SPARC (secreted protein, acidic and rich in cysteine)is a matricellular protein that modulates cell adhesion and proliferation and is thought to function in tissue remodeling and angiogenesis. In this study, we demonstrate that SPARC inhibits DNA synthesis by >90% in human microvascular endothelial cells (HMEC) stimulated by the endothelial cell mitogen vascular endothelial growth factor (VEGF). Peptides derived from SPARC domain IV, which contains a disulfide-bonded EF-hand sequence and binds to endothelial cells, mimicked the effect of native SPARC. The inhibition was also observed with a peptide from the follistatin-like domain II, whereas peptides from SPARC domains I and III had no effect on VEGF-stimulated DNA synthesis. The inhibition of HMEC proliferation was mediated in part by the binding of VEGF to SPARC. The binding of 125 I-VEGF to HMEC was reduced by SPARC and SPARC peptides from domain IV in a concentration-dependent manner. In a radioimmune precipitation assay, peptides from SPARC domains II and IV each competed with native SPARC for its binding to VEGF. It has been reported that VEGF stimulates the tyrosine phosphorylation and activation of mitogen-activated protein kinases Erk1 and Erk2. We now show that SPARC reduces this phosphorylation in VEGF-stimulated HMEC to levels of unstimulated controls. SPARC thus modulates the mitogenic activity of VEGF through a direct binding interaction and reduces the association of VEGF with its cell-surface receptors. Moreover, an additional diminution of VEGF activity by SPARC is accomplished through a reduction in the tyrosine phosphorylation of mitogen-activated protein kinases.

Abstract-The heparin-binding protein vascular endothelial growth factor (VEGF) is a highly specific growth factor for endothelial cells. VEGF binds to specific tyrosine kinase receptors, which mediate intracellular signaling. We investigated 2 hypotheses: (1) VEGF affects intracellular calcium [Ca 2ϩ ] i regulation and [Ca 2ϩ ] i -dependent messenger systems; and (2) these mechanisms are important for VEGF's proliferative effects. [Ca 2ϩ ] i was measured in human umbilical vein endothelial cells using fura-2 and fluo-3. Protein kinase C (PKC) activity was measured by histone-like pseudosubstrate phosphorylation. PKC isoform distribution was observed with confocal microscopy and Western blot. Inhibition of PKC isoforms was assessed by specific antisense oligonucleotides (ODN) for the PKC isoforms. VEGF (10 ng/mL) induced a transient increase in [Ca 2ϩ ] i followed by a sustained elevation. The sustained [Ca 2ϩ ] i plateau was abolished by EGTA. Pertussis toxin also abolished the plateau phase, whereas the initial peak was not affected. The PKC isoforms ␣, ␦, ⑀, and were identified in endothelial cells. VEGF induced a translocation of PKC-␣ and PKC-toward the nucleus and the perinuclear area, whereas cellular distribution of PKC-␦ and PKC-⑀ was not influenced. Cell exposure to TPA led to a down-regulation of PKC-␣ and reduced the proliferative effect of VEGF. VEGF-induced endothelial cell proliferation also was reduced by the PKC inhibitors staurosporine and calphostin C. Specific down-regulation of PKC-␣ and PKC-with antisense ODN reduced the proliferative effect of VEGF significantly. Our data show that VEGF induces initial and sustained Ca 2ϩ influx. VEGF leads to the translocation of the [Ca 2ϩ ] i -sensitive PKC isoform ␣ and the atypical PKC isoform . Antisense ODN for these PKC isoforms block VEGF-induced proliferation. These findings suggest that PKC isoforms ␣ and are important for VEGF's angiogenic effects.

Binding of the poorly hydrolyzable GTP analog, guanosine 5'-[y-thioltriphosphate (GTP [S]), to purified guanine-nucleotide-binding regulatory proteins (G proteins) has been shown to be nonreversible-in the presence of millimolar concentrations of Mg2 Heart muscarinic acetylcholine (mACh) receptors are coupled via guanine-nucleotide-binding regulatory proteins (G proteins) to various effector systems [l -91. Agonist-liganded receptors interacting with G proteins initiate signal transduction apparently by facilitating the release of GDP from inactive G proteins, which can then bind GTP. In the active, GTPbound state, the G proteins are dissociated from the receptors and interact with and regulate effector systems. The signal is switched off by the hydrolysis of bound GTP by the intrinsic GTPase activity of G proteins. The resulting GDP-bound G proteins can re-enter the cycle of activation and deactivation (for reviews see [lo, 111).This process of receptor-induced G-protein activation has been analyzed in cardiac and other membrane systems primarily by demonstrating receptor-stimulated GTPase activity of G proteins [I2 -141 and receptor-induced binding of the poorly

No abstract