HIP/RPL29 antagonizes VEGF and FGF2 stimulated angiogenesis by interfering with HS‐dependent responses

HIP/RPL29 is a heparan sulfate (HS) binding protein with diverse activities including modulation of heparanase (HPSE) activity. We examined HIP/RPL29's ability to modulate actions of HS‐binding growth factors (HBGFs) in angiogenesis. Between 1 and 2.5 µg/ml (ca. 60–150 nM), HIP/RPL29 inhibited HBGF‐stimulated endothelial cell tube formation. Aortic explant outgrowth also was inhibited, but at higher concentrations (40 µg/ml). At this concentration, HIP/RPL29 had no effect on HBGF‐stimulated MAPK phosphorylation or VEGF‐stimulated receptor‐2 phosphorylation at site Y‐996. Partial inhibition occurred at VEGF receptor‐2 site Y951, associated with cell migration. HBGF displacement from HS‐bearing perlecan domain I showed that HIP/RPL29 released 50% of bound HBGF at 20 µg/ml, a dose where endothelial tube formation is inhibited. Similar FGF2 release occurred at pH 5.0 and 7.0, conditions where HPSE is highly and residually active, respectively. We considered that HIP/RPL29 inhibits HPSE‐dependent release of HS‐bound HBGFs. At pH 5.0, release of soluble HS was inhibited by 64% at concentrations of 5 µg/ml and by 77% at 40 µg/ml, indicating that HIP/RPL29 antagonizes HPSE activity. At concentrations up to 40 µg/ml (ca. 2.5 µM) where angiogenic processes are inhibited, release of FGF2 occurred in the presence of HPSE and HIP/RPL29. The majority of this FGF2 is not bound to soluble HS. Studies of HIP/RPL29 binding to HS indicated that many structural features of HS are important in modulation of HBGF activities. Our findings suggest that inhibition of angiogenic processes by HIP/RPL29 involves attenuation of the formation of soluble, biologically active HBGF:HS complexes that activate HBGF receptors. J. Cell. Biochem. 105: 1183–1193, 2008. © 2008 Wiley‐Liss, Inc.