Proliferation and angiogenesis in mammary glands, tumors, and tumor microenvironments in mice fed on high fat and low fat diets . (A) Proliferation: fold increases in proliferation in normal mammary epithelium at 4 and 10 weeks, hyperplastic foci at 10 weeks, and in tumor epithelium from mice on a high fat diet (HFD) versus low fat diet (LFD). At 4 weeks, mice fed HFD exhibited increased cellular proliferation as measured by proliferating cell nuclear antigen (PCNA); *P <0.05. Note, the 4-week data are a re-analysis of data published in Olson et al. (2008). At 10 weeks, mice fed HFD exhibited increased cellular proliferation in both normal epithelium and hyperplastic foci, as measured by 5-bromo-2’-deoxyuridine (BrdU) incorporation; *P <0.05. Early developing tumors on HFD (HFD-E) also exhibited increased cellular proliferation, as measured BrdU incorporation; *P <0.05. (B) Angiogenesis: blood vessel density was measured, as described in Materials and Methods, by the area occupied by CD31-positive vessels near normal mammary epithelium at 3, 4, and 10 weeks, and in tumor epithelium from mice on HFD versus LFD. At 10 weeks, CD31-stained vessels were significantly increased adjacent to normal mammary gland structures (*P = 0.01), hyperplastic foci (**P = 0.04) and microscopic tumors (tumors versus hyperplasia; ***P = 0.02) in HFD-fed compared to low fat diet (LFD)-fed mice. CD31 staining was also greater in HFD-E tumors (#P = 0.01) compared to LFD tumors. (C) Insets show increased CD31 staining of (ii) an HFD-E tumor compared with (i) an LFD tumor. Scale bars = 50 mm.