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Importance: Primary open angle glaucoma, an age-related, retinal neurodegenerative disease of unknown etiology, is treated by lowering intraocular pressure, even though elevated intraocular pressure is present in only about 60% of patients. Since we found that tolbutamide, which inhibits the opening of ATP-sensitive potassium channels, modulates aqueous dynamics with a significant increase in outflow, and since aquaporin-9 is essential for retinal ganglion cells survival, gene variants coding for the ATP-sensitive potassium channels and aquaporin-9 may participate in the development and progression of glaucoma. Objective: To identify gene variants involved in ion and water transport in the trabecular meshwork of glaucoma donors. Design: The study is a gene association study; since gene variants can be somatic or germline, the following genes KCNJ8, KCNJ11, ABCC8, and ABCC9, QP1, AQP4, AQP9, ATP1A1, KCNMA11, and CLCN3 associated to ATP-sensitive potassium channels and water transport in the trabecular meshwork were sequenced from DNA isolated from trabecular meshwork of glaucoma donors. Setting: The study is a gene association study carried out with samples obtained through the Cooperative Human Tissue Network of which the DNA was isolated by the technical personnel at the Lions Vision Gift eye bank and analyzed by Admera Health LLC. Participants: The only criteria for the ten donors (5 male, 5 females; 70-91 years of age) in the study was a diagnosis of primary open angle glaucoma and a consent form signed at their lifetime or by responsible relatives. Main Outcomes and Measures: Of several missense variants, one was found in all 10 and three were found in nine trabecular meshwork samples. All variants, whether synonymous or missense, were germline. Results: The AQP9 missense variant, rs1867380 (Aca/Gca, 279T/A), was found in all 10 trabecular meshwork samples. Two common missense variants in the KCNJ11 gene, rs5215 (Gtc/Atc, 337V/I) and 5219 (Aag/Gag, 23K/E), and one missense variant in the ABCC8 gene, rs757110 (Gcc/Tcc, 1369AS), were found in the same nine trabecular meshwork samples. Several other missense variants were found in some, but not in the majority of trabecular meshwork samples. Conclusions and Relevance: Variants of the KCNJ11 and ABCC8 genes that code for subunits of ATP-sensitive potassium channels and a variant of the AQP9 gene may be implicated in the development of elevated intraocular pressure and glaucoma. Understanding how these genes impact the energetics of the neural retina may provide further insights into the pathogenic nature of these variants, as well as offer clues for developing novel therapeutic targets.

A continuous source of osteoblasts for normal bone maintenance, as well as remodeling and regeneration during fracture repair, is ensured by the mesenchymal osteoprogenitor stem cells of the bone marrow (BM). The differentiation and maturation of osteoprogenitor cells into osteoblasts are thought to be modulated by transforming growth factors-beta (TGF-beta1 and TGF-beta2) and TGF-beta-related bone morphogenetic proteins (BMPs). To define the responses of mesenchymal osteoprogenitor stem cells to several growth factors (GFs), we cultured Fischer 344 rat BM cells in a collagen gel medium containing 0.5% fetal bovine serum for prolonged periods of time. Under these conditions, survival of BM mesenchymal stem cells was dependent on the addition of GFs. Recombinant hTGF-beta1-F2, a fusion protein engineered to contain an auxiliary collagen binding domain, demonstrated the ability to support survival colony formation and growth of the surviving cells, whereas commercial hTGF-beta1 did not. Initially, cells were selected from a whole BM cell population and captured inside a collagen network, on the basis of their survival response to added exogenous GFs. After the 10-day selection period, the surviving cells in the rhTGF-beta1-F2 test groups proliferated rapidly in response to serum factors (10% FBS), and maximal DNA synthesis levels were observed. Upon the addition of osteoinductive factors, osteogenic differentiation in vitro was evaluated by the induction of alkaline phosphatase (ALP) expression, the production of osteocalcin (OC), and the formation of mineralized matrix. Concomitant with a down-regulation of cell proliferation, osteoinduction is marked by increased ALP expression and the formation of colonies that are competent for mineralization. During the induction period, when cells organize into nodules and mineralize, the expression of OC was significantly elevated along with the onset of extracellular matrix mineralization. Differentiation of BM mesenchymal stem cells into putative bone cells as shown by increased ALP, OC synthesis, and in vitro mineralization required the presence of specific GFs, as well as dexamethasone (dex) and beta-glycerophosphate (beta-GP). Although rhTGF-beta1-F2-selected cells exhibited the capacity to mineralize, maximal ALP activity and OC synthesis were observed in the presence of rhBMPs. We further report that a novel rhTGF-beta1-F2 fusion protein, containing a von Willebrand's factor-derived collagen binding domain combined with a type I collage matrix, is able to capture, amplify, and stimulate the differentiation of a population of cells present in rat BM. When these cells are subsequently implanted in inactivated demineralized bone matrix (iDBM) and/or diffusion chambers into older rats they are able to produce bone and cartilage. The population of progenitor cells captured by rhTGF-beta1-F2 is distinct from the committed progenitor cells captured by rhBMPs, which exhibit a considerably more differentiated phenotype.

