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

Abstract

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 1–2.5 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 occured 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 mM) 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.

Soluble perlecan domain i enhances vascular endothelial growth factor-165 activity and receptor phosphorylation in human bone marrow endothelial cells

“…The HS dependent binding of VEGF 165 to immobilized PlnDI described herein is consistent with recent reports [5,6]. In contrast, a new communication has reported PlnDI does not bind immobilized VEGF 165 [36].…”

Multifunctionality of extracellular and cell surface heparan sulfate proteoglycans

“…Its patterns of expression are consistent with a role in extracellular matrix remodeling as well as growth factor release. Two proteins have been identified that are naturally occurring inhibitors of heparanase activity, namely eosinophil major basic protein [68] and heparin/HS-binding protein/RPL29 [69]. In addition to inhibiting heparanase-1 activity, HIP/RPL29 also displaces HS-binding growth factors from HS-bearing substrates [69].…”

“…Two proteins have been identified that are naturally occurring inhibitors of heparanase activity, namely eosinophil major basic protein [68] and heparin/HS-binding protein/RPL29 [69]. In addition to inhibiting heparanase-1 activity, HIP/RPL29 also displaces HS-binding growth factors from HS-bearing substrates [69]. Thus, a complex scenario exists where HIP/RPL29 displays seemingly antagonistic actions in terms of HS-binding growth factor release.…”

Endogenous ribosomal protein L29 (RPL29): a newly identified regulator of angiogenesis in mice

“…However, in mouse cells the surface expression of RPL29 has not been demonstrated (Kirn-Safran et al, 2007) and our data corroborate this finding. At the surface of human cells, the heparin/heparan sulphate binding property of RPL29 can regulate responses to growth factors (Rohde et al, 1996; Jacobs et al, 1997; Liu et al, 1997; Rohde et al, 1998; Lander and Selleck, 2000; D’Souza et al, 2008). Indeed, application of synthetic RPL29 peptides has been shown to interfere with heparan sulphate binding to VEGF and FGF thus inhibiting angiogenesis ex vivo (D’Souza et al, 2008).…”

“…Rpl29 -null embryonic fibroblasts have decreased rates of proliferation and protein synthesis (Kirn-Safran et al, 2007). Although a synthetic RPL29 peptide has been shown to inhibit VEGF-and FGF-induced angiogenesis via its interaction with cell surface heparin and/or heparan sulphate proteoglycans (D’Souza et al, 2008), endogenous, cellular RPL29 has not been shown to be involved in angiogenesis.…”