Thrombospondin: a new attachment protein in preretinal traction membranes.

Thrombospondin (TSP), an adhesive integrin-binding protein of plasma and platelets, was detected in preretinal traction membranes from patients with idiopathic (8/8) and traumatic (7/8) proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR) (6/8). TSP immunoreactivity was compared to the pattern of von Willebrand factor, plasma transglutaminase (blood coagulation factor XIII), fibronectin, and mononuclear phagocytes, using double-label immunofluorescence microscopy. TSP was partially colocalised with the endothelial cell marker, von Willebrand factor, in PDR. The codistribution of catalytic factor XIII and two cross-linking substrates, fibronectin and TSP, suggests a functional role of the enzyme in the extracellular matrix build-up in PVR and PDR. No significant TSP synthesis by mononuclear phagocytes was observed. Western blotting indicated a plasmin-mediated intravitreal breakdown of presumably plasmatic TSP in PVR and PDR.

[Protein composition of the vitreous body in proliferative diabetic retinopathy. An analysis with 2-D-electrophoresis]. / Die Proteinzusammensetzung des Glaskörpers bei proliferativer diabetischer Retinopathie. Eine Analyse durch 2-D-Elektrophorese.

Gradient SDS-polyacrylamide gel electrophoresis is a useful technique for characterization of soluble human vitreous protein. However, this technique is limited in the case of suboptimal discrimination power. In this study, we used 2-D electrophoresis to analyse the protein composition of proliferative diabetic retinopathy (PDR) intraocular fluid (n = 10), and compared it with normal vitreous (n = 10) and serum (n = 10). In normal vitreous, the protein content consisted mainly of albumin, transferrin, alpha 1-antitrypsin, IgG, and prealbumin as confirmed by the comparison with protein standards. Compared to vitreous controls, all PDR samples were shown to have lower amounts of transferrin, alpha 1-antitrypsin, and prealbumin. However, the amounts of IgG were higher than in the controls. This reflects a shift to a serum-like protein profile, indicating a blood-retinal barrier breakdown. However, we also detected in PDR vitreous three protein spots in a low-molecular-weight range (5-10 kDa) none of which could be found in native vitreous or in serum. Therefore, additional local protein synthesis appears to be present in the pathogenesis of PDR. 2-D electrophoresis permits precise characterization of the soluble vitreous proteins which may be associated with the fibrovascular proliferative vitreoretinal response.

[Idiopathic macular foramen: new aspects of staging and possible therapeutic concepts]. / Das idiopathische Makulaforamen: Neue Aspekte zur Stadieneinteilung und mögliche Therapiekonzepte.

Idiopathic macular hole and its early stages raises one of the most fascinating subjects in retinal disease. In this review, we aim to summarize the current knowledge of pathogenesis, the recent classification and the strategies for therapy. Recent evidence strongly suggests tangential traction induced by a thin epiretinal membrane to be the main cause for idiopathic senile macular holes. A thickening of the epicortical vitreous membrane or the internal limiting membrane of the retina could be demonstrated in these cases. When such a precursor situation (stage I) is present, in many cases there will be a progression to a full thickness macular hole (stage II). Further traction usually causes an enlarging of the defect in these eyes and the classic appearance of macular holes (stage III-IV) can then be observed. The clinical appearance, the diagnosis and the strategies of treatment are discussed for all stages of idiopathic senile macular holes. Based on the deeper insight into macular hole development, prophylactic vitrectomy and removal of the epiretinal membrane in cases of stage I macular holes, has been considered in order to prevent a further progression of the disease. There is also evidence that in stages II-IV macular holes, a closure of the hole and a visual improvement can be gained by vitrectomy, removal of the epiretinal membrane and fluid-gas exchange. The additional application of biological modifiers (transforming growth factor-beta, human autologous serum or tissue glue) may enhance the adhesion of the detached retina and therefore lead to better anatomical and functional success rates. The results of the pilot studies are reviewed and the surgical techniques as well as the possible complications are discussed.

Vitreous body-derived mitogenic activity for retinal pigment epithelial cells. A further characterization.

To obtain a better insight into the possible role of vitreous body in proliferative vitreoretinopathy (PVR), we examined the influence of bovine vitreous body fractions separated by fast protein liquid chromatography (FPLC) on the growth of porcine retinal pigment epithelial (RPE) cells in vitro. Tetrazolium colorimetric assay (MTT assay) and cell counting were used for quantification. After fractionating proteins of the physiological vitreous body by FPLC gel chromatography (Superose 12/Superdex 75), we determined the mitogenic effect of the resulting fractions by either cell counting or MTT assay. With both columns we were capable of separating two fractions (20-40 kDa and 1-2 kDa) that induced RPE cell proliferation. A comparison of the mitogenesis and the protein content of the fractions indicated that we were not dealing with a nonspecific protein effect. The low-molecular fraction weighing less than 2 kDa was of particular interest to us. This was further separated by FPLC reversed-phase chromatography.