Normal endochondral bone formation follows a temporal sequence: immature or resting chondrocytes move away from the resting zone, proliferate, flatten, become arranged into columns, and finally become hypertrophic, disintegrate, and are replaced by bone. The mechanisms that guide this process are incompletely understood, but they include programmed cell death, a stage important in development and some disease processes. Using immunofluorescence we have studied the distribution of various complement proteins to examine the hypothesis that this sequence of events, particularly cell disintegration and matrix dissolution, are complement mediated. The results of these studies show that complement proteins C3 and Factor B are distributed uniformly in the resting and proliferating zones. Properdin is localized in the resting and hypertrophic zone but not in the proliferating zone. Complement proteins C5 and C9 are localized exclusively in the hypertrophic zones. This anatomically segregated pattern of distribution suggests that complement proteins may be important in cartilage-bone transformation and that the alternate pathway is involved.

Macrophages are usually present in epiretinal membranes from eyes with proliferative vitreoretinopathy (PVR). Information on the kinetics of macrophages in the eye may be of help in identifying their role in this disease. To determine the half-life of macrophages in the vitreous, peritoneal macrophages were labeled by allowing them to phagocytose 141Cerium (gamma-emitter) labeled microspheres, and were then injected into the vitreous of the same rabbit from which they were obtained. The animals were sacrificed at various times post-injection and the radioactivity remaining in the vitreous was measured. Using this procedure, the half-life was found to be 4.8 days.

Acylated derivatives of ascorbic acid were found to be active in a number of biochemical and physiological processes. In the present study we investigated the effects of 6-O-palmitoyl ascorbate on collagen synthesis by cultured foreskin human fibroblasts. Our observations indicate a marked stimulatory effect on collagen synthesis by 6-O-palmitoyl ascorbate in the concentration range of 5-20 microM, while the synthesis stimulated by ascorbic acid was maximal at concentrations of 20-100 microM. Cells treated with 10 microM palmitoyl ascorbate for 36 h exhibited a production of collagen threefold greater than those in the presence of 10 microM ascorbic acid, and it was about the same as in cells treated with 100 microM ascorbic acid. By 48 h differences were not significant. Acylated ascorbate impaired vitality of the treated fibroblasts at concentrations exceeding 20 microM in media supplemented with 0.5% FCS. However, most of the cytotoxic effect was neutralized by FCS at a concentration of 10%. The resistance of acylated ascorbate against oxidative degradation as well as the role of free radicals in the modulation of collagen synthesis by ascorbic acid and by its derivatives is discussed.

