A recombinant human TGF-beta1 fusion protein with collagen-binding domain promotes migration, growth, and differentiation of bone marrow mesenchymal cells.

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.

Acylated ascorbate stimulates collagen synthesis in cultured human foreskin fibroblasts at lower doses than does ascorbic acid.

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.

Nitric oxide mediates hyperglycemia-induced defective migration in cultured endothelial cells.

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 mono-layers 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. CONCLUSIONS: 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.

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.

Complement proteins are present in developing endochondral bone and may mediate cartilage cell death and vascularization.

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.

Ultrastructures of the glial epiretinal membrane induced by activated macrophages.

A rabbit model of glial epiretinal membrane was established following the injection of activated macrophages into the vitreous. The membrane was composed entirely of cells with glial characteristics, ie, abundant intermediate filaments, microvilli, junctional complexes and basement membranes. The extracellular matrix of the mature membranes contained collagen fibrils of 10 to 15 and 20 to 25 nm in diameter. Fusiform densities were seen adjacent to the cell membrane and cells with indented nuclei were found in thick membranes. These observations demonstrate that glial cells in epiretinal membranes may synthesize collagen and possess myofibroblast-like properties.

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.

Heterotypic and homotypic cell-cell adhesion molecules in endothelial cells.

Sickle red blood cells display an abnormal propensity to adhere to cultured bovine aortic endothelial cells when compared to normal red blood cells. The adherence was potentiated three-fold by endothelial cell derived conditioned medium, enriched in multimers of von Willebrand factor. Such adherence was ablated by 80% by either the synthetic peptide (RGDS) or antibody to GPIIb/IIIa, indicating the presence of RGD peptide recognition domain/receptor in either endothelial cells or sickle cells or both. The adherence was also inhibited by 70% by phosphatidylserine, but not by other phospholipids, indicating the presence of putative receptors for this phospholipid in endothelial cells. The labeling of cultured bovine aortic endothelial cells with monoclonal antibodies revealed the localization of MAB D2 to regions of cell-cell contact. The antigen on endothelial cells which cross-reacts with this antibody has a Mr of 130,000. The addition of such an antibody during the plating of endothelial cells disrupted monolayer formation. It appears that a 130-kDa polypeptide antigen in endothelial cells which is recognized by MAB D2, may be a cell-cell adhesion molecule.

Clearance rate of macrophages from the vitreous in rabbits.

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.

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.

Fibrovascular proliferation and retinal detachment after intravitreal injection of activated macrophages in the rabbit eye.

Injection of activated macrophages into the posterior vitreous of the rabbit induced vigorous fibrovascular proliferation over the optic disk and medullary rays, as demonstrated by 3H-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 3H-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.

Experimental subretinal neovascularization in the rabbit.

Subretinal neovascularization (SRN) in the rabbit was induced by subretinal injection of vitreous without rupture of Bruch's membrane. Eight of 26 eyes developed SRN. The incidence of SRN rose from 33% to 57% in a period of 4-40 weeks. Because of the absence of any fluorescein angiographic indication of SRN, these occult new vessels were identified by light and transmission electron microscopy. Histological examination showed that these newly formed vessels are composed of continuous capillaries with the morphologic characteristics of choriocapillaris, including diaphragmed fenestrations, basement membranes, and junctional complexes. The new vessels originated from the choriocapillaris and penetrated through Bruch's membrane into the subretinal space, where they were associated with the degenerated sensory retina and proliferating glial and/or RPE cells. This experiment provides a model of SRN without breaks in Bruch's membrane.

Pathogenesis of proliferative vitreoretinopathy. Modulation of retinal pigment epithelial cell functions by vitreous and macrophages.

RPE cell migration and proliferation are believed to play a role in the pathogenesis of 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 in vitro under serum-free conditions. A significant vitreous growth factor activity for RPE cells and fibroblasts, however, could be released by 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 (fig. 22). Since macrophages are a ubiquitous component of periretinal membranes, we sought to determine whether they modulate proliferation and/or migration of RPE cells, functions that may be essential for the development of PVR. Using an in vitro assay, we found that macrophage supernatant contains factors that stimulate proliferation and migration of cultured human RPE cells. Since 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 (fig. 22). 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 (fig. 22).

Morphologic observations of retinal pigment epithelial proliferation and neovascularization in the rabbit.

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.

Investigations on contractile properties of retinal pigment epithelial cells.

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.

Macrophage modulation of retinal pigment epithelial cell migration and proliferation.

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.

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.

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.

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.

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.