[Immunohistology of proliferative vitreoretinopathy following giant tear detachment]. / Zur Immunhistologie der proliferativen Vitreoretinopathie nach Riesenriss-Amotio.

A patient with multiple bilateral retinal holes and a history of several prior surgical interventions was treated by pars plana vitrectomy for total traction retinal detachment in the right eye. The preretinal membrane specimen obtained surgically was analyzed using an immunological label for macrophages, vimentin, cytokeratin, fibronectin, fibrinogen, transferrin, the transferrin receptor, blood coagulation factor XIII-A and XIII-S, and haptoglobin. The results of the study suggest that factor XIII and fibronectin play an important role in idiopathic PVR. Proliferating cells of fibroblastic appearance invade a preretinal exudate and restructure the injured vitreoretinal tissue, forming a contracted scar. Macrophages, known to be characteristic of early posttraumatic PVR development, were not present in this case of PVR, where traction retinal detachment was presumably induced by the formation of multiple retinal holes.

Vitronectin and proliferative intraocular disorders. II. Expression of cell surface receptors for fibronectin and vitronectin in periretinal membranes.

Several cell types participate in the formation of vitreoretinal traction membranes in proliferative intraocular disorders. The communication between these cells involves hormones, growth factors, and the interaction with extracellular matrix molecules. We have previously demonstrated a partial colocalisation of two potent mediators of cell attachment, fibronectin and vitronectin, in periretinal membranes from patients with proliferative vitreoretinopathy (PVR). We found a similar pattern of vitronectin and fibronectin deposition in proliferative diabetic retinopathy (PDR) (n = 6). Now we show the expression of the corresponding cell surface receptors, integrins, for fibronectin and vitronectin by proliferating cells in 22 periretinal membranes, including traumatic (n = 8) and idiopathic (n = 8) PVR as well as PDR membranes (n = 6). Integrins are membrane receptors for extracellular matrix macromolecules which are involved in such basic biological phenomena as embryogenesis and metastasis. Future studies on the pathogenesis of vitreoretinal proliferation will have to focus on the initiation, maintenance, and regulation of this intercellular communication network involving attachment proteins and integrins.

Vitronectin and proliferative intraocular disorders. I. A colocalisation study of the serum spreading factor, vitronectin, and fibronectin in traction membranes from patients with proliferative vitreoretinopathy.

The presence of a scaffold for cellular spreading and proliferation is a precondition for the development of traction membranes in proliferative vitreoretinopathy (PVR). This study shows the presence of the serum spreading factor, vitronectin, in the extracellular matrix of periretinal membranes removed during vitreoretinal surgery. By means of a double label immunofluorescence protocol, a partial colocalisation of vitronectin with fibronectin at the magnification of light microscopy can be detected. Fibronectin is a high-molecular glycoprotein with multiple biological functions including the mediation of cell attachment and migration. Both proteins share a special cell recognition site which could be a target for experimental pharmacological approaches to PVR. Preliminary studies of vitreous aspirates using electrophoresis and Western blotting indicate that vitronectin may play a more important role in post-traumatic PVR as compared to PVR following rhegmatogenous retinal detachment.

Blood coagulation factor XIII contributes to the development of traction retinal detachment.

The blood coagulation factor XIII catalyzes the crosslinking of fibrin monomers at the end of the coagulation cascade. Additional functions are the enzymatic coupling of fibrinectin to itself, fibrin, and collagen. We located the two subunits of factor XIII in 20 surgically obtained periretinal membranes, using double label immunofluorescence microscopy. Both subunits of factor XIII could be detected in all specimens. The positive staining in all specimens examined prompted us to determine the source of factor XIII. The abundant fibroblastic cells did not contain factor XIII. Macrophages, half of which stained for the alpha-subunit of factor XIII could not account for the presence of factor XIII because these cells were not present in all specimens, and did not stain for the beta-subunit. Factor XIII is probably derived from the exudation of plasma and platelets through disrupted blood-ocular barriers. This is confirmed by the detection of both subunits in vitreous aspirates from patients with proliferative intraocular disorders (n = 15) by Western blotting.

[Detection of coagulation factor XIII in the vitreous body and periretinal membranes in proliferative retinal diseases]. / Nachweis von Gerinnungsfaktor XIII im Glaskörper und periretinalen Membranen bei proliferativen Netzhauterkrankungen.

