Expression of extradomain-B-containing fibronectin in subretinal choroidal neovascular membranes.
ABSTRACT To investigate the presence of the fibronectin isoform containing the extradomain B (B-FN), a marker-protein of angiogenesis, in surgically excised human choroidal neovascular membranes (CNVM) to evaluate whether B-FN could be used as a therapeutic target for specific antibody-photosensitizer immunoconjugates.
The setting was an institutional practice. The study population consisted of 15 eyes (15 patients) with CNVM undergoing membrane excision (four eyes with age-related macular degeneration, seven with pathologic myopia and four with multifocal choroiditis). The control group consisted of eight eye bank eyes (four subjects) without choroidal neovascularization. Light microscopic immunohistochemistry on cryostat sections of tissues was obtained. B-FN was detected by a human recombinant antibody, CGS-1, and compared with immunostaining for endothelial cells with factor VIII-related antigen. The main outcome measure was the presence of CGS-1 positively stained cells or areas of the extracellular matrix. Staining of CGS-1 was scored on a scale from 0 to 3.
Fourteen of 15 neovascular membranes stained strongly with CGS-1 (score 2 or 3). One membrane from a patient with pathologic myopia was negatively stained (score 0). CGS-1 positive staining was detected around endothelial cells and in the extracellular matrix of CNVMs. The retina of eyes without choroidal neovascularization was negative with CGS-1 in all eight donor eyes, while the choroid contained some weakly CGS-1 positive cells (score 0 and 1, respectively).
The extradomain B is abundantly expressed in CNVMs, but its expression is more restricted in eyes harboring no apparent choroidal neovascularization. In the future, B-FN might serve as a target for the delivery of antibody-photosensitizer immunoconjugates to newly developed vessels to enhance the selectivity of photodynamic therapy.
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ABSTRACT: Because several polypeptide growth factors are known to influence capillary endothelial cell mitogenesis, the authors investigated the presence of some of these molecules in choroidal neovascular membranes (CNVMs) removed surgically from human subjects with age-related macular degeneration (ARMD). The authors performed immunoelectron microscopic studies on surgically removed submacular CNVMs from nine subjects with ARMD and from one subject with ARMD whose eye was studied after death. These were compared with retinal pigment epithelial (RPE) and choroidal tissue from eight normal subjects whose eyes were received after death and one received after massive trauma. RPE cells from the CNVMs were strongly immunoreactive for acidic and basic fibroblast growth factor (aFGF and bFGF) and for transforming growth factor beta (TGF beta). Some of the immunoreactivity was intracytoplasmic, but most was intralysosomal. In addition, some choriocapillary endothelial cells located close to the RPE layer in these CNVMs were immunopositive for bFGF and for FGF receptor. Reaction product for these two substances was located at regular intervals along the endothelial plasma membrane on both the anteluminal and the luminal side of the cells, suggesting a physiological reaction between the growth factor and its receptor. Choriocapillary endothelial cells deeper within the stroma were unreactive to bFGF and FGF receptor antibodies. There was little immunoreactivity for the growth factors in RPE or choriocapillary endothelial cells from normal eyes. The aFGF and bFGF immunoreactivity was highly specific because aFGF positivity was abolished when the antibody was incubated with 10(-6) M aFGF but not a with the same concentration of bFGF, whereas bFGF immunoreactivity was abolished by incubation of the antibody with bFGF but not with aFGF. RPE cells from normal eyes and from eyes affected by ARMD showed strong cytoplasmic immunoreactivity to antibodies for cytoplasmic retinaldehyde-binding protein and superoxide dismutase and weak reactivity to antibodies for vimentin. These results are consistent with the hypothesis that one or both FGFs are causally related to the development of choroidal neovascularization. The authors have reported similar observations in experimental choroidal neovascularization in pigmented rats after red krypton laser photocoagulation. TGF beta may serve to modulate the effects of these mitogens. The authors suggest that growth factor production is induced in RPE cells after physical or chemical damage. Because of the damage to these cells, FGF molecules can be released from the cells despite the absence of a "signal sequence" in the DNA coding for FGF production.Investigative Ophthalmology & Visual Science 08/1994; 35(8):3178-88. · 3.44 Impact Factor
