Vanessa F. Merino

Genome-wide Methylation Analysis Identifies Genes Specific to Breast Cancer Hormone Receptor Status and Risk of Recurrence

To better understand the biology of hormone receptor-positive and-negative breast cancer and to identify methylated gene markers of disease progression, we carried out a genome-wide methylation array analysis on 103 primary invasive breast cancers and 21 normal breast samples, using the Illumina Infinium HumanMethylation27 array that queried 27,578 CpG loci. Estrogen and/or progesterone receptor-positive tumors displayed more hypermethylated loci than estrogen receptor (ER)-negative tumors. However, the hypermethylated loci in ER-negative tumors were clustered closer to the transcriptional start site compared with ER-positive tumors. An ER-classifier set of CpG loci was identified, which independently partitioned primary tumors into ER subtypes. A total of 40 (32 novel and 8 previously known) CpG loci showed differential methylation specific to either ER-positive or ER-negative tumors. Each of the 40 ER subtype-specific loci was validated in silico, using an independent, publicly available methylome dataset from the Cancer Genome Atlas. In addition, we identified 100 methylated CpG loci that were significantly associated with disease progression; the majority of these loci were informative particularly in ER-negative breast cancer. Overall, the set was highly enriched in homeobox containing genes. This pilot study shows the robustness of the breast cancer methylome and illustrates its potential to stratify and reveal biological differences between ER subtypes of breast cancer. Furthermore, it defines candidate ER-specific markers and identifies potential markers predictive of outcome within ER subgroups.

Mice deficient for both kinin receptors are normotensive and protected from endotoxin-induced hypotension

Kinins play a central role in the modulation of cardiovascular function and in the pathophysiology of inflammation. These peptides mediate their effects by binding to two specific G‐protein coupled receptors named B1 and B2. To evaluate the full functional relevance of the kallikrein‐kinin system, we generated mice lacking both kinin receptors (B1B2‐/‐). Because of the close chromosomal position of both kinin receptor genes, B1B2‐/‐ mice could not be obtained by simple breeding of the single knockout lines. Therefore, we inactivated the B1 receptor gene by homologous recombination in embryonic stem cells derived from B2‐deficient animals. The B1B2‐/‐ mice exhibited undetectable levels of mRNAs for both receptors and a lack of response to bradykinin (B2 agonist) and des‐Arg9‐bradykinin (B1 agonist), as attested by contractility studies with isolated smooth muscle tissues. B1B2‐/‐ mice are healthy and fertile, and no sign of cardiac abnormality was detected. They are nor‐motensive but exhibit a lower heart rate than controls. Furthermore, kinin receptor deficiency affects the pathogenesis of endotoxin‐induced hypotension. While blood pressure decreased markedly in wild‐type mice and B2‐/‐ and moderately in B1 ‐/‐ mice after bacterial lipopolysaccharide (LPS) injection, blood pressure remained unchanged in B1B2‐/‐ mice. These results clearly demonstrate a pivotal role of kinins and their receptors in hypotension induced by endotoxemia in mice.—Cayla C., Todiras, M., Iliescu, R., Saul, V. V., Gross, V., Pilz, B., Chai, G., Merino, V. F., Pesquero, J. B., Baltatu, O. C., Bader M. Mice deficient for both kinin receptors are normotensive and protected from endotox‐in‐induced hypotension. FASEB J. 21, 1689–1698 (2007)

Epigenetic regulation of cell type-specific expression patterns in the human mammary epithelium

Differentiation is an epigenetic program that involves the gradual loss of pluripotency and acquisition of cell type–specific features. Understanding these processes requires genome-wide analysis of epigenetic and gene expression profiles, which have been challenging in primary tissue samples due to limited numbers of cells available. Here we describe the application of high-throughput sequencing technology for profiling histone and DNA methylation, as well as gene expression patterns of normal human mammary progenitor-enriched and luminal lineage-committed cells. We observed significant differences in histone H3 lysine 27 tri-methylation (H3K27me3) enrichment and DNA methylation of genes expressed in a cell type–specific manner, suggesting their regulation by epigenetic mechanisms and a dynamic interplay between the two processes that together define developmental potential. The technologies we developed and the epigenetically regulated genes we identified will accelerate the characterization of primary cell epigenomes and the dissection of human mammary epithelial lineage-commitment and luminal differentiation.

