CYR61, a product of a growth factor-inducible immediate early gene, promotes angiogenesis and tumor growth

Abstract

CYR61 is a secreted, cysteine-rich, heparin-binding protein encoded by a growth factor-inducible immediate-early gene. Acting as an extracellular, matrix-associated signaling molecule, CYR61 promotes the adhesion of endothelial cells through interaction with the integrin alphaVbeta3 and augments growth factor-induced DNA synthesis in the same cell type. In this study, we show that purified CYR61 stimulates directed migration of human microvascular endothelial cells in culture through an alphaV beta3-dependent pathway and induces neovascularization in rat corneas. Both the chemotactic and angiogenic activities of CYR61 can be blocked by specific anti-CYR61 antibodies. Whereas most human tumor-derived cell lines tested express CYR61, the gastric adenocarcinoma cell line RF-1 does not. Expression of the CYR61 cDNA under the regulation of a constitutive promoter in RF-1 cells significantly enhances the tumorigenicity of these cells as measured by growth in immunodeficient mice, resulting in tumors that are larger and more vascularized than those produced by control RF-1 cells. Taken together, these results identify CYR61 as an angiogenic inducer that can promote tumor growth and vascularization; the results also suggest potential roles for CYR61 in physiologic and pathologic neovascularization.

Figure 1

Chemotactic activity of CYR61. The migration of HMVECs was measured in a modified Boyden chamber assay. The cells placed in the lower chamber that migrated into the upper chamber were counted in three high power fields for each condition after a 4-hr incubation at 37°C. CYR61 was used at 1 μg/ml (except for dose response in A), vitronectin was used at 5 μg/ml, bFGF was used at 10 ng/ml, and VEGF was used at 1 ng/ml as chemoattractants. (A) CYR61-stimulated cell migration is dose-dependent. Cells that migrated into the upper well, where the indicated amount of purified CYR61 (▪) or the equivalent amount of CYR61 storage buffer (○) was placed, were counted. In parallel, HMVEC migration was measured by using bFGF as a chemoattractant, placed in the upper well with various amounts of CYR61 storage buffer corresponding to the amounts used in CYR61-stimulated migration experiment. Cells migrated are expressed as a percentage of bFGF-induced migration ± SD (B) Specific inhibition of CYR61-stimulated cell migration by anti-Cyr61 antibodies (25 μg/ml). HMVEC migration was monitored as described above by using either CYR61 or bFGF as a chemoattractant. Where indicated, these proteins were preincubated with anti-Cyr61 antibodies before use. (C) Directed migration of HMVEC tested in a checkerboard-type analysis. CYR61 or bFGF was added to the upper chamber, the lower chamber, neither chamber, or both chambers as indicated. In B and C, results are shown as the number of cells that migrated per three high power fields ± SD (D) Specific inhibition of CYR61-stimulated migration by an antibody against integrin α_Vβ₃ but not α_Vβ₅. HMVEC migration was monitored by using CYR61, vitronectin, bFGF, or VEGF as chemoattractants. Where indicated, cells were preincubated with 50 μg/ml LM609 for 1 hr before addition to the lower chamber. Results are expressed as percentage of cells migrated to VEGF. Neg., background migration in the absence of chemoattractant.

Figure 2

CYR61 stimulates neovascularization in rat cornea. Hydron pellets containing test (or control) samples were formulated and implanted into corneas of rats as described in Materials and Methods (also see Table 1). New blood vessels that formed were visualized by perfusion with colloidal carbon 7 days after implantation. Hydron pellets contained (A) CYR61; (B) bFGF; (C) CYR61 storage buffer; or (D) CYR61 protein preincubated with anti-Cyr61 antibodies.

Figure 3

Expression of CYR61 and effect in cell growth. (A) Immunoblot analysis of lysates from RF-1 cells transfected with a cyr61-expression vector (RF1–61SN) or with the empty vector (RF1-XSN). Lysates were electrophoresed on a 10% SDS/PAGE gel followed by immunoblotting with anti-CYR61 antibodies. The lane marked “CYR61” displays a sample of purified CYR61 protein. Molecular weights of marker proteins are indicated on the left in kDa. (B) The growth of RF1–61SN (▪) and RF1-XSN (▿) was monitored in culture. Cells were seeded at 1 × 10⁵ cells per well in 24-well plates, and the medium was changed every 3 days. Cell numbers were counted in triplicates on indicated days.

Figure 4

Effect of cyr61 expression on tumor size and vessel density. Tumors grown 32 days after s.c. injection of RF-1 cells, which were transfected either with a cyr61-expressing vector (RF1–61SN) or with the empty vector (RF1-XSN), into severe combined immunodeficiency/beige mice were harvested and examined. (A) Tumor weights and (B) number of blood vessels per high power field were determined as described in Materials and Methods. Histograms show the mean values, and error bars show the SEM. Statistical analysis was performed by using the Student’s t test, as well as ANOVA and the Tukey–Kramer multiple comparison test. ∗, P < 0.05; ∗∗, P < 0.001.

Figure 5

Immunohistochemical analysis. Detection of CYR61 in tumors derived from (A) RF1-XSN or (B) RF1–61SN was carried out by immunostaining with anti-Cyr61 antibodies. Immunostaining for CYR61 in an RF1–61SN-derived tumor was abolished when the anti-CYR61 antibodies were preincubated with purified CYR61 protein (not shown). To visualize blood vessels, (C) RF1-XSN and (D) RF1–61SN tumor sections were stained with anti-CD31 antibodies.