Isolation and characterization of a factor from calf serum that promotes the pigmentation of embryonic and transformed melanocytes.

A protein (Mr = 63,000) from calf serum that promotes the pigmentation of cultured chick neural crest and mouse melanoma cells has been partially isolated and characterized in this study. The stimulation of melanin synthesis in cultured cells was used to follow its activity during purification. The pigment-promoting factor was isolated by sequential column chromatography on dye-agarose matrices followed by hydroxyapatite and high pressure molecular sieve chromatography. The factor was found to stimulate melanin biosynthesis at 2-4 micrograms/ml and was specific for melanin-producing cells and their precursors. Antibodies raised in rabbits against the factor inhibited its pigment-promoting activity as well as that of whole calf serum. Enzyme-linked immunoadsorbent assays demonstrated that calf and bovine sera contain molecules that cross-react with the pigment-promoting factor. Horse, human, rat, and chicken sera, which lack the biological activity, also lacked immunological cross-reactivity. Extracts of certain tissues, particularly the submaxillary gland, were observed to be rich sources of pigment-promoting activity.

Retinal microvessel extracellular matrix: an immunofluorescent study.

The vasculature of the retina functions within a sheath of extracellular matrix (ECM). Unfortunately, little is known about the biochemical composition of this matrix. Abnormalities in the ECM of the retinal microvasculature are important in diabetic retinopathy as well as vasculopathies associated with connective tissue disorders. The ECM of unfixed frozen human retinal blood vessels was examined by indirect immunofluorescence using antibodies raised against collagen types I, II, III, IV, and V as well as the structural glycoproteins laminin and fibronectin. Antisera against collagen types I and IV as well as laminin and fibronectin stained a broad spectrum of retinal vessels, from large thick-walled vessels down to microvessels less than 10 micron in diameter. In contrast, antibodies against types III and V collagen were seen to stain primarily the walls of the larger vessels. Antibodies against type II collagen did not react with retinal vessels. Preincubation with the appropriate antigen or preimmune serum eliminated staining of the vessels by the antisera.

Effects of TGF-beta and TGF-beta neutralizing antibodies on fibroblast-induced collagen gel contraction: implications for proliferative vitreoretinopathy.

PURPOSE: The main cause of failure after retinal reattachment surgery is proliferative vitreoretinopathy (PVR), in which contractile fibrocellular membranes form on the retinal surface and vitreous base. Recently, elevated levels of transforming growth factor-beta 2 (TGF-beta 2) were measured in the vitreous of patients with PVR, suggesting a possible association with the disease. Because neutralizing TGF-beta may prove useful in controlling this blinding disease process, the authors examined the effect of anti-TGF-beta 1 and TGF-beta 2 antibodies in TGF-beta-mediated fibroblast-induced collagen gel contraction. METHOD: Rabbit dermal fibroblasts were combined with type I collagen in an in vitro model of collagen gel contraction. The authors evaluated the effect of TGF-beta 1, TGF-beta 2, and their antibodies on fibroblast-induced gel contraction. RESULTS: TGF-beta 1 and TGF-beta 2 equally enhanced gel contraction to an average of 6% to 7% of the control area by day 4. In contrast, gels without TGF-beta contracted only to an average of 38% of the control gels. Several anti-TGF-beta antibodies neutralized this TGF-beta-enhanced contraction, whereas control IgGs had no effect. A dose-dependent response was detected with TGF-beta 1, TGF-beta 2, and anti-TGF-beta. CONCLUSION: Because TGF-beta levels have been shown to correlate with the severity of PVR, the neutralizing action of anti-TGF-beta on TGF-beta-mediated contraction may offer further insights into the structure and function of PVR membranes and may provide clues to possible therapeutic solutions for controlling this disease process.

Retinal pigment epithelium cells can influence endothelial cell plasminogen activators.

Endothelial cells from both human retinal microvessels (HME) and fetal bovine aortic endothelium (FBAE) were grown in aggregate cultures alone, or with either retinal pigment epithelium (RPE) cells or fibroblasts. The levels of plasminogen activator (PA) and plasminogen activator inhibitor (PAI) in the conditioned media of the various aggregate types were measured. High PA levels were detected in the conditioned medium of pure endothelial cell aggregates (equal to 140% and 124% of urokinase control for HME and FBAE, respectively), and high PAI levels were associated with pure RPE aggregates (inhibiting 93% of the urokinase control). The conditioned medium of pure fibroblast aggregates had very low levels of either PA or PAI. When RPE cells were aggregated with FBAE or HME cells into mixed (heterogenous) aggregates, the PA measured in the conditioned medium was equal to 22% and 30% of the urokinase control, respectively. The PA level in the conditioned medium of mixed fibroblast-FBAE cell aggregates was higher, 104% of the control, and the difference was statistically significant (P less than 0.001). Co-incubation of pure RPE aggregates with pure FBAE aggregates or with pure HME aggregates resulted in PA activity in the conditioned medium that was equal to 110% and 96% of the control, respectively. The PA level found when pure FBAE cell aggregates were co-incubated with pure fibroblast aggregates was higher, 134% of the control, and the difference was statistically significant (P less than 0.001). Our results indicate that RPE cells can reduce endothelial cell PA, probably through both direct contact between the cells and PAI production. Fibroblasts did not have this influence on endothelial cell PA.