Loss of Myocardial Ischemic Postconditioning in Adenosine A1 and Bradykinin B2 Receptors Gene Knockout Mice

Background— Ischemic postconditioning (PostC) is a recently described cardioprotective modality against reperfusion injury, through series of brief reflow interruptions applied at the very onset of reperfusion. It is proposed that PostC can activate a complex cellular signaling cascade, in which cell membrane receptors could serve as the upstream triggers of PostC. However, the exact subtypes of such receptors remain controversial or uninvestigated. To this context, the purpose of present study was to determine the definitive role of adenosine A1 and bradykinin B1 and B2 receptors in PostC. Methods and Results— The hearts isolated from adult male C57BL/6J wild-type mice or the mice lacking adenosine A1, or bradykinin B1 or B2 receptors subjected to zero-flow global ischemia and reperfusion in a Langendorff model. PostC, consisting of 6 cycles of 10 seconds of reperfusion and 10 seconds of ischemia, demonstrated significantly reduced myocardial infarct size (22.8±3.1%, mean±SEM) as compared with the non-PostC wild-type controls (35.1±2.8%, P<0.05). The infarct-limiting protection of PostC was absent in adenosine A1 receptor knockout mice (34.9±2.7%) or bradykinin B2 receptor knockout mice (33.3±1.7%) and was partially attenuated in bradykinin B1 receptor–deficient mice (25.6±2.9%; P>0.05). On the other hand, PostC did not significantly alter postischemic cardiac contractile function and coronary flow. Conclusions— With the use of three distinctive strains of gene knockout mice, the current study has provided the first conclusive evidence showing PostC-induced infarct-limiting cardioprotection could be triggered by activation of multiple types of cell membrane receptors, which include adenosine A1 and bradykinin B2 receptors.

Molecular profiling of human mammary gland links breast cancer risk to a p27(+) cell population with progenitor characteristics.

Early full-term pregnancy is one of the most effective natural protections against breast cancer. To investigate this effect, we have characterized the global gene expression and epigenetic profiles of multiple cell types from normal breast tissue of nulliparous and parous women and carriers of BRCA1 or BRCA2 mutations. We found significant differences in CD44(+) progenitor cells, where the levels of many stem cell-related genes and pathways, including the cell-cycle regulator p27, are lower in parous women without BRCA1/BRCA2 mutations. We also noted a significant reduction in the frequency of CD44(+)p27(+) cells in parous women and showed, using explant cultures, that parity-related signaling pathways play a role in regulating the number of p27(+) cells and their proliferation. Our results suggest that pathways controlling p27(+) mammary epithelial cells and the numbers of these cells relate to breast cancer risk and can be explored for cancer risk assessment and prevention.

Kinin B1 Receptor Deficiency Leads to Leptin Hypersensitivity and Resistance to Obesity

OBJECTIVE—Kinins mediate pathophysiological processes related to hypertension, pain, and inflammation through the activation of two G-protein–coupled receptors, named B1 and B2. Although these peptides have been related to glucose homeostasis, their effects on energy balance are still unknown. RESEARCH DESIGN AND METHODS—Using genetic and pharmacological strategies to abrogate the kinin B1 receptor in different animal models of obesity, here we present evidence of a novel role for kinins in the regulation of satiety and adiposity. RESULTS—Kinin B1 receptor deficiency in mice (B1−/−) resulted in less fat content, hypoleptinemia, increased leptin sensitivity, and robust protection against high-fat diet–induced weight gain. Under high-fat diet, B1−/− also exhibited reduced food intake, improved lipid oxidation, and increased energy expenditure. Surprisingly, B1 receptor deficiency was not able to decrease food intake and adiposity in obese mice lacking leptin (ob/ob-B1−/−). However, ob/ob-B1−/− mice were more responsive to the effects of exogenous leptin on body weight and food intake, suggesting that B1 receptors may be dependent on leptin to display their metabolic roles. Finally, inhibition of weight gain and food intake by B1 receptor ablation was pharmacologically confirmed by long-term administration of the kinin B1 receptor antagonist SSR240612 to mice under high-fat diet. CONCLUSIONS—Our data suggest that kinin B1 receptors participate in the regulation of the energy balance via a mechanism that could involve the modulation of leptin sensitivity.

Absence of diabetic hyperalgesia in bradykinin B1 receptor-knockout mice

Experimental evidence has shown that the inducible bradykinin (BK) B1 receptor (BKB1-R) subtype is involved in the development of hyperalgesia associated with type 1 diabetes. Selective BKB1-R antagonists inhibited, whereas selective agonists increased the hyperalgesic activity in diabetic mice in thermal nociceptive tests. Here we evaluate the development of diabetic hyperalgesia in a BKB1-R-knockout (KO) genotype compared to wild-type (WT) mice. The BKB1-R-KO mice were backcrossed for 10 generations to C57BL/6 genetic background before use in the experiments. Diabetes was induced by streptozotocin (STZ) and thermal nociception was assessed by the hot plate and tail immersion tests. The hyperalgesia observed in wild type mice was totally absent in the BKB1-R-KO mice. Furthermore, the selective BKB1-R agonist, desArg9BK, significantly increased the hyperalgesic activity in diabetic WT mice but had no effect on nociceptive responses in diabetic BKB1-R-KO mice. Taken together, the results confirm the crucial role of the BKB1-R, upregulated alongside inflammatory diabetes, in the development of diabetes-induced hyperalgesia.

