Endostatin-induced tyrosine kinase signaling through the Shb adaptor protein regulates endothelial cell apoptosis

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.

The angiogenesis inhibitor endostatin impairs blood vessel maturation during wound healing

“…These morphological changes are a typical sign of cell damage that might eventually lead to cell death. This finding is consistent with the effect of endostatin on endothelial cell apoptosis in vitro (18,33). Interestingly, the observed blood vessel abnormalities were at least partially reversible, because only few abnormal vessels were observed within 7 days after endostatin withdrawal.…”

“…We have recently prepared recombinant endostatin in highly soluble form and determined its crystal structure (15,16). This material was shown to inhibit cytokine-induced angiogenesis and to affect various parameters of endothelial cell growth and migration (12,17,18). The data also showed that proteolytically released endostatin is a distinct component of the extracellular matrix of various normal tissues (15,19).…”

AIDS‐related Kaposi's sarcoma cells rapidly internalize endostatin, which co‐localizes to tropomysin microfilaments and inhibits cytokine‐mediated migration and invasion

“…Our functional assay data are in agreement with the findings of Rehn et al [2001], which showed soluble endostatin served as an integrin antagonist for endothelial origin cells. In addition, the endostatin concentration [10 mg/ml (4.71 Â 10 À7 M)] used in our KS cell studies is comparable or lower than endostatin concentrations that have demonstrated efficacy during in vitro endothelial cell studies [Dhanabal et al, 1999;Dixelius et al, 2000;Rehn et al, 2001;Shichiri and Hirata, 2001]. This dose consistency implies that KS cells have similar thresholds of endostatin responsiveness as nontransformed endothelial cells.…”

Intraocular expression of endostatin reduces VEGF‐induced retinal vascular permeability, neovascularization, and retinal detachment

“…Low-and high-affinity endostatin binding sites are present on endothelial cells, but so far only the low-affinity receptors, glypicans, have been identified (37). Some of the molecules implicated in intracellular signaling are the Shb adaptor (38), c-myc (39), phosphorylated retinoblastoma gene product (40), cyclin D1 (40), and tropomyosin (41). One consequence of endostatin-induced altered intracellular signaling is enhanced apoptosis of endothelial cells participating in neovascularization, but not quiescent endothelial cells (38,42).…”