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. 2005 Aug 2:3:9.
doi: 10.1186/1478-811X-3-9.

Anti-lipid phosphate phosphohydrolase-3 (LPP3) antibody inhibits bFGF- and VEGF-induced capillary morphogenesis of endothelial cells

Kishore K Wary  1 , Joseph O Humtsoe
Affiliations

Anti-lipid phosphate phosphohydrolase-3 (LPP3) antibody inhibits bFGF- and VEGF-induced capillary morphogenesis of endothelial cells

Kishore K Wary et al. Cell Commun Signal. .

Abstract

Background: Angiogenesis, or the remodeling of existing vasculature serves as a lifeline to nourish developing embryos and starved tissues, and to accelerate wound healing, diabetic retinopathy, and tumor progression. Recent studies indicate that angiogenesis requires growth factor activity as well as cell adhesion events mediated by alpha5beta1 and alphavbeta3 integrins. We previously demonstrated that human lipid phosphate phosphohydrolase-3 (LPP3) acts as a cell-associated ligand for alpha5beta1 and alphavbeta3 integrins. Here, we test the hypothesis that an anti-LPP3 antibody can inhibit basic fibroblast growth factor (bFGF)-and vascular endothelial growth factor (VEGF)-induced capillary morphogenesis of endothelial cells (ECs).

Results: We report that bFGF and VEGF up-regulate LPP3 protein expression in ECs. Immunoprecipitation analyses show that LPP3 is a cell surface protein and undergoes N-glycosylation. Fluorescent activated cell sorting (FACS) data suggest that anti-LPP3-RGD detects native neoepitope on the surface of activated ECs. Moreover, we demonstrate LPP3 protein expression in tumor endothelium alongside VEGF. The embedding of ECs into three-dimensional type I collagen in the presence of bFGF and VEGF induce capillary formation. Importantly, we show that the addition of an anti-LPP3 antibody specifically and significantly blocks bFGF- and VEGF-induced capillary morphogenesis of ECs.

Conclusion: These data suggest that activated ECs as well as tumor endothelium express LPP3 protein. In an in vitro assay, the anti-LPP3-RGD specifically blocks bFGF and VEGF induced capillary morphogenesis of ECs. Our results, therefore, suggest a role for LPP3 in angiogenesis.

