Figure 5

Chemokine (C–X–C motif) ligand 1 (CXCL1) regulates angiogenesis in a xenograft model. (a) Thirty Balb/c mice were injected with 500 μl of Matrigel containing recombinant vascular endothelial growth factor (VEGF) (200 ng/ml), plus either 50 μg/ml of control immunoglobulin G (IgG) (n=10), 50 μg/ml of anti-CXCL1-neutralizing antibody (n=10) or 50 μg/ml of bevacizumab (n=10). After 7 days, Matrigel plugs were resected and stained with hematoxylin and eosin. Vasculature identified on hematoxylin and eosin staining was counted in five random fields. Less vasculature was noted in the both the anti-CXCL1 and bevacizumab groups. *P<0.05, original magnification, × 100. (b) The population of endothelial cells within the Matrigel plugs was assessed by anti-CD31 immunofluorescence staining. The number of fluorescent cells was counted per high-power field (hpf). CD31-positive cells were reduced in both the anti-CXCL1 and bevacizumab groups (*P<0.05), original magnification, × 400. (c) The total hemoglobin (Hb) content of the Matrigel plugs was used as another quantifiable surrogate measure of angiogenesis. Hb was extracted from the Matrigel plugs and detected colorimetrically. A significant reduction in Hb content was evident in the Matrigel plugs treated with anti-CXCL1 antibodies (*P<0.05). (d) Double immunofluorescence staining analysis shows the expression of CXCL1 (red) and CD31 (green) in Matrigel plug from control animals. DAPI, 4′,6-diamidino-2-phenylindole (DAPI) is used for nuclear counterstain. CD31-positive vascular endothelial cells express CXCL1, original magnification, × 1000.