The human blood coagulation factor catalyses the cross-linking of fibrin monomers at the end of the coagulation cascade. Additional functions are the coupling of fibronectin and collagen to each other and fibrin. Therefore we tried to investigate the significance of factor XIII in the development of intraocular membranes. Using gel electrophoresis and western blotting, both subunits (A and B) of factor XIII could be detected in vitreous aspirates from patients with "idiopathic" proliferative vitreoretinopathy (PVR) (n = 5), traumatic PVR (n = 5), and proliferative diabetic retinopathy (n = 5). In contrast the vitreous of five human "normal" post mortem eyes did not contain the subunits of factor XIII. Furthermore, we observed immunofluorescence staining for both subunits of factor XIII in 20 surgically obtained periretinal membranes. In early cellular as opposed to late hypocellular membranes we observed stronger labeling for both subunits of factor XIII. With double label staining techniques, the fibroblastic cells recognized by vimentin staining did not contain factor XIII. About 50% of the macrophages stained positive for the A-subunit of factor XIII. We observed no labeling for the B-subunit in macrophages. Therefore, we hypothesize that factor XIII in proliferative vitreoretinal disorders (PVR, PDR) is derived from the exudation of plasma and platelets through disrupted blood-retinal barriers.

Giant preretinal membrane formation behind a silicone oil bubble in a hypotensive eye.

A patient who suffered a severe contusion injury with scleral rupture and subsequent peracute development of proliferative vitreoretinopathy was referred after wound closure at the local ophthalmology center. Initial treatment of the almost blind eye consisted of vitrectomy, silicone oil tamponade, and intraocular daunorubicin. A vitreous aspirate obtained during surgery was analyzed biochemically by electrophoresis and Western blotting. After a few days, the patient needed further surgical intervention because silicone oil was leaking through the primary sutured scleral wound. A giant fibrinous membrane extending over the posterior pole was removed. Double-label immunofluorescence examination of this specimen showed a positive reaction for transferrin, the cell surface transferrin receptor, fibrinogen, fibronectin, macrophages, and vimentin. No staining was obtained for proliferating cell antigen and T-lymphocyte antigen. The analysis of this case with a very well-defined clinical course offers valuable insight into the early stages of proliferative vitreoretinopathy.

[Vitronectin: mediator of cell adhesion in proliferative vitreoretinopathy?]. / Vitronektin: Mediator der Zelladhäsion bei der proliferativen Vitreoretinopathie?

Vitronectin, also known as protein S, the "serum spreading factor", or epibolin, was detected as an essential mediator of adhesion and spreading in many cells in vitro. The relatively low molecular weight of 65 kDa and a high plasma level of 200 mg/l implicate vitronectin as a possibly important factor in the pathogenesis of proliferative vitreoretinopathy (PVR) which is characterized by a breakdown of blood-ocular barriers. In a study of 15 periretinal membranes, using double label immunofluorescence techniques, we found vitronectin to be a significant component of the specimens' extracellular matrix in 13 cases. Vitronectin is co-localized with fibronectin, a much larger glycoprotein suggested as being involved in the pathogenesis of PVR. Among the biological properties of fibronectin are a role in cellular migration, adhesion, and proliferation. Both proteins share a unique cell recognition amino acid sequence, which mediates the receptor-dependent interaction between the extracellular matrix macromolecules and proliferating cells. Plasma is suggested as the major source of vitronectin in PVR because vitronectin could be detected in vitreous aspirates from patients with PVR, using electrophoresis and immunoblotting, but not in physiological human vitreous.

[Analysis of the protein pattern in physiologic and pathologic vitreous bodies by electrophoresis and immunologic identification]. / Analyse des Proteinmusters in physiologischen und pathologischen Glaskörpern durch Elektrophorese und immunologische Identifizierung.

The protein composition of normal and pathological human vitreous was analyzed using gradient SDS-PAGE and western blot. By electroblotting and immunodetection, the presence of ten proteins was proven in the normal vitreous. The major proteins included: albumin, transferrin, alpha-1-antitrypsin, and immunoglobulin G. For the detection of a distinct protein in the vitreous, western blot analysis seems to be an appropriate method. Using densitometric analysis of silver-stained gels, we compared the protein pattern of post-mortem vitreous, surgically obtained samples (from proliferative vitreous-retinopathy, proliferative diabetic retinopathy, and macular pucker patients), and serum. The protein composition of the pathological intraocular fluid was uniform, but different from that of normal vitreous and serum. These results suggest that a breakdown of the blood-retina barrier is the main reason for the increase of soluble proteins in the vitreous cavity under pathological conditions. The methods described allow detailed analysis of the vitreous protein pattern in intraocular proliferative disorders with a small sample volume (10 microliters).

The effects of basic fibroblast growth factor on bovine retinal pigment epithelium in vitro.