Purpose: To examine the effects of elevated glucose on the migration and proliferation of vascular endothelial cells in an in vitro wound model and to investigate whether nitric oxide (NO) mediates the effects of elevated glucose. Methods: Migration was investigated in monolayers of bovine aortic endothelial cells wounded by scraping and measuring the distance, the number of cells migrating, and the area covered by the migrating cells in the presence of various concentrations of glucose. The effects of NO were evaluated by adding to the cultures NG-monomethyl arginine (NMMA), an inhibitor of NO synthase, or S-nitrosylated penicillamine, which is a slow-release agent of NO. Proliferation was investigated in the presence of various concentrations of serum, glucose, or both. Results: Elevated glucose levels (16.5 and 27.7 mmol/L) inhibited endothelial cell migration in a dose-dependent manner compared with cells cultured in the presence of 5.5 mmol/L glucose. Inhibition of migration was also observed when wounded monolayers cultured in 5.5 mmol/L glucose were treated with S-nitrosylated penicillamine, which generates NO. Inhibition of NO synthase by NMMA prevented the inhibition of migration observed in media containing 27.7 mmol/L glucose. Elevated glucose levels did not affect cell proliferation except in the presence of 20% fetal bovine serum. Conclusion: An elevated glucose level inhibits endothelial cell migration in an in vitro wound model, and the inhibition appears to be mediated by increased levels of NO. (J Vasc Surg 1997 26:319-26.)

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.

• 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 maxtrix components by the culture population was increased.

Lysozyme (mucopeptide- N -acetylmuramylhydrolase, EC 3.2.1.17) is present in mammalian cartilage. Lysozyme was isolated and purified from bovine and canine cartilage and from dog serum using various chromatographic steps and affinity chromatography on carboxymethylated chitin. Amino acid analysis of bovine cartilage lysozyme showed that it is similar to other mammalian lysozymes. Anti-canine lysozyme antibodies cross-react with calf lysozyme, but not with hen egg white or embryonic chick cartilage lysozyme. In the epiphyseal plate of the dog, 90-μm sections were analyzed for lysozyme and its was found that in the hypertrophic zone its concentration is approximately six times higher than it is in the resting zone. Using immunocytochemical techniques at the electromicroscopic level, lysozyme in the epiphyseal plate of the dog was localized extracellularly, mainly in the immediate vicinity of the chondrocytes, the territorial matrix.

We used an experimental model of proliferative vitreoretinopathy (PVR) and cell-induced traction retinal detachment to study the therapeutic value of six cytotoxic drugs (actinomycin C, colchicine, cytosine arabinoside hydrochloride, 5-fluorodeoxyuridine, vinblastine sulfate, and daunomycin). Pigmented rabbits were injected with 2.5 X 10(5) cultured homologous dermal fibroblasts. At the same time, cytotoxic drugs were injected into the vitreous in an attempt to inhibit cellular proliferation. The sensitivity of the cells to the drug was also tested in vitro before injection. Daunomycin at a dose of 10 nmol per eye stopped cellular proliferation and subsequent traction retinal detachment in vivo. The electroretinogram (ERG) showed no evidence of drug-induced retinal toxicity with this dose of daunomycin. No alteration in frequency or severity of vitreal membranes and retinal detachment was observed after injection of equivalent doses of the other drugs.

Injection into the rabbit vitreous of cultured cells results in a condition that mimics some of the features of proliferative Vitreoretinopathy (PVR) in man. Because of its simplicity and reproducibility, this model is especially useful for screening drugs that may control or inhibit intraocular cell proliferation. The cell injection model of PVR is reviewed, and some modifications are described.

Previous work demonstrated that micropuncture aspirates from rat epiphysical plate cartilage contain a nucleating agent for Ca 3 (PO 4 ) 2 mineral growth, and that the nucleation is inhibited by proteoglycan aggregates. In this report data are described which show that mammalian lysozyme inactivates the inhibition. When micropuncture aspirates are incubated in vitro with mammalian lysozyme, a rapid, spontaneous initiation of mineral growth occurs. Incubation of proteoglycan aggregate preparations in the presence of cartilagea lysozyme, but not hen egg white lysozyme, causes a marked decrease of the sedimentation coefficients of the proteoglycans, usually to values close to those obtained with proteoglycan monomer preparations. The inhibition of this effect of mammalian lysozyme by a specific inhibitor of the enzyme tri( N -acetyl-D-glucosamine) suggests that it may be enzymatic in nature.

