Demonstration of vasoproliferative activity from mammalian retina.

Vasoproliferative activity has been demonstrated in extracts of retinas from human, bovine, and feline sources. These retinal extracts are capable of stimulating (a) proliferation and thymidine uptake of bovine vascular endothelial cells in culture and (b) neovascularization on the chick chorioallantoic membrane. Extracts of skeletal muscle, cardiac muscle, and liver lack similar stimulatory activity. The activity is nondialyzable, stable at 56 degrees C, and inactivated at 100 degrees C. Retinal extracts stimulate the proliferation of corneal fibroblasts but have no effect on the proliferation of vascular smooth muscle cells. Indirect evidence suggests the liberation of a vasoproliferative factor from retina in several ocular disorders. The data in this report represent the first direct demonstration of vasoproliferative activity from mammalian retina.

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.

Stimulation of endothelial cell prostacyclin release by retina-derived factors.

An angiogenic extract of bovine retina as well as two purified angiogenic growth factors, acidic and basic fibroblast growth factor, stimulate vascular endothelial cell prostacyclin (PGI2) release in vitro as measured by radioimmunoassay of its stable metabolite 6-keto PGF1 alpha (6kPGF). After incubating fetal bovine aortic endothelial cells with 10% retinal extract (RE) for 24 hr, 6.5 ng of 6-kPGF/10(5) cells were released compared to 0.8 ng of 6kPGF/10(5) cells for unstimulated endothelium. Similar qualitative results were obtained using human retina-derived microvessel endothelium. PGI2 release in response to RE depended on endothelial cell density with subconfluent cultures releasing six-fold more 6kPGF compared to confluent monolayers. Thin layer radiochromatography of endothelial cell conditioned media demonstrated enhanced release of 6-kPGF, PGE2 and arachidonic acid after RE addition. Cycloheximide and actinomycin D inhibited PGI2 release but hydroxyurea had no effect. Most of the PGI2-stimulating activity of RE was adsorbed to heparin-Sepharose and eluted by 2 M NaCl. Purified acidic (10 ng/ml) and basic (1 ng/ml) fibroblast growth factors caused seven-fold and four-fold stimulation of endothelial PGI2 release, respectively. An initial event in the regression of new blood vessels following the removal of an angiogenic stimulus is the formation of intraluminal platelet aggregates. PGI2 is a potent inhibitor of platelet aggregation.(ABSTRACT TRUNCATED AT 250 WORDS)

The extracellular matrix of human retinal pigment epithelial cells in vivo and its synthesis in vitro.

The production of extracellular matrix material by retinal pigment epithelium (RPE) may influence or mediate some of the many important functions of this tissue. Using immunohistochemical staining techniques, the extracellular matrix surrounding the RPE in vivo and the components produced by RPE in vitro have been investigated. Frozen sections of eye bank eyes showed antigen specific staining for collagen types I, III, and IV, and for fibronectin and laminin in Bruch's membrane and surrounding the RPE. Only very slight staining of the membrane was seen with antiserum against type II collagen, and there was no staining for type V collagen. Specific staining was demonstrated for the four collagens and glycoproteins in the extracellular matrix of RPE cells grown in culture. Once again, there was no staining for type V collagen. This study demonstrates that the RPE is capable of producing many of the components of the extracellular matrix found in Bruch's membrane and surrounding the RPE in vivo. This function may be important in the maintenance of a physical barrier to subretinal neovascularization, and may also play a role in such pathologic states as proliferative vitreoretinopathy.

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.

Platelet-derived growth factor is chemotactic for human retinal pigment epithelial cells.

The presence of blood or serum in the vitreous cavity has been associated with the formation of cellular membranes in proliferative vitreoretinopathy and after penetrating ocular trauma. Retinal pigment epithelial (RPE) cells are an important component of these membranes. For RPE cells to effectively spread throughout the vitreous cavity and form contractile membranes, cell migration must occur. Serum has been shown to initiate RPE cell migration. Fibronectin (FN), a glycoprotein found in serum, stimulates RPE cell migration but accounts for only part of the stimulatory effect of serum. We report that another serum component, platelet-derived growth factor (PDGF), also stimulates RPE cell migration. Furthermore, the effect of PDGF and FN are additive and together probably account for a large part of the chemotactic activity found in serum.