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Figures

Figure 1
Figure 1
Expression of LPP3 protein in monolayer ECs. Confluent ECs (passage 4) were starved for 6 h in conditioned medium (M199 supplemented with 0.2% BSA + 1× ITS), and then stimulated with VEGF165 (100 ng/ml) or bFGF (20 ng/ml) for various durations, as indicated. Cells were solubilized, clarified by centrifugation, and the protein concentrations were determined. Samples were subjected to SDS-PAGE and analyzed by immunoblotting with: (A) Rabbit anti-LPP3-c-cyto polyclonal antibody (2.0 μg/ml). Anti-LPP3-c-cyto antibody detects unprocessed LPP3 protein that appears as ~36 kDa, and two major polypeptides migrate below the ~52 kDa molecular weight marker, LPP3 polypeptides are indicated by arrowheads; (B) Anti-PCNA monoclonal antibody (0.5 μg/ml). (C) Anti-Fak monoclonal antibody (1.0 μg/ml). (D) Cell surface biotinylation of intact cells and immunoprecipitation analysis. K562 (lane 1, unstimulated), HUVECs (lane 2, stimulated with VEGF, 100 ng/ml 6 hours), and HUVECs (lane 3, stimulated with bFGF, 20 ng/ml for 6 h) subjected to cell surface biotinylation, lysed in RIPA buffer, clarified by centrifugation and immunoprecipitated using anti-LPP3-c-cyto (5 μg) antibody. Immunocomplexes were resolved by 10% SDS-PAGE under reducing condition and analyzed by ligand blotting with streptavidin-HRP (1:10000). Data shown are representative of those obtained in at least three separate experiments, with similar results. (E) De-N-glycosylation of LPP3 protein. Monolayer HUVECs (passage 4) were stimulated with VEGF165 for 6 h and subjected to cell surface biotinylation, lysed in RIPA buffer, clarified by centrifugation, and immunoprecipitated by either rabbit IgG (control) or anti-LPP3-c-cyto antibodies, as indicated. Following immunoprecipitation with anti-LPP3-c-cyto antibodies, the contents were equally divided into two tubes. One tube was left untreated, and the second tube treated with N-Glyganase (50 units of PNGaseF) enzyme at 37°C for 3 h. Immunocomplexes were analyzed by ligand blotting with streptavidin-HRP (1:10000). Arrowheads indicate LPP3 polypeptides. Arrows indicate unknown polypeptides. Molecular weights are given in kiloDaltons (kDa). Data shown are representative of those obtained in at least three separate experiments, with similar results.
Figure 2
Figure 2
LPP3 is a cell surface antigen. A) HUVECs (passage 4) were serum-starved for 6 h, thereafter, left untreated (-) or treated with VEGF165 (100 ng/ml) for 6 h and subjected to fluorescent activated cell sorting (FACS) using indicated antibodies. B) Schematic representation of human LPP3 protein showing 6-transmembrane organization. Proposed 3 extracellular loops (L-l, -2, and -3) are as shown. One lipid phosphatase motif, a cell adhesion sequence, a putative proton donor sequence and a dityrosine basolateral targeting sequence are as shown. Both N-and C-terminals of LPP3 protein are located inside the cytoplasm.
Figure 3
Figure 3
Tumor endothelium express LPP3 protein alongside VEGF. Paraffin-embedded angioma (A-C) and hemangioma (D-I) tumor tissue sections (4 μm) were subjected to antigen retrieval, and sequentially incubated with the indicated antibodies. After washing with PBS, sections were incubated with donkey anti-goat/rabbit IgG conjugated to Texas-red (red) and goat anti-mouse IgG conjugated to FITC (green). C, F, and I images represent overlays of A, B; D, E; and G, H respectively. Images were captured below saturation level. Merged yellow represents co-expression. Data shown are representative of those obtained in at least three separate experiments, with similar results. (L, lumen; Magnification, 100×; Bars, 50 μM).
Figure 4
Figure 4
Effect of specific antibodies on pre-formed capillaries. ECs were cultured in 3D collagen matrices in the presence of bFGF and VEGF165. Cultures were treated with the indicated antibodies at 24 h and processed at various time points (indicated). The 3D cultures were fixed, serial sections prepared and stained with eosin. The capillaries were counted as described in methods section. Values represent the mean ± SEM obtained from three independent experiments that used five wells in each case. * P < 0.02.
Figure 5
Figure 5
Inhibition of bFGF and VEGF induced capillary morphogenesis of ECs in 3D type I collagen matrix. The samples shown in the upper panels (A-E) were treated with anti-MHC class II mAbs, whereas those in the lower panels (F-J) with anti-αvβ3 integrin antibodies. Cross sections were stained with acidified eosin as described in methods. Magnification, 100×. Bar, 200 μM.
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References

    1. Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med. 1995;1:27–31. doi: 10.1038/nm0195-27. - DOI - PubMed
    1. Hanahan D, Folkman J. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell. 1996;86:353–364. doi: 10.1016/S0092-8674(00)80108-7. - DOI - PubMed
    1. Ferrara N, Davis-Smyth T. The biology of vascular endothelial growth factor. Endocr Rev. 1997;8:4–25. doi: 10.1210/er.18.1.4. - DOI - PubMed
    1. Carmeliet P, Jain RK. Angiogenesis in cancer and other diseases. Nature. 2000;407:249–257. doi: 10.1038/35025220. - DOI - PubMed
    1. Sato Y, Kanno S, Oda N, Abe M, Ito M, Shitara K, Shibuya M. Properties of two VEGF receptors, Flt-1 and KDR, in signal transduction. Ann N Y Acad Sci. 2000;902:201–205. - PubMed

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