Isolated matrix vesicles from 26-day embryonic New Zealand White rabbit incisor tooth organs were incubated on 14 C labeled reconstituted Type I collagen at 25°C for 18 hours. Under these conditions the matrix vesicles demonstrated the ability to degrade collagen as demonstrated by radio-activity solubilized. Analysis of the released radioactivity using SDS-PAGE demonstrated the presence of two molecular species with molecular weights of 67,000 and 32,000 daltons. These results are in accordance with the presence of a mammalian-like collagenase associated with the matrix vesicles, whose function may be the degradation of the basal lamina.

Macrophages are fully differentiated cells that do not synthesize an extracellular matrix and do not contract; they do, however, produce substances that modify the behavior and functions of other cells, particularly those involved in the inflammatory and immune responses. Since macrophages are a ubiquitous component of periretinal membranes, we sought to determine whether they modulate proliferation and/or migration of retinal pigment epithelial (RPE) cells, functions that may be essential for the development of proliferative vitreoretinopathy (PVR). Using an in vitro assay, we found that macrophage supernatant contains factors that stimulate proliferation and migration of cultured human RPE cells. Since interleukin-1 (IL-1) is a product of activated macrophages that modulates a number of cellular functions, we also examined its effect on RPE proliferation and migration. We found that IL-1 increased migration but did not affect proliferation, and thus could not duplicate the effect of macrophage supernatant. Injection of activated macrophages into the vitreous of rabbits which had a retinal hole stimulated RPE cell proliferation in the area of the retinal hole, where the RPE cells were exposed. These findings suggest the ability of macrophages to modulate functions of RPE cells that are thought to be critical for the development of PVR. Macrophages may thus be an important part of the vitreous environment that favors the development of PVR.

Poly(A+)-messenger RNA was isolated from bovine lens as well as poly(A+)-mRNA from crude and sucrose-purified plasma membrane. Bovine lens MP26 antisera was also propagated in rabbits, from which anti-MP26 IgG was partially purified and used to assay for the specific synthesis of MP26 during in vitro translation of the isolated poly(A+)-mRNAs. We found that membrane-associated poly(A+)-mRNA supported the synthesis of proteins which were identical to those translated from total lens fiber cell poly(A+)-mRNA. These proteins included MP26, as assayed by immunoprecipitation of [35S]-labeled MP26.

Neovascularization and proliferation of the retinal pigment epithelium (RPE) was induced in the rabbit by subretinal injection of vitreous without rupture of Bruch's membrane. New vessels developed between the layer of RPE and photoreceptor outer segments, but were enveloped in proliferating RPE. For this reason they were occult; no fluorescein leakage was visible by angiography. The vessels were identified only by histologic examinations. Endothelial cell budding was the initial stage of vessel development, first seen two weeks after injection. The new vessels grew from the choriocapillaris, penetrated Bruch's membrane, and spread into the subretinal space, despite the absence of subretinal fluid. Fenestrations with diaphragms were found in the endothelial walls during the earliest stages of vessel formation, and were also present in the fully matured vessels. Intermediate junctional complexes were frequently observed among the endothelial cells. During maturation of these plexi, junctions changed from open to putative tight junctions. The mature vessels were ultimately completely enveloped by collagen and RPE cells. Our results show that all new vessels in this animal model have the morphologic characteristics of choriocapillaris. We assume that they leak fluorescein, as does the choriocapillaris, but that the dye has no opportunity to pool in the subretinal space and thus cannot be seen during angiography.

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.

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.

Water and glycosaminoglycan contents were measured in upper and lower thoracic aortas of calves 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.

We have demonstrated cell surface-associated and released proteolytic activity on bovine aorta endothelial cells, representing normal cells with regulated invasive properties. To demonstrate these proteolytic activities on viable cells grown in monolayer cultures, a new method was developed. The method consists of rolling modified plastic beads carrying covalently-linked [125I]-labeled casein in contact with the cell surfaces or adjacent to the cell monolayers without actual contact. The rate of radioactive peptide release is proportional to the proteolytic activity. Released proteases are detected when no contact occurs between the substrate and the cells. When the bead-substrate complex is rolled over the surface of endothelial cells, a significant increase in released labeled peptides is observed which represents a cell surface-associated proteolytic activity. These activities may be relevant to the endothelial cell's invasive capacity and appear to be similar to the neutral proteolytic activity of transformed cells.