Endothelial cells release a chemoattractant for retinal pigment epithelial cells in vitro.

Though the pathogenesis of choroidal neovascular membranes is uncertain, there is evidence to support a primary dysfunction in the retinal pigment epithelium (RPE). This suggests the possibility that a healthy RPE may provide a physical and/or chemical barrier to subretinal endothelial cell invasion. It has recently been shown that RPE cells in culture produce an inhibitor of neovascularization. Histopathologic evidence suggests that RPE cells tend to surround new blood vessels and contain them. We therefore investigated the possibility that RPE cells are guided toward endothelial cells by chemoattractants. Using a modified Boyden chamber technique, we showed that endothelial cells in culture produce a chemoattractant for RPE cells. The active component is trypsin sensitive, stable at extremes of pH (3 through 10), and nondialyzable (12,000- to 14,000-dalton cutoff). It is partially heat stable but becomes completely heat stable in the presence of 1% sodium dodecyl sulfate. These are all characteristics of the previously described endothelial cell-derived growth factor, suggesting that this mitogen might be the chemoattractant. The ability of RPE cells to be attracted to sites of new blood vessel formation may enhance their potential function as inhibitors of neovascularization.

Retinal pigment epithelial cells release a chemoattractant for astrocytes.

Astrocyte migration was studied using a modified Boyden chamber. Astrocytes migrated in response to a concentration gradient (chemotaxis) of retinal pigment epithelial cell-conditioned medium. In contrast, smooth muscle cell- and corneal fibroblast-conditioned media stimulated random migration (chemokinesis) of astrocytes but not chemotaxis. Furthermore, retinal pigment epithelial cell-conditioned medium stimulates only random migration of smooth muscle cells and corneal fibroblasts. The chemotactic factor produced by retinal pigment epithelial cells appears to have a molecular weight between 10,000 and 30,000 determined by ultrafiltration. Activity is lost after boiling but is stable, with a pH between 2 and 7. These results suggest that in proliferative vitreoretinopathy, retinal pigment epithelial cells on the retina and within the vitreous cavity may release a chemoattractant that stimulates astrocytes to migrate into the vitreous cavity.

Mechanisms involved in retinal pigment epithelial cell chemotaxis.

Retinal pigment epithelial (RPE) cell chemotaxis may play an important role in proliferative vitreoretinopathy (PVR). Fibronectin and platelet-derived growth factor are chemoattractants for RPE cells. In this study, we used these chemoattractants to examine mechanisms involved in RPE cell chemotaxis. Our results demonstrate that inhibition of RNA and protein synthesis, but not DNA synthesis, are associated with reduced chemotaxis. Microtubules and microfilaments are involved since cytochalasin B and colchicine are potent inhibitors of RPE cell migration. Dexamethasone, to which a beneficial effect has been attributed in an animal model of PVR, had no effect on RPE cell migration. Information concerning mechanisms involved in RPE cell migration may help to explore new avenues of treatment in PVR.

Inhibition of retinal pigment epithelial cell attachment by a synthetic peptide derived from the cell-binding domain of fibronectin.

Retinal pigment epithelial (RPE) cells are dispersed into the vitreous cavity during retinal reattachment surgery and attach to the vitreous gel and internal limiting membrane. Subsequent migration and cell-mediated contraction can result in traction retinal detachment. Fibronectin (FN) can enhance the attachment of various cell types. We now report that FN enhances the attachment of human and porcine RPE cells. A synthetic tetrapeptide, arg-gly-asp-ser (RGDS), derived from the FN cell-binding domain, has been shown to interfere with attachment of some cell types. We now report that RGDS inhibits RPE cell attachment to FN in a dose-dependent manner with 70% inhibition at 1 mg/mL. Also, RGDS inhibits attachment to types I and II collagen, as well as to lens capsule basement membrane by 40%, 50%, and 25%, respectively. Therefore, RGDS or its derivatives may have some eventual role in the management of proliferative vitreoretinopathy.