Predisposition to atherosclerosis and aortic aneurysms in mice deficient in kinin B1 receptor and apolipoprotein E

Kinin B1 receptor is involved in chronic inflammation and expressed in human atherosclerotic lesions. However, its significance for lesion development is unknown. Therefore, we investigated the effect of kinin B1 receptor deletion on the development of atherosclerosis and aortic aneurysms in apolipoprotein E-deficient (ApoE−/−) mice. Mice deficient both in ApoE and in kinin B1 receptor (ApoE−/−-B1−/−) were generated and analyzed for their susceptibility to atherosclerosis and aneurysm development under cholesterol rich-diet (western diet) and angiotensin II infusion. Kinin B1 receptor messenger RNA (mRNA) expression was significantly increased in ApoE−/− mice after Western-type diet. Although no difference in serum cholesterol was found between ApoE−/−-B1−/− and ApoE−/− mice under Western-type diet, aortic lesion incidence was significantly higher in ApoE−/−-B1−/− after this treatment. In accordance, we observed increased endothelial dysfunction in these mice. The mRNA expression of cyclic guanosine monophosphate-dependent protein kinase I, CD-11, F4/80, macrophage colony-stimulating factor, and tumor necrosis factor-alpha were increased in the aorta of double-deficient mice following Western-type diet, whereas the levels of peroxisome proliferator-activated receptor gamma protein and the activity of matrix metalloproteinase-9 activity were decreased. In addition to the increased atherosclerotic lesions, the lack of kinin B1 receptor also increased the incidence of abdominal aortic aneurysms after angiotensin II infusion. In conclusion, our results show that kinin B1 receptor deficiency aggravates atherosclerosis and aortic aneurysms under cholesterolemic conditions, supporting an antiatherogenic role for the kinin B1 receptor.

Increased susceptibility to endotoxic shock in transgenic rats with endothelial overexpression of kinin B1 receptors

Two kinin receptors have been described, the inducible B1 and the constitutive B2. B1 receptors are important in cardiovascular homeostasis and inflammation. To further clarify their vascular function, we have generated transgenic rats (TGR(Tie2B1)) overexpressing the B1 receptor exclusively in the endothelium. Endothelial cell-specific expression was confirmed by B1-agonist-induced relaxation of isolated aorta, which was abolished by endothelial denudation of the vessel. This vasodilatation was mediated by nitric oxide (NO) and K+ channels. TGR(Tie2B1) rats were normotensive but, in contrast to controls, reacted with a marked fall in blood pressure and increased vascular permeability after intravenous injection of a B1 agonist. After lipopolysaccharide treatment, they present a more pronounced hypotensive response and marked bradycardia associated with increased mortality when compared to non-transgenic control animals. Thus, the transgenic rats overexpressing kinin B1 receptors exclusively in the endothelium generated in this study support an important role of this receptor in the vasculature during the pathogenesis of endotoxic shock.

Molecular and pharmacological evidence for modulation of kinin B1 receptor expression by endogenous glucocorticoids hormones in rats

The effect of endogenous glucocorticoid hormones on the expression of rat B1 receptors was examined by means of molecular and pharmacological functional approaches. Rats were adrenalectomized (ADX), and 7 days after this procedure the intradermal injection of B1 receptor agonist des‐Arg9‐BK produced a significant increase in the paw volume, while only a weak effect was observed in sham‐operated animals. A similar increase in the contractile responses mediated by B1 agonist des‐Arg9‐BK was also observed in the rat portal vein in vitro. Chemical ADX performed with mitotane (a drug that reduces corticosteroid synthesis) produced essentially the same up‐regulation of B1 receptors as that observed in ADX rats. The modulation of B1 receptor expression was evaluated by ribonuclease protection assay, employing mRNA obtained from the lungs and paw of ADX rats. Additionally, both paw oedema and contraction of portal vein mediated by B1 agonist des‐Arg9‐BK in ADX rats, were markedly inhibited by treatment with dexamethasone, or COX‐2 inhibitor meloxican, or with the NF‐κB inhibitor PDTC. Interestingly, the same degree of inhibition was achieved when the animals were treated with a combination of submaximal doses of dexamethasone and PDTC. The involvement of NF‐κB pathway was further confirmed by mobility shift assay using nuclear extracts from lung, paw and heart of ADX rats. It was also confirmed that the treatment of ADX rats with dexamethasone, PDTC or dexamethasone plus PDTC completely inhibit NF‐κB activation caused by absence of endogenous glucucorticoid. Together, the results of the present study provide, for the first time, molecular and pharmacological evidence showing that B1 kinin receptor expression can be regulated through endogenous glucocorticoids by a mechanism dependent on NF‐κB pathway. Clinical significance of the present findings stem from evidence showing the importance of B1 kinin receptors in the mediation of inflammatory and pain related responses.