Obesity and metabolic syndrome are linked to an increased prevalence of breast cancer among postmenopausal women. A common feature of obesity, metabolic syndrome, and a Western diet rich in saturated fat is a high level of circulating cholesterol. Epidemiological reports investigating the relationship between high circulating cholesterol levels, cholesterol-lowering drugs, and breast cancer are conflicting. Here, we modeled this complex condition in a well-controlled, preclinical animal model using innovative isocaloric diets. Female severe combined immunodeficient mice were fed a low-fat/no-cholesterol diet and then randomized to four isocaloric diet groups: low-fat/no-cholesterol diet, with or without ezetimibe (cholesterol-lowering drug), and high-fat/high-cholesterol diet, with or without ezetimibe. Mice were implanted orthotopically with MDA-MB-231 cells. Breast tumors from animals fed the high-fat/high-cholesterol diet exhibited the fastest progression. Significant differences in serum cholesterol level between groups were achieved and maintained throughout the study; however, no differences were observed in intratumoral cholesterol levels. To determine the mechanism of cholesterol-induced tumor progression, we analyzed tumor proliferation, apoptosis, and angiogenesis and found a significantly greater percentage of proliferating cells from mice fed the high-fat/high-cholesterol diet. Tumors from hypercholesterolemic animals displayed significantly less apoptosis compared with the other groups. Tumors from high-fat/high-cholesterol mice had significantly higher microvessel density compared with tumors from the other groups. These results demonstrate that hypercholesterolemia induces angiogenesis and accelerates breast tumor growth in vivo.

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Objective To examine a series of candidate markers for urological chronic pelvic pain syndrome (UCPPS), selected based on their proposed involvement in underlying biological processes so as to provide new insights into pathophysiology and suggest targets for expanded clinical and mechanistic studies. Methods Baseline urine samples from Multidisciplinary Approach to the Study of Chronic Pelvic Pain (MAPP) Research Network study participants with UCPPS (n = 259), positive controls (PCs; chronic pain without pelvic pain, n = 107) and healthy controls (HCs, n = 125) were analysed for the presence of proteins that are suggested in the literature to be associated with UCPPS. Matrix metalloproteinase (MMP)‐2, MMP‐9, MMP‐9/neutrophil gelatinase‐associated lipocalin (NGAL) complex (also known as Lipocalin 2), vascular endothelial growth factor (VEGF), VEGF receptor 1 (VEGF‐R1) and NGAL were assayed and quantitated using mono‐specific enzyme‐linked immunosorbent assays for each protein. Log‐transformed concentration (pg/mL or ng/mL) and concentration normalized to total protein (pg/μg) values were compared among the UCPPS, PC and HC groups within sex using the Student's t‐test, with P values adjusted for multiple comparisons. Multivariable logistic regression and receiver‐operating characteristic curves assessed the utility of the biomarkers in distinguishing participants with UCPPS and control participants. Associations of protein with symptom severity were assessed by linear regression. Results Significantly higher normalized concentrations (pg/μg) of VEGF, VEGF‐R1 and MMP‐9 in men and VEGF concentration (pg/mL) in women were associated with UCPPS vs HC. These proteins provided only marginal discrimination between UCPPS participants and HCs. In men with UCCPS, pain severity was significantly positively associated with concentrations of MMP‐9 and MMP‐9/NGAL complex, and urinary severity was significantly positively associated with MMP‐9, MMP‐9/NGAL complex and VEGF‐R1. In women with UCPPS, pain and urinary symptom severity were associated with increased normalized concentrations of MMP‐9/NGAL complex, while pain severity alone was associated with increased normalized concentrations of VEGF, and urinary severity alone was associated with increased normalized concentrations of MMP‐2. Pain severity in women with UCPPS was significantly positively associated with concentrations of all biomarkers except NGAL, and urinary severity with all concentrations except VEGF‐R1. Conclusion Altered levels of MMP‐9, MMP‐9/NGAL complex and VEGF‐R1 in men, and all biomarkers in women, were associated with clinical symptoms of UCPPS. None of the evaluated candidate markers usefully discriminated UCPPS patients from controls. Elevated VEGF, MMP‐9 and VEGF‐R1 levels in men and VEGF levels in women may provide potential new insights into the pathophysiology of UCPPS.

The hypothesis that bone marrowderived, circulating endothelial cells incorporate into tumor blood vessels is unresolved. We have measured the numbers of bone marrow-derived versus resident endothelial cells in spontaneous prostate cancers during different stages of tumor progression and in age-matched normal prostates. Bone marrow-derived endothelial cells were rare in dysplasia IntroductionBone marrow has been proposed as a depot for tumor endothelial cells (TECs). 1 However, there is controversy surrounding the temporal and quantitative aspects of bone marrow-derived endothelial cell (BMDEC) recruitment in different tumors. 2,3 There are reported differences in BMDEC numbers depending on tumor grade 4 and between humans and mice. 5 Adding to these uncertainties is a lack of standardization in the methodology and markers used to identify and quantify BMDECs. 6 For example, hematopoietic stem cells (HSCs) and bona fide endothelial cells (ECs) share common markers (eg, CD31, CD34, and VEGFR-2), creating confusion over their identity. 7 In the present study, we have quantified BMDECs from spontaneous tumors at different stages of progression using transgenic adenocarcinoma of the mouse prostate (TRAMP) mice. 8 Our results show a paucity of BMDECs in both tumors and normal prostates. On the other hand, marrow-derived CD45 ϩ myelomonocytic cells were recruited in substantial numbers to tumors where they reside in a perivascular position in vivo and stimulate angiogenesis in vitro. MethodsMethods are described in the online data supplement (available on the Blood Web site; see the Supplemental Materials link at the top of the online article). All animal procedures were approved by the Animal Care and Use Committee of Children's Hospital, Harvard Medical School. Results and discussionWe first established the threshold for detecting rare cells in vivo by titrating increasing numbers of CD31 ϩ TECs 9 with 1 ϫ 10 6 CD31 Ϫ TRAMP C1 tumor cells. Fluorescence-activated cell sorting (FACS) showed a linear relationship between numbers of TECs added and the percentage of TECs detected (r 2 ϭ 0.99; Figure 1A). We could detect as few as 100 CD31 ϩ TECs per 1 million CD31 Ϫ tumor cells, approximately 0.01% of the total cellular pool. Thus, values approximating 0.01% were considered the technical detection limit using this methodology.To enumerate BMDECs in tumors or normal prostates, mice were lethally irradiated followed by bone marrow reconstitution with green fluorescent protein (GFP) ϩ marrow. Using FACS, CD45 ϩ hematopoietic cells were gated out and the GFP ϩ /CD31 ϩ and GFP Ϫ /CD31 ϩ populations were analyzed. Figure 1B shows the 3 CD45 Ϫ populations detected in a well differentiated tumor: GFP Ϫ /CD31 ϩ (5.05%), GFP ϩ /CD31 ϩ (0.027%), and GFP ϩ / CD31 Ϫ (1.45%). Multiple mice analyzed in this way showed that average numbers of CD45 Ϫ /GFP ϩ /CD31 ϩ were unchanged irrespective of tumor stage ( Figure 1C). Moreover, the range of CD45 Ϫ / GFP ϩ /CD31 ϩ ECs detected in all tissues (normal prostate or prostate tumors) was o...