source of osteoblasts throughout life. Its remodeling and regeneration during fracture repair is ensured by osteoprogenitor stem cells which are part of the stroma of the bone marrow (BM). Many investigators have reported that in cultured BM stromal cells there is a cell population that will differentiate along an osteogenic lineage if stimulated by the addition of osteogenic inducers, such as dexamethasone (dex), beta-glycerophosphate (beta-GP), transforming growth factor beta-1 (TGF-beta 1) and bone morphogenetic protein-2 (BMP-2). Here we report the effects of demineralized bone matrix (DBM) on the osteogenic differentiation of BM stromal cells in vitro, using morphological criteria, alkaline phosphatase (AP) activity, and calcium accumulation. DBM and DBM-conditioned medium (DBMcm) enhanced bone formation in the presence of dex and beta-GP, whereas DBM particles caused changes in the cell phenotype. Temporal expression of total and skeletal AP by BM stromal cells from 4-week-old rats showed a biphasic pattern enhanced by DBM and suggesting the presence of two cell populations. In one population, AP synthesis reaches a maximum during the first week in culture, following which cells either die or loose their ability to synthesize AP. A second, less abundant population begins to proliferate and synthesize AP during the second and third weeks. The synthesis of AP, which often decreases by the third week, can be maintained at high levels only if DBM is added to the cultures. BM stromal cells isolated from 24- and 48-week-old rats showed a decrease or loss of this biphasic AP expression pattern compared with cells isolated from 4-week-old rats. The addition of DBM to cultures derived from 24- and 48-week-old rats stimulated mostly the second cell population to synthesize AP, suggesting that DBM contains a factor(s) that acts on a specific bone marrow cell population by increasing the proliferation of active cells or inducing the differentiation of dormant cells.

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.

The ground substance of the arterial wall. Part 1. Extractability of glycosaminoglycans and the isolation of a proteoglycan from bovine aorta.

Water and glycosaminoglycan contents were measured in upper and lower thoracic aortas of claves and steers. The ability of various molarities of guanidine hydrochloride to extract glycosaminoglycans from these tissues was assessed. Some glycosaminoglycans seem to be more resistant to extraction than others. A procedure is described for the isolation of a proteoglycan. The molecule appears to contain both dermatan sulfate and chondroitin sulfate. It also seems to be less dense than cartilage proteoglycans extracted by similar methods as assessed by its behavior in centrifugal fields. The properties, locus and biological activities of this molecule are currently being studied.

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.

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.

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.

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.

Vitreous modulation of migration and proliferation of retinal pigment epithelial cells in vitro.

Retinal pigment epithelial (RPE) cell migration and proliferation are believed to play a role in the pathogenesis of proliferative vitreoretinopathy (PVR). Since PVR develops in situations where vitreous contacts the RPE, we sought to determine whether human vitreous contains factors that stimulate proliferation and migration of RPE cells. We found that postmortem human vitreous stimulates migration but not proliferation of human RPE cells under serum-free conditions in vitro. Stimulation of proliferation of RPE cells and fibroblasts was observed, however, following admixture of albumin with the vitreous. These findings suggest that vitreous contributes modulators that stimulate some functions of RPE cells that are believed to play a role in the pathogenesis of PVR.

Daunomycin in the treatment of experimental proliferative vitreoretinopathy. Effective doses in vitro and in vivo.

In previous studies the authors have shown that daunomycin, an anthracycline antibiotic, when injected into the vitreous effectively controls experimental proliferative vitreoretinopathy. Here we show that by administering daunomycin intravitreally it is possible to achieve in vivo concentrations that prevent fibroblast proliferation in vitro. The authors have also determined that the half-life of daunomycin in the vitreous is 131 min, indicating that a critical concentration is maintained in the eye for longer than 4 hr after a single injection. Using 3H-daunomycin, the authors have found that the drug is eliminated across the retina; no significant binding of the drug to vitreous components occurs. These studies demonstrate that it is possible to define the kinetics of drugs injected into the vitreous; and a knowledge of the distribution of any drug in ocular tissues is necessary to effectively determine whether such drug is of therapeutic value.

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.

Surgical removal of vitreous. Its effect on intraocular fibroblast proliferation in the rabbit.

The effect of surgical removal of vitreous on intraocular fibroblast proliferation was studied in an animal model. Forty-eight young adult pigmented rabbits were vitrectomized; four groups of eight animals subsequently received intravitreal injection of tissue-cultured rabbit dermal fibroblasts in doses of 50,000, 100,000, 250,000, and 750,000 cells. As controls, eight vitrectomized rabbits received intravitreal culture medium alone, and the remaining eight vitrectomized rabbits were left uninjected. An additional 40 normal rabbits received the same injections of the same four doses and served as nonvitrectomized controls. All animals were observed for 28 days after injection. The results were dose related and showed that vitrectomy aggravated intraocular fibroblast proliferation in that traction retinal detachment occurred earlier and was more severe in the vitrectomized than in the nonvitrectomized controls.

Synthesis of extracellular matrix by macrophage-modulated retinal pigment epithelium.

In proliferative vitreoretinopathy, macrophages and retinal pigment epithelial cells are associated with microfibrillar matrix proteins in the vitreous cavity, but the contribution of this extracellular matrix to the pathophysiology is not known. We used radiolabeling techniques on cultured human retinal pigment epithelial cells to correlate the secretion of extracellular matrix proteins with macrophage-induced modulation of cell proliferation and morphologic features. Retinal pigment epithelial cells incubated in a macrophage-conditioned medium assumed fibrocytelike morphologic characteristics, grew faster, and exhibited a decreased cellular release of fibrillar and nonfibrillar matrix components. However, due to a simultaneous greater increase in cell numbers in these modulated cultures, the total production of fibrillar and nonfibrillar matrix components by the culture population was increased.

Glial epiretinal membranes and contraction. Immunohistochemical and morphological studies.

It has been suggested that glial cells do not contribute substantially to the contractile forces generated by epiretinal membranes. We have established a rabbit model in which epiretinal membranes form on the inferior peripheral retina after the injection of activated macrophages into the vitreous. By two months, the membranes were extensive but without evidence of traction. At four months, however, full-thickness retinal folds were present beneath the thick epiretinal membrane. A homogeneous glial cell composition was suggested by light microscopic examination of serial sections through several membranes. Immunohistochemical staining with anti-glial fibrillary acidic protein and antivimentin and immunoelectron microscopy confirmed that these thick epiretinal membranes were composed entirely of glial cells, which may cause mild traction on the retina; this traction is associated with cell alignment and the tissue bridges connecting the membrane and the retina. The fusiform densities and indented nuclei suggested that the glial cells within the membrane may possess some characteristics of myofibroblasts.

Cellular proliferation induced by subretinal injection of vitreous in the rabbit.

A new experimental model of subretinal cellular proliferation, based on injection of autologous vitreous into the subretinal space of rabbits, was studied by light and electron microscopy. As early as five days after injection, proliferation of retinal pigment epithelial (RPE) and retinal glial cells was observed in the subretinal space. These morphologically distinct proliferating cells were sometimes joined by junctional complexes. Morphologically, the proliferating RPE cells resembled either RPE cells or fibroblasts. Some proliferating RPE cells also retained their epithelial characteristics (ie, basement membranes and cell junctions), while others were partially dedifferentiated and showed some embryonic features. New formation of melanin could be identified within the proliferated RPE cells, which could account, in part, for the hyperpigmentation at the site of the bleb caused by the injection of vitreous. The results demonstrated that injection of autologous vitreous into the subretinal space can lead to subretinal proliferation of retinal glial and RPE cells in the rabbit.

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.