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ABSTRACT: The cornea is a simple, nonvascularized structure, advantageous for studying the molecular components of epithelial and stromal wound repair. Fibronectin (Fn), of uncertain source and composition, accumulates in healing corneas. We postulated that local synthesis of Fn occurs, as exogenous plasma/tear-derived Fn, which lack the embryonic EIIIA and EIIIB segments, have no consistent beneficial effect on healing. Two contrasting corneal wounds were examined by in situ hybridization: a wound of the anterior stroma, basement membrane, and epithelium (anterior excimer laser keratectomy) and a superficial wound restricted to the epithelium that preserved the basement membrane (mechanical scrape). Both wounds heal without scarring. In normal corneas, only the endothelium had detectable Fn mRNA, containing the V and EIIIB domains, sporadically and at low levels. After anterior keratectomies, extensive expression of Fn mRNA occurred in a specific distribution that changed during the phases of healing. Before re-epithelialization (days 1 and 2) V+, EIIIA+, and EIIIB+ isoforms were diffusely found in stromal cells under and adjacent to the wound. After re-epithelialization (days 3 to 42) and reconstitution of laminin in the regenerating basement membrane zone, V+, EIIIA+, and EIIIB+ isoform synthesis was largely restricted to subepithelial stromal cells at the epithelial/stromal interface. In addition, the corneal epithelial cells focally expressed Fn mRNA. The endothelium showed increased levels of V+, EIIIA+, and EIIIB+ Fn mRNA in open and recently re-epithelialized wounds. At 12 weeks after keratectomy, Fn mRNA expression returned to control levels. In contrast, scrape wounds had only a modest increase of stromal and endothelial Fn mRNA (EIIIA+, EIIIB+, and V+) during the first 7 days and no evidence of epithelial Fn synthesis. Embryonic Fn isoforms are synthesized transiently by the cornea in response to even the most superficial wounds and are likely to be relevant to corneal healing and restoration of structure without scar formation.American Journal Of Pathology 09/1996; 149(2):549-58. · 4.52 Impact Factor
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ABSTRACT: Fibronectin (FN) represents the mixture of a number of structurally different molecules (isoforms) whose make-up varies depending on the FN sources. FN from cultured transformed human cells has a very different isoform composition with respect to its normal counterpart. In fact, SV-40-transformed WI-38VAI3 human fibroblasts produce high levels of a FN isoform (B-FN) which is very poorly expressed in their normal, WI-38, counterpart. We have recently demonstrated that the B-FN isoform derives from a differential splicing pattern of the FN primary transcript which leads, in transformed cells, to a high level expression of the exon ED-B (Zardi, L., B. Carnemolla, A. Siri, T. E. Petersen, G. Paolella, G. Sebastio, and F. E. Baralle. 1987. EMBO (Eur. Mol. Biol. Organ.) J. 6:2337-2342). Here we report on the production and characterization of a monoclonal antibody (BC-1) which recognizes an epitope within the protein sequence coded for by the ED-B exon. This monoclonal antibody makes it possible to carry out immunohistochemical analysis of the distribution of the ED-B-containing FN isoform (B-FN) in human tissues. The results show that while in normal, adult, human tissues total FN has a widespread distribution, the B-FN isoform is restricted only to synovial cells, to some vessels and areas of the interstitium of the ovary, and to the myometrium. On the contrary, the B-FN isoform has a much greater expression in fetal and tumor tissues. These results demonstrate that, in vivo, different FN isoforms have a differential distribution and indicate that the B-FN isoform may play a role in ontogenesis and oncogenetic processes.The Journal of Cell Biology 04/1989; 108(3):1139-48. · 10.82 Impact Factor