Expression of endostatin in human choroidal neovascular membranes secondary to age-related macular degeneration.
ABSTRACT Endostatin is an endogenous angiogenesis inhibitor which requires E-selectin for its antiangiogenic activity. The aim of this study was to investigate the expression of endostatin in human choroidal neovascular membranes (CNV) secondary to age-related macular degeneration (AMD) with regard to vascularization and proliferative activity. An interventional case series of 36 patients who underwent removal of CNV were retrospectively investigated. Thirty-six CNV were analyzed by light microscopic immunohistochemistry for the expression of CD34 (endothelial cells, EC), CD105 (activated EC), Ki-67 (cell proliferation), Cytokeratin 18 (epithelial cells), VEGF (vascular endothelial growth factor), E-selectin and endostatin. Donor eyes (n=7) including one with AMD were used as controls. Endostatin immunoreactivity was present in choroidal vessels of five as well as in the retinal pigment epithelium (RPE)-Bruch's membrane complex of two donor eyes without AMD. In one eye with AMD, endostatin was detected in RPE, Bruch's membrane and choroidal vessels. Ninety-two percent (33/36) of CNV disclosed endostatin staining. RPE-Bruch's membrane complex, choroidal vessels and stroma were positive in 50% (18/36), 72% (26/36), and 78% (28/36) of the membranes, respectively. Both control eyes and CNV expressed all the investigated markers except E-selectin being positive only in membranes. Endostatin, an endogenous angiogenesis inhibitor, is expressed in CNV and its therapeutic up-regulation may be a new strategy in the treatment of neovascular AMD.
- SourceAvailable from: bloodjournal.hematologylibrary.org[show abstract] [hide abstract]
ABSTRACT: Endostatin, which corresponds to the C-terminal fragment of collagen XVIII, is a potent inhibitor of angiogenesis. Fibroblast growth factor-2 (FGF-2)-induced angiogenesis in the chicken chorioallantoic membrane was inhibited by endostatin, but not by an endostatin mutant R158/270A, lacking heparin-binding ability. Endostatin was internalized by endothelial cells, but not by mouse fibroblasts. Treatment of murine brain endothelial (IBE) cells with endostatin reduced the proportion of cells in S phase, whereas growth-arrested IBE cells in collagen gels treated with endostatin displayed enhanced tubular morphogenesis. IBE cells overexpressing Shb, an adaptor protein implicated in angiostatin-induced apoptosis, displayed elevated apoptosis and decreased tubular morphogenesis in collagen gels in response to endostatin when added together with FGF-2. Induction of apoptosis was dependent on the heparin-binding ability of endostatin and the expression of Shb with a functional Src homology 2 (SH2)-domain. Endostatin treatment for 10 minutes or 24 hours induced tyrosine phosphorylation of Shb and formation of multiprotein complexes. An Shb SH2 domain fusion protein precipitated a 125-kd phosphotyrosyl protein in endostatin-treated cells. The 125-kd component either contained intrinsic tyrosine kinase activity or occurred in complex with a tyrosine kinase. In conclusion, our data show that endostatin induces tyrosine kinase activity and enhanced apoptosis in FGF-treated endothelial cells.Blood 07/2000; 95(11):3403-11. · 9.06 Impact Factor
- [show abstract] [hide abstract]
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
- [show abstract] [hide abstract]
ABSTRACT: Here we report the inhibition of cellular invasion by a recombinant mouse endostatin and the possible mechanism of the inhibition. Endostatin significantly reduced endothelial as well as tumor cellular invasion into the reconstituted basement membrane in vitro. Gelatin zymographic analysis revealed that the activation of promatrix metalloproteinase-2 (proMMP-2) that was secreted from endothelial cells was blocked upon endostatin treatment. Studies with recombinant MMPs confirmed that endostatin inhibited proMMP-2 activation, mediated by both membrane-type 1 MMP and 4-aminophenylmercuric acetate. Furthermore, enzymatic assays using a peptide substrate demonstrated that endostatin inhibited the catalytic activities of both MMP-2 and membrane-type 1 MMP. Finally, coimmunoprecipitation experiments revealed that endostatin formed a stable complex with proMMP-2. These novel findings would, at least in part, explain the mechanism of the potent antiangiogenic and antitumor activities of endostatin.Cancer Research 11/2000; 60(19):5410-3. · 8.65 Impact Factor