Abstract
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 125I-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.
Highlights
Angiogenesis, the sprouting of new capillaries from established vasculature, occurs in many physiological and pathological processes [1, 2] and involves several distinct steps that include proteolysis of the extracellular matrix (ECM),1 cell migration, proliferation, and changes in capillary permeability
Peptide 3.4, had no significant effect on vascular endothelial growth factor (VEGF)-stimulated DNA synthesis (Fig. 1C). These results demonstrate that SPARC and SPARC peptides from domains II and IV abrogated significantly the mitogenic effect of VEGF on human microvascular endothelial cells (HMEC)
Endothelial cells from the microvasculature participate in angiogenesis during wound healing, a process during which both SPARC and VEGF are expressed [31, 32]
Summary
SPARC Protein and Synthetic Peptides—SPARC was purified from the culture medium of murine parietal yolk sac carcinoma cells [19]; SPARC peptides were synthesized and purified by high performance liquid chromatography [20]. The cells were washed three times with PBS containing 1 mg/ml BSA and 0.25 mM CaCl2, and were incubated for 5 min in the presence of 1% Triton X-100, 1 mg/ml BSA, and 0.16% NaN3 to remove bound VEGF. Immunoreactivity was detected by incubation with goat anti-mouse IgG coupled to horseradish peroxidase and was subsequently visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech). The proteins were separated by SDS-PAGE (10% gels), transferred to a nitrocellulose membrane, blocked with 5% milk, 0.05% Tween 20 in PBS, and incubated with rabbit anti-human enolase IgG followed by incubation with goat anti-rabbit IgG conjugated to horseradish peroxidase. For assessment of differences in protein loading, the membrane was incubated at 50 °C for 30 min with 2-mercaptoethanol (100 mM) and SDS (2%) in Tris-HCl (62 mM, pH 6.7) (“stripping buffer”), blocked, and incubated with rabbit anti-␣-enolase IgG as described above. Immunoreactivity was detected by avidin-rhodamine (Vector Laboratories) and was visualized by fluorescence microscopy
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