The mechanism of thrombin-induced angiogenesis is poorly understood. Using a gene chip array to investigate the pro-malignant phenotype of thrombin-stimulated cells we observed that thrombin (0.5–1 u/ml/24 hr) upregulates GRO-α 168 fold in an undifferentiated mouse cell line (UMCL). Thrombin-induced GRO-α upregulation was also observed in several tumor cell lines [murine B16F10 melanoma and 4T1 breast CA, human DU145 and PC3 prostate CA, human MCF7 breast CA, as well as human umbilical vein (HUVEC) and brain microvascular endothelial cells] by mRNA and protein analysis. Thrombin enhanced the secretion of GRO-α from PC3 and MCF7 cells 25–64 fold at 24 hrs. GRO-α is a CXC chemokine oncogene that binds to a seven transmembrane receptor, CXCR2 and enhances growth, chemotaxis and metastasis of several tumor cell lines. GRO-α induced metastasis is associated with increased angiogenesis. To obtain a better understanding of neoangiogenesis mechanisms involved in the thrombin-stimulated malignant phenotype we examined the effect of anti-GRO-α Ab on thrombin-induced angiogenesis in the CAM assay at 72 hrs. GRO-α (5ug/egg/day) enhanced angiogenesis 2.2 fold (n=19, p=0.005), providing direct evidence for GRO-α as an angiogenic growth factor. Thrombin also enhanced angiogenesis 2.7 fold (n=9, p=0.01), however, anti-GRO-α Ab inhibited thrombin-induced angiogenesis to basal control levels whereas IgG control Ab had no effect (n=9). Similar results were obtained with an endothelial (HUVEC) cord formation assay in matrigel. Thrombin-induced cord formation increased 1.5 fold (n=20, p<0.0001) at 24 hrs which was inhibited by anti-GRO-α Ab to basal control levels (n=6, p<0.01). We next examined the relationship between thrombin and GRO-α on the upregulation of various vascular growth factors and receptor genes in HUVEC at 24 hrs. Thrombin as well as its PAR-1 receptor activation peptide (TRAP, 100 uM) as well as GRO-α (3 ug/ml) all markedly upregulated (>2-4 fold) KDR, Ang-2, MMP-1, MMP-2, CXCR2 and CD31 (angiogenesis marker) protein compared to control cells (n=3). Similar upregulation of GRO-α was also noted in tumor cells (PC3, B16F10 and 4T1 cells) (n=3). All of the thrombin/TRAP gene upregulations were completely inhibited by anti-GRO-α Ab and unaffected by irrelevant Ab-treated cells (n=3). bFGF and VEGF had no effect on upregulation of GRO-α (immunoblot) yet they upregulated other vascular growth factors, demonstrating specificity for thrombin. Similar inhibition of thrombin upregulation of vascular growth factors was noted in siRNA GRO-α knock down (KD) HUVEC as well as 4T1 and B16F10 cells. In vivo tumor growth studies in wild-type mice injected with subcutaneous GRO-α KD 4T1 cells revealed 2-4 fold impaired tumor growth, spontaneous metastasis and angiogenesis which was not affected by endogenous thrombin (hirudin treatment). Thus thrombin-induced angiogenesis requires the upregulation of GRO-α. Thrombin upregulation of GRO-α in tumor cells as well as endothelial cells, contributes to tumor angiogenesis.

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