The extracellular matrix composition of the monkey optic nerve head.

The presence and distribution of laminin, heparan sulfate proteoglycan and collagen types I, III and IV were immunohistologically determined in cynomolgus monkey optic nerve heads using the avidin-biotin-peroxidase complex technique. Collagen types I and III were detected within the collagenous plates of the scleral lamina cribrosa, in the septa and pia mater of the postlaminar optic nerve and in blood vessel walls in all regions of the optic nerve head. Collagen type IV, laminin and heparan sulfate proteoglycans were all localized to the margins of the collagenous laminar plates of the scleral lamina cribrosa and along the margins of the optic nerve septa and the pia mater. All three components also appeared beneath the blood vessel endothelium throughout the optic nerve head. Within the lamina cribrosa, collagen types I and III occupy the core of the scleral laminar plates and may provide structural support for optic nerve bundles exiting the eye. The distribution of collagen type IV, laminin and heparan sulfate proteoglycan corresponds to basement membranes from two sources: vascular endothelial cells and glial cells lining the axonal bundles. Abnormalities of these substances may influence optic nerve function and susceptibility to elevated intraocular pressure by altering their mechanical support functions within the nerve head, by interfering with axonal nutrition, or both.

Diabetic preretinal membranes. An immunohistochemical study.

Fibrovascular membranes removed during vitreous surgery from patients with proliferative diabetic retinopathy were examined using indirect immunofluorescence. Antisera against collagen types I, III, IV, and V, attachment factors fibronectin and laminin, endothelial marker factor VIII, and the glial-marker glial-fibrillary acidic protein were used to examined the membranes. Seven of ten specimens contained cells staining positively for glial-fibrillary acidic protein, either singly or in large clusters within the membrane. Four of six membranes showed a well-developed vascular bed surrounded by a dense collagenous matrix. The matrix stained strongly with antiserum against both interstitial type I collagen and its associated attachment factor, fibronectin. In contrast, only localized staining was observed when antisera against both type IV collagen and its associated attachment factor, laminin, were used, and this was concentrated in regions of blood vessels and associated internal limiting membrane.

Serum contains chemoattractants for human retinal pigment epithelial cells.

Blood or serum in the vitreous cavity is associated with cellular membrane formation in proliferative vitreoretinopathy and following severe trauma. Retinal pigment epithelial cells (RPE) are important components of these membranes. For RPE cells to effectively spread throughout the vitreous cavity and form contractile membranes, cell migration must occur. We found that fetal bovine serum and human serum increase human RPE cell migration in a dose-dependent manner; fibronectin (FN) caused a dose-dependent increase in RPE cell migration of similar magnitude. This effect was independent of any effect on cell attachment. The RPE cell migration occurred along a gradient from low to high concentrations of FN and thus represents chemotaxis. Serum depleted of FN lost much of its ability to stimulate RPE cell migration. These results demonstrate that serum contains chemoattractants for human RPE cells and that FN accounts for much of this activity.

Vitreous aspirates from patients with proliferative vitreoretinopathy stimulate retinal pigment epithelial cell migration.

Several studies have focused on retinal pigment epithelial (RPE) cell proliferation as an important event in proliferative vitreoretinopathy (PVR). Little attention has been given to the question of how RPE cells gain access to the vitreous cavity where proliferation occurs. We have recently demonstrated that the serum components fibronectin and platelet-derived growth factor stimulate and direct RPE migration in vitro. In this study, we used this same in vitro technique to examine vitreous aspirates from 13 eyes with PVR, five eyes with macular puckers, and three eyes with uncomplicated retinal detachments for their ability to stimulate RPE migration. We found that aspirates from eyes with PVR stimulated RPE migration to a much greater extent than aspirates from eyes with macular pucker and uncomplicated retinal detachments. The ability to stimulate RPE cell migration correlated with high levels (mean +/- SEM, 178 +/- 67 mg/L) of immunoreactive fibronectin.

Ultrastructural location of extracellular matrix components in the optic nerve head.