New and previously unidentified retinal breaks in eyes with recurrent retinal detachment with proliferative vitreoretinopathy.

The location of retinal breaks found on preoperative examination was studied in 68 eyes of 68 patients with recurrent retinal detachment and proliferative vitreoretinopathy. Twelve eyes had 23 open breaks that were known to exist previously, no open break was detected in 18 eyes, and 72 new or previously unidentified breaks were found in 41 eyes. Forty-seven (65.3%) of the 72 breaks were located on previous buckles, and 31 of these were on the posterior slope of the buckle. Twenty-nine (40.2%) of all new or previously unidentified breaks were on the border of a cryopexy-induced chorioretinal scar, and of these, 25 breaks (86%) were on the posterior slope of the buckle. Our results indicate that the retina that borders chorioretinal scars is vulnerable and prone to develop retinal tears secondary to traction from preretinal membranes. The vicinity of cryopexy-induced scars should be closely observed for retinal breaks in cases of recurrent retinal detachment with proliferative vitreoretinopathy.

Role of lensectomy and posterior capsule in movement of tracers from vitreous to aqueous.

The rate of movement of 20,000- and 70,000-dalton dextran from vitreous to aqueous was determined after extracapsular lensectomy in rabbits with an intact posterior capsule. Dextran was injected into the vitreous, and 3.5 hours later the aqueous concentration of 20,000-dalton dextran was 10.3-fold greater in the aphakic than the phakic eye. The aqueous concentration of the 70,000-dalton dextran was 3.9-fold greater in the aphakic eye after 3.5 hours. Thus, extracapsular lensectomy with an intact posterior capsule increases the rate of movement of 20,000- and 70,000-dalton dextran from the vitreous to the anterior chamber. In addition, the movement of tracers from vitreous to aqueous is increased further when the posterior capsule is opened.

Effect of lensectomy on the movement of tracers from vitreous to aqueous.

To investigate the effect of extracapsular lensectomy with posterior capsulotomy, 20,000- and 70,000-dalton dextran was injected into the vitreous of rabbits. The concentration of the 20,000-dalton dextran in the aqueous of aphakic eyes was 14-fold greater than in phakic eyes 3.5 hours after injection of dextran into the vitreous. The aqueous concentration of the 70,000-dalton dextran was four times greater in aphakic eyes after 3.5 hours. The aqueous concentration of a 20,000-dalton dextran in phakic and aphakic eyes became equal between 15.5 and 22.0 hours after injection. Thus, extracapsular lensectomy with posterior capsulotomy increased the rate of movement of a single bolus of both 20,000- and 70,000-dalton dextran preparations from the vitreous to the anterior chamber.

Low-dose colchicine inhibits astrocyte, fibroblast, and retinal pigment epithelial cell migration and proliferation.

We studied the effect of low-dose colchicine on cell proliferation and migration. Colchicine at doses well below the level of ocular toxicity was found to be a potent inhibitor of astrocyte, fibroblast, and retinal pigment epithelial (RPE) cell proliferation and migration. A colchicine concentration of 1.3 X 10(-8) mol/L, 10(-8) mol/L, and 1.7 X 10(-8) mol/L showed a 50% inhibition of proliferation for RPE, astrocytes, and fibroblasts, respectively. At 10(-7) mol/L, colchicine inhibited 44%, 46%, and 93% of RPE, astrocyte, and fibroblast migration, respectively. Low-dose colchicine appears to be an effective inhibitor of RPE, astrocyte, and fibroblast migration and proliferation in vitro.

Oral colchicine for the treatment of experimental traction retinal detachment.