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 macrophates produce a morphoplastic substance for RPE cells, and suggest that vitreous also contains a factors(s) that affects RPE cell shape, and that requires mediation by serum components.

We modified a preexisting experimental model of massive periretinal proliferation by injecting cells of differing origins into the rabbit vitreous cavity. These cells included autologous and homologous fibroblasts, homologous chondrocytes, homologous retinal pigment epithelial cells, heterologous bovine endothelial cells, and heterologous murine embryonal cells. All cell injections caused vitreous and retinal membrane formation that resulted in a process similar to massive periretinal proliferation. Clinically the character of the membranes formed and the time course in the development of traction retinal detachments was similar for all the different cell types. The initial cell dosage injected was the critical factor in determining the severity of the traction retinal detachments leading to massive periretinal proliferation.

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.

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.

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 cell injection model of proliferative vitreoretinopathy (PVR) in the rabbit was used to study the therapeutic value of intravitreal adriamycin (doxorubicin). Adriamycin in a dose of 10 nmol per eye, if injected at the same time as the cells, controls PVR. At the cell doses used, PVR is not affected if there is a time interval between cell and drug injection. Because of retinal toxicity, as evidenced by electroretinographic and histopathologic changes, the beneficial effects of adriamycin on membrane formation cannot be exploited at this time.

The effects of adriamycin, 5-fluorouracil, methotrexate, triamcinolone and Viroptic on an experimental model of proliferative vitreoretinopathy (PVR) produced by the injection of homologous dermal fibroblasts into the rabbit vitreous were studied. Adriamycin and fluorouracil inhibited fibroblast proliferation and prevented the formation of membranes as well as did triamcinolone, whereas methotrexate and Viroptic had no beneficial effect in this model of PVR.

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.

Drusen-like changes In Bruch's membrane following subretlnal Injection of vitreous In the rabbit were studied by electron microscopy. A sequence of changes Is seen that closely panel lets 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.

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 - 3 H]methionine into alkali-labile, [ 3 H]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 μg/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.

Extrakte verschiedener Bindegewebe wurden auf ihre Proteasehemmung hin elektrophoretisch untersucht. Alle Gewebe zeigten kationisch wandernde Aktivitat und ihre Anwesenheit im zellfreien Dentin lasst den Schluss zu, dass das Material extrazellular lokalisiert ist.

A condition similar to massive periretinal proliferation in man can be produced in rabbits by injecting homologous fibroblasts into the vitreous. We have studied the effect of daunomycin, a cytotoxic drug, in this model to determine a dose which would not be toxic to the retina but would be effective in preventing proliferation of the injected fibroblasts and eventual retinal detachment. The results of this study demonstrate that daunomycin at a dose of 9 nmol per eye reduces the incidence of retinal detachment by over 50%. Doses higher than 30 nmol per eye are toxic to the retina.

• 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.

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.

• To elucidate the role of inflammation in the occurrence of experimental subretinal neovascularization caused by highintensity 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.

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.

The retinal toxicity of daunomycin, a drug that effectively suppresses experimental proliferative vitreoretinopathy (PVR), was studied in the rabbit by clinical examination, electroretinography (ERG), light microscopy, and electron microscopy. Although no toxicity was observed at the therapeutically effective dose of 10 nmol per eye, the safety margin is too small to recommend this drug for therapy of proliferative vitreoretinopathy in man.

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. These monoclonal antibodies will be useful for monitoring the separation of luminal and abluminal membrane domains and in the immunoaffinity purification of these domains.