Basic fibroblast growth factor (bFGF) has been reported to initiate DNA synthesis in a variety of cells involved in the pathogenesis of proliferative vitreoretinal disorders, e.g., glial cells and fibroblasts. We analyzed the mitogenic effects of bFGF on cultured bovine retinal pigment epithelial cells in relation to time and dose response regulations and culture conditions. Maximum stimulatory effect of bFGF (+70% compared to control group) was found on day 3 following treatment of cultures with 80 ng/ml bFGF. The action of bFGF seems to depend on the serum concentration in the culture media, which means that cofactors may be present in the serum and potentiate the effects of bFGF on cell proliferation.

The significance of vitronectin in proliferative diabetic retinopathy.

Vitronectin, an integrin-binding a-1-glycoprotein with potent cell-adhesion and proliferation-mediating properties, has been shown to be incorporated in surgically removed membranes from patients with proliferative vitreoretinopathy (PVR), proliferative diabetic retinopathy (PDR) and macular pucker. Therefore we developed an ELISA technique to quantify levels of vitronectin in human vitreous and plasma samples in order to be able to evaluate the significance of vitronectin in these different vitreoretinal diseases. Our results indicate a significant increase of vitronectin in all proliferative disorders except idiopathic macular pucker. Adjustment of all probes to equal total protein content yielded a significant increase only in PDR (type II diabetes). Plasma samples demonstrated a significant increase of vitronectin in patients with type II diabetes suffering from PDR. Therefore, break-down of the blood-retina barrier appears to be the most likely explanation for the increased levels of vitronectin in the vitreous. Our results indicate that vitronectin may not only be involved in the regulation of epiretinal membrane formation at significantly higher levels in patients with type II diabetes, but the increase of vitronectin in diabetic plasma may also help to explain the pathological alteration of the coagulation cascade in diabetics.

Immunoglobulin G, complement factor C3 and lymphocytes in proliferative intraocular disorders.

This study examines a possible immunological contribution to the development of proliferative intraocular disorders (PID) with traction retinal detachment. We analysed 24 periretinal membranes and 35 vitreous aspirates from patients with idiopathic proliferative vitreoretinopathy (PVR), traumatic PVR, and proliferative diabetic retinopathy (PDR). Lymphocytes and complement factor C3 deposits could not be detected in any of the membrane specimens. IgG was present in all but one of the PVR membranes but in less than half of the PDR specimens and there to a lesser extent. The IgG immunoreactivity was not collocalized with macrophages but instead located to the extracellular matrix. The intravitreal levels of IgG (ELISA) and protein were elevated in PID but the range of these biochemical changes was so wide that there were no significant differences of the IgG levels between the single types of PID. Using electrophoresis and Western blotting, C3 was detected in normal and pathologic vitreous but smaller C3 fragments indicative of C3 breakdown were only found in PID.

Intercellular adhesion molecule-1 levels in plasma and vitreous from patients with vitreoretinal disorders.

Intercellular adhesion molecule 1 (ICAM-1) is a member of the immunoglobulin superfamily with important functions in immune activation and inflammation. Its interaction with different cytokines [interleukin 1 (IL-1), tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma)] is important for lymphocyte migration into inflammatory sites. We used a sandwich enzyme immunoassay (EIA) for the quantitative determination of soluble ICAM-1 (cICAM-1) in vitreous and plasma from patients undergoing vitrectomy for a variety of proliferative vitreoretinal disorders. The values obtained were compared with the total vitreal protein. The respective concentrations of cICAM-1 in vitreous were as follows control samples, 3.47 +/- 1.84 ng/ml; proliferative diabetic retinopathy (PDR) of diabetes type I 27.43 +/- 14.72 ng/ml; PDR of diabetes type II, 32.46 +/- 10.31 ng/ml; idiopathic proliferative vitreoretinopathy 35.74 +/- 15.30 ng/ml; and traumatic PVR, 45.23 +/- 24.24 ng/ml. Plasma samples yielded the following concentrations: controls, 415 +/- 43.4 ng/ml; PDR of diabetes type I, 469 +/- 96.9 ng/ml; PDR of diabetes type II, 425 +/- 65.4 ng/ml; idiopathic PVR, 402 +/- 119.9 ng/ml; and traumatic PVR, 434 +/- 118.6 ng/ml. Our results demonstrate high levels of ICAM-1 in most proliferative vitreoretinal disorders. In PDR and in traumatic PVR, cICAM-1 levels were elevated significantly more than were total vitreal protein levels. In traumatic PVR, patients with a short interval between previous surgery or traumatic event demonstrated the highest levels of cICAM. Since plasma levels were not significantly altered, we suggest that local cICAM-1 production, possibly from macrophages, may be of importance in the early phase of PVR and PDR by enhancing immune activation and inflammation.