In proliferative vitreoretinopathy and trauma, long-term reattachment of the retina is often prevented by the formation of contractile cellular membranes on the retinal surface and within the vitreous cavity. Colchicine, a well-documented inhibitor of microtubule assembly, is a potent inhibitor of retinal pigment epithelium cell, astrocyte, and fibroblast proliferation and migration. To study the therapeutic value of orally administered colchicine, we used an experimental animal model in which intravitreally injected platelet-derived growth factor and fibronectin produced traction retinal detachments in rabbits. With oral administration of colchicine, we were able to decrease the incidence and severity of traction retinal detachment from 74% in controls (20 of 27 eyes) to 29.6% in the treated animals (eight of 27 eyes) at five weeks (P less than .0009). These results, and the apparent lack of retinal and systemic toxicity, suggest that oral therapy with colchicine may prove to be of value in the treatment of human disease.

Effect of fibrin on morphologic characteristics of retinal pigment epithelial cells.

Human retinal pigment epithelial (RPE) cells in culture, when overlaid by a fibrin clot, lose their normal epithelial morphologic features and migrate into the overlying clot as fibrocytelike cells. The behavior of human RPE cells on exposure to fibrin correlates well with the observed response of RPE in several ocular disorders in which fibrin deposition within the eye is an important feature. The mechanism of recognition and interaction between fibrin and RPE cells is unknown. The in vitro system used in our current studies allows the investigation of this interaction in a controlled environment. Further study of the interaction between human RPE cells and fibrin offers the possibility of improving our understanding and treatment of several blinding ocular disorders, including the disciform phase of senile macular degeneration, proliferative vitreoretinopathy, and the sequelae of ocular trauma.

Effect of vitreous on morphologic characteristics of retinal pigment epithelial cells. A new approach to the study of proliferative vitreoretinopathy.

Normally, human retinal pigment epithelial (RPE) cells in confluent monolayers maintain a hexagonal to oval shape. However, when these monolayers were overlaid with autologous vitreous, the RPE cells elongated and migrated into the vitreous gel as bipolar fibrocytelike cells. We studied the effect of sodium hyaluronate and collagen individually. Retinal pigment epithelial cells overlaid with hyaluronate did not change their morphologic features. In contrast, when RPE monolayers were overlaid with a collagen gel, the cells changed from their normal epithelial characteristics to bipolar fibrocytelike cells that later migrated into the collagen gel. Exposure of RPE cells to vitreous may play a role in the pathogenesis of proliferative vitreoretinopathy.

Vitrectomy for macular traction caused by incomplete vitreous separation.

Pars plana vitrectomy was performed in 16 eyes to release vitreomacular traction. All patients had a symptomatic preoperative decrease in visual acuity, most often to 20/200. Partial detachment of the posterior vitreous surface was associated with vitreoretinal attachment remaining in the area of the macula and sometimes to the optic nerve head. The posterior vitreous traction on the macula was released by tangential traction on the posterior vitreous surface, causing it to separate from the retina. The postoperative vision was the same or improved in each case. No operative complications occurred, but progressive nuclear sclerosis developed postoperatively in five of the 15 phakic eyes.

Effect of epsilon-aminocaproic acid on postvitrectomy hemorrhage.

We performed a prospective study involving 96 patients undergoing vitrectomy for proliferative diabetic retinopathy to determine the effect of epsilon-aminocaproic acid on the occurrence of postoperative intraocular hemorrhage. epsilon-Aminocaproic acid significantly reduced postoperative vitreous hemorrhage during the immediate postoperative period. Follow-up examinations two to six weeks after discharge from the hospital disclosed no statistically significant difference in the severity of vitreous hemorrhage between the treated and untreated groups. The loss of drug effect at this stage was in part due to spontaneous repeated bleeding in the treated group and in part to spontaneous clearing of hemorrhage in the untreated group. There was no statistically significant difference in the rate of repeated bleeding between the two groups or in rate of spontaneous clearing.