The distributions of laminin and collagen types I through V were determined in optic nerve heads of cynomolgus monkeys using colloidal gold immunoelectron microscopy. Collagen types I and III localized to striated collagen fibrils in arterial walls throughout the optic nerve head and to fibrils comprising the core of the lamina cribrosa beams and optic nerve septa. Collagen type IV and laminin localized to basement membranes of all blood vessels and to basement membranes of astrocytes that lie between the laminar beams and line the optic nerve septa and pia mater. Collagen type V was detected both in striated collagen fibrils and in vascular and astrocyte basement membranes. These results provide a detailed understanding of the optic nerve head extracellular matrix distribution and illustrate a sensitive method for future study of its role in glaucomatous optic nerve damage.

Structural proteins of the neonatal and adult lamina cribrosa.

Optic nerve heads from three premature infants and six adults were studied immunohistochemically to compare the extracellular proteins in the lamina cribrosa of young and old human eyes. In both age groups, antibodies to the basement membrane components laminin and collagen type IV were associated with blood vessels and laminar beam margins. In the adult eyes, interstitial collagen types I and III were heavily distributed within the laminar beams. Antibodies to fibrillin, the microfibrillar portion of elastin, labeled discrete, heavy bands oriented longitudinally within these beams. The beams of the neonatal lamina cribrosa contained much less interstitial collagen, with a predominance of collagen type III. Neonatal elastic tissue bands were less numerous and distinct within the laminar beams. These biochemical differences between the young and old lamina cribrosa may, in part, explain different clinical behaviors of the optic nerve head in congenital and adult glaucoma.

Extracellular matrix of newly forming vessels--an immunohistochemical study.

During wound healing, embryological development, and solid tumor growth, the established vasculature gives rise to large numbers of new blood vessels. This neovascular response occurs at the level of the capillary bed, where endothelial cells divide rapidly, locally remodel the surrounding stroma, and migrate away from existing vessels to form capillary sprouts. In order to examine the environment of these newly forming vessels, actively growing blood vessels in neovascularized rabbit and guinea pig corneas were examined immunohistochemically using antibodies against laminin, type IV collagen, heparan sulfate proteoglycans, entactin, and factor VIII-related antigen. Sequential serial 5-microns sections taken from the unfixed frozen corneas in a plane perpendicular to the direction of vessel growth were stained with these antibodies. It was possible to follow well-defined lumenized vessels out through sequential sections to the point where they became single factor VII-R positive cells in the region of the capillary sprout. Examination of these stained sections has shown the presence of four important basement membrane components--laminin, type IV collagen, heparan sulfate proteoglycan, and entactin--associated with actively migrating and invading capillary sprouts. These results suggest that the extracellular matrix of the actively invading capillary sprouts does not differ qualitatively from that of the established vasculature.

Retinal pigment epithelial cells release inhibitors of neovascularization.

Human retinal pigment epithelial (RPE) cells in culture were found to release a substance (or substances) that causes the regression of new blood vessels on the chick embryonic yolk sac and inhibits proliferation of fetal bovine aortic endothelial cells and human retinal microvessel endothelial cells in vitro. Neither astrocytes nor fibroblasts under identical test conditions released detectable inhibitors of neovascularization or endothelial cell growth. Subconfluent and superconfluent cultures of human RPE cells released higher levels of inhibitor than confluent cultures.

15-Hydroxyeicosatetraenoic acid stimulates migration of human retinal microvessel endothelium in vitro and neovascularization in vivo.

We evaluated 15-hydroxyeicosatetraenoic acid (15-HETE), a major arachidonic acid product of vascular endothelium and leukocytes, for its effect on neovascularization. In a modified Boyden chamber assay, 15-HETE (10-7 M) stimulated human retinal microvessel endothelial cell migration by 42 +/- 10% (mean +/- S.E.M., p less than 0.01). 12-HETE, a major arachidonic acid metabolite of platelets, had no such effect. Further studies in the rabbit corneal pocket assay revealed that 15-HETE stimulated neovascularization in vivo. Concentrations at which the in vivo effects were observed are within the range generated by several cell types and are achievable in human serum. 15-HETE stimulation of human endothelial cell migration in vitro and neovascularization in vivo suggests that it may play a role in vasoproliferative disorders.

Proliferative vitreoretinopathy membranes. An immunohistochemical study.

Proliferative vitreoretinopathy (PVR) is the leading cause of failure after retinal detachment surgery. Therefore, both the extracellular matrix and cellular components of preretinal membranes from 23 eyes with PVR were characterized immunohistochemically. The membrane stroma was composed primarily of types I, II, and III collagen. Laminin and both heparan sulfate proteoglycans and collagens types IV and V were co-distributed in discrete regions within the stroma. Glial and retinal pigment epithelial (RPE) cell populations were identified in these membranes using specific immunohistochemical markers as was a small but significant macrophage population. Double-labeling experiments indicated that RPE cells in these membranes expressed the class II histocompatibility antigen HLA-DR, although neither the RPE monolayer in situ nor cultured RPE cells was HLA-DR positive unless induced by gamma interferon. Only rare isolated vascular endothelial cells were detected in 5 of the 23 membranes.