We have investigated the contractile properties of human and bovine retinal pigment epithelial (RPE) cells in culture. In collagen gels, RPE cells sent out processes which were able to retract the gels. In a glycerinated model of contraction, RPE cells contracted and aggregated into clusters in response to the addition of ATP to the media. This contraction was very likely mediated by contractile proteins which are arranged in circumferential microfilament bundles in RPE cells. SDS-PAGE analysis of the RPE cell cytoskeletal elements showed protein bands of 200 kD and 43 kD, probably heavy-chain myosin and actin, respectively; in human RPE, cells bands of 58 kD and 52 kd, representing intermediate filament subunits, were evident; in bovine cells the 58-kD band was observed.

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.

Using light and electron microscopy, we studied the sequence of events that lead to the formation of new vascular lumens after laser photocoagulation of the retina and choroid of primates. 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.

Bovine aortic endothelial cells (BAEC) in culture have the ability to regulate their own proliferation. We have found that a fraction below 100,000 daltons obtained from the media of confluent cultures of BAEC inhibits tritiated thymidine [3H]TdR incorporation as well as their proliferation. The inhibition is dose- and time-dependent; maximum inhibition of [3H]TdR incorporation occurs 8 hr after cells are released from synchronization and the inhibitory fraction is added. Inhibition is evident at concentrations as low as 50 micrograms/ml and reaches a maximum at 600 micrograms/ml. The blockage of [3H]TdR incorporation is reflected in the inhibition of cell proliferation. In the presence of 400 micrograms of endogenous inhibitor per ml of media, added at the time of plating, the average population doubling time increases from 19 to 41 hr. These findings indicate that, in culture, BAEC can regulate their own proliferation by synthesizing an endogenous inhibitor(s) of proliferation.

Injection of activated macrophages into the posterior vitreous of the rabbit induced vigorous fibrovascular proliferation over the optic disk and medullary rays, as demontrated by 3 H-thymidine autoradiography. One week after injection, endothelial cells and pericytes of the capillaries near the inner surface of the optic disk and rays were labeled; fibroblast-like cells, which were also labeled, migrated and formed vitreous strands. By the second week after injection, the fibrovascular tissue proliferated most actively, and traction medullary ray detachment and peripapillary retinal fold formation were observed. The cellular proliferation was accompanied by inflammatory cell infiltration. Glial cells within the optic disk, as well as retinal pigment epithelial cells beneath the detached retina, were labeled by 3 H-thymidine. These results demonstrate that the fibrovascular proliferation originates from the vessel complex of the optic disk and medullary rays in this experimental model of retinal detachment.

An endothelial cell growth inhibitor (EGI), which affects proliferation of vascular endothelial cells, has been isolated from a 1 M guanidine hydrochloride extract of bovine cartilage by ion exchange chromatography on CM-Sephadex C-25. Two peaks of EGI activity were observed, both of which eluted in positions separate from a peak of trypsin inhibitory activity. Using bovine endothelial cells in culture, we have demonstrated that EGI is not cytotoxic, but rather affects the cell cycle. The average population doubling time increased from 24 h for cells grown in control media to 40 h when cells were grown in the presence of 2 μg EGI/ml of culture medium. The trypsin inhibitor isolated from cartilage had no effect on endothelial cell proliferation. As predicted by segmented regression analysis of the growth curves, we have shown that EGI remains active in the media for a period of 3 days. An appendix of the statistical treatment of the data by segmented regression analysis is presented.

• 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.

In proliferative vitreoretinopathy, retinal pigment epithelial cells and glial cells migrate into the vitreous, proliferate, and assume characteristics of myofibroblasts. The addition of vitreous to the culture media stimulates the proliferation of porcine retinal pigment epithelial cells, bovine and lapine dermal fibroblasts but not the proliferation of bovine aortic endothelial cells and smooth muscle cells. The mitogenic activity is not species-specific, since vitreous from various species stimulates the proliferation of these cells. The mitogenic activity is destroyed by heating at 100°C for 10 min or by trypsin treatment. Since the vitreous, under our assay conditions, was not mitogenic for endothelial cells or smooth muscle cells, the mitogenic activity is probably not derived from leakage into the vitreous of circulating fibroblast, epidermal or platelet-derived growth factor.