MABC87 Sigma-AldrichAnti-CD148/DEP-1 Antibody, clone Ab1 (Azide Free)

Angiogenesis contributes to a wide range of neoplastic, ischemic, and inflammatory disorders. Definition of the intrinsic molecular controls in angiogenic vessel growth promises novel therapeutic approaches for angiogenesis-related diseases. CD148 (also named DEP-1/PTP eta) is a receptor-like protein tyrosine phosphatase that is abundantly expressed in vascular endothelial cells. To explore a role of CD148 in endothelial vessel formation, we generated a monoclonal antibody, Ab1, against the ectodomain sequence of CD148 and examined its effects on endothelial-cell growth and vessel formation. Here we report that a bivalent, but not a monovalent, form of the Ab1 antibody inhibits endothelial-cell growth and blocks angiogenesis in mouse cornea in vivo. We further demonstrate that (1) bivalent Ab1 arrests cell-cycle progression of CD148-transfected CHO cells at G(0)/G(1) phase, (2) coexpression of catalytically inactive CD148 mutants attenuates the Ab1-cell growth inhibition, and (3) bivalent Ab1 suppresses phosphorylation of ERK1/2 kinases and Met tyrosine kinase as activated CD148 does, with an increase in CD148-associated tyrosine phosphatase activity. Taken together, these findings demonstrate that Ab1-induced ectodomain oligomerization arrests endothelial-cell growth through catalytic activity of the CD148 cytoplasmic domain. The present study defines CD148 as a valuable molecular target for antiangiogenesis therapy.

Vascularization defects in genetic recombinant mice have defined critical roles for a number of specific receptor tyrosine kinases. Here we evaluated whether an endothelium-expressed receptor tyrosine phosphatase, CD148 (DEP-1/PTPeta), participates in developmental vascularization. A mutant allele, CD148(DeltaCyGFP), was constructed to eliminate CD148 phosphatase activity by in-frame replacement of cytoplasmic sequences with enhanced green fluorescent protein sequences. Homozygous mutant mice died at midgestation, before embryonic day 11.5 (E11.5), with vascularization failure marked by growth retardation and disorganized vascular structures. Structural abnormalities were observed as early as E8.25 in the yolk sac, prior to the appearance of intraembryonic defects. Homozygous mutant mice displayed enlarged vessels comprised of endothelial cells expressing markers of early differentiation, including VEGFR2 (Flk1), Tal1/SCL, CD31, ephrin-B2, and Tie2, with notable lack of endoglin expression. Increased endothelial cell numbers and mitotic activity indices were demonstrated. At E9.5, homozygous mutant embryos showed homogeneously enlarged primitive vessels defective in vascular remodeling and branching, with impaired pericyte investment adjacent to endothelial structures, in similarity to endoglin-deficient embryos. Developing cardiac tissues showed expanded endocardial projections accompanied by defective endocardial cushion formation. These findings implicate a member of the receptor tyrosine phosphatase family, CD148, in developmental vascular organization and provide evidence that it regulates endothelial proliferation and endothelium-pericyte interactions.

Developmental assembly of the renal microvasculature requires spatially and temporally coordinated migration, assembly, differentiation, and maturation of endothelial cells in the context of adjacent epithelial and mesangial cells. In this study, endothelial expression and distribution of the receptor tyrosine phosphatase ECRTP/DEP-1 were evaluated during and after developmental assembly of the renal microvasculature. Monoclonal antibodies against ECRTP/DEP-1 ectodomain epitopes localize its expression to membrane surfaces of endothelial cells in glomerular, peritubular capillary, and arterial renal sites of mature human and murine kidney. During kidney development, ECRTP/DEP-1 immunostaining is evident on a subpopulation of metanephric mesenchymal cells and on putative progenitors of glomerular capillary endothelial cells early in their recruitment to developing glomeruli. ECRTP/DEP-1 is prominently displayed on luminal membrane surfaces with punctate accumulations at inter-endothelial contacts that overlap with vascular endothelial-cadherin staining. ECRTP/DEP-1 is recruited to inter-endothelial contacts in confluent cultured human renal and dermal microvascular endothelial cells, yet experimental dissociation of vascular endothelial-cadherin from endothelial junctional complexes fails to redistribute ECRTP/DEP-1. These findings indicate that ECRTP/DEP-1 is expressed in anticipation of glomerular capillary endothelial recruitment during development, and suggest that ECRTP/DEP-1 ectodomain interacts with endothelial surface ligands that are engaged by cell-cell contact.