The therapeutic potential of an allosteric non-competitive CXCR1/2 antagonist for diabetic nephropathy.

AIMS: Diabetic nephropathy is a major consequence of inflammation developing in type 1 diabetes, with interleukin-8 (IL-8)-CXCR1/2 axis playing a key role in kidney disease progression. In this study, we investigated the therapeutic potential of a CXCR1/2 non-competitive allosteric antagonist (Ladarixin) in preventing high glucose-mediated injury in human podocytes and epithelial cells differentiated from renal stem/progenitor cells (RSC) cultured as nephrospheres. MATERIALS AND METHODS: We used human RSCs cultured as nephrospheres through a sphere-forming functional assay to investigate hyperglycemia-mediated effects on IL-8 signalling in human podocytes and tubular epithelial cells. RESULTS: High glucose impairs RSC self-renewal, induces an increase in IL-8 transcript expression and protein secretion and induces DNA damage in RSC-differentiated podocytes, while exerting no effect on RSC-differentiated epithelial cells. Accordingly, the supernatant from epithelial cells or podocytes cultured in high glucose was able to differentially activate leucocyte-mediated secretion of pro-inflammatory cytokines, suggesting that the crosstalk between immune and non-immune cells may be involved in disease progression in vivo. CONCLUSIONS: Treatment with Ladarixin during RSC differentiation prevented high glucose-mediated effects on podocytes and modulated either podocyte or epithelial cell-dependent leucocyte secretion of pro-inflammatory cytokines, suggesting CXCR1/2 antagonists as possible pharmacological approaches for the treatment of diabetic nephropathy.

Serotoninergic receptor ligands improve Tamoxifen effectiveness on breast cancer cells.

BACKGROUND: Serotonin (or 5-Hydroxytryptamine, 5-HT) signals in mammary gland becomes dysregulated in cancer, also contributing to proliferation, metastasis, and angiogenesis. Thus, the discovery of novel compounds targeting serotonin signaling may contribute to tailor new therapeutic strategies usable in combination with endocrine therapies. We have previously synthesized serotoninergic receptor ligands (SER) with high affinity and selectivity towards 5-HT2A and 5-HT2C receptors, the main mediators of mitogenic effect of serotonin in breast cancer (BC). Here, we investigated the effect of 10 SER on viability of MCF7, SKBR3 and MDA-MB231 BC cells and focused on their potential ability to affect Tamoxifen responsiveness in ER+ cells. METHODS: Cell viability has been assessed by sulforhodamine B assay. Cell cycle has been analyzed by flow cytometry. Gene expression of 5-HT receptors and Connective Tissue Growth Factor (CTGF) has been checked by RT-PCR; mRNA levels of CTGF and ABC transporters have been further measured by qPCR. Protein levels of 5-HT2C receptors have been analyzed by Western blot. All data were statistically analyzed using GraphPad Prism 7. RESULTS: We found that treatment with SER for 72 h reduced viability of BC cells. SER were more effective on MCF7 ER+ cells (IC50 range 10.2 µM - 99.2 µM) compared to SKBR3 (IC50 range 43.3 µM - 260 µM) and MDA-MB231 BC cells (IC50 range 91.3 µM - 306 µM). This was paralleled by accumulation of cells in G0/G1 phase of cell cycle. Next, we provided evidence that two ligands, SER79 and SER68, improved the effectiveness of Tamoxifen treatment in MCF7 cells and modulated the expression of CTGF, without affecting viability of MCF10A non-cancer breast epithelial cells. In a cell model of Tamoxifen resistance, SER68 also restored drug effect independently of CTGF. CONCLUSIONS: These results identified serotoninergic receptor ligands potentially usable in combination with Tamoxifen to improve its effectiveness on ER+ BC patients.

Adipocyte precursor cells from first degree relatives of type 2 diabetic patients feature changes in hsa-mir-23a-5p, -193a-5p, and -193b-5p and insulin-like growth factor 2 expression.

First-degree relatives (FDRs) of type 2 diabetics (T2D) feature dysfunction of subcutaneous adipose tissue (SAT) long before T2D onset. miRNAs have a role in adipocyte precursor cells (APC) differentiation and in adipocyte identity. Thus, impaired miRNA expression may contribute to SAT dysfunction in FDRs. In the present work, we have explored changes in miRNA expression associated with T2D family history which may affect gene expression in SAT APCs from FDRs. Small RNA-seq was performed in APCs from healthy FDRs and matched controls and omics data were validated by qPCR. Integrative analyses of APC miRNome and transcriptome from FDRs revealed down-regulated hsa-miR-23a-5p, -193a-5p and -193b-5p accompanied by up-regulated Insulin-like Growth Factor 2 (IGF2) gene which proved to be their direct target. The expression changes in these marks were associated with SAT adipocyte hypertrophy in FDRs. APCs from FDRs further demonstrated reduced capability to differentiate into adipocytes. Treatment with IGF2 protein decreased APC adipogenesis, while over-expression of hsa-miR-23a-5p, -193a-5p and -193b-5p enhanced adipogenesis by IGF2 targeting. Indeed, IGF2 increased the Wnt Family Member 10B gene expression in APCs. Down-regulation of the three miRNAs and IGF2 up-regulation was also observed in Peripheral Blood Leukocytes (PBLs) from FDRs. In conclusion, APCs from FDRs feature a specific miRNA/gene profile, which associates with SAT adipocyte hypertrophy and appears to contribute to impaired adipogenesis. PBL detection of this profile may help in identifying adipocyte hypertrophy in individuals at high risk of T2D.

In severe obesity, subcutaneous adipose tissue cell-derived cytokines are early markers of impaired glucose tolerance and are modulated by quercetin.

BACKGROUND: Excessive adiposity provides an inflammatory environment. However, in people with severe obesity, how systemic and local adipose tissue (AT)-derived cytokines contribute to worsening glucose tolerance is not clear. METHODS: Ninty-two severely obese (SO) individuals undergoing bariatric surgery were enrolled and subjected to detailed clinical phenotyping. Following an oral glucose tolerance test, participants were included in three groups, based on the presence of normal glucose tolerance (NGT), impaired glucose tolerance (IGT), or type 2 diabetes (T2D). Serum and subcutaneous AT (SAT) biopsies were obtained and mesenchymal stem cells (MSCs) were isolated, characterized, and differentiated in adipocytes in vitro. TNFA and PPARG mRNA levels were determined by qRT-PCR. Circulating, adipocyte- and MSC-released cytokines, chemokines, and growth factors were assessed by multiplex ELISA. RESULTS: Serum levels of IL-9, IL-13, and MIP-1ß were increased in SO individuals with T2D, as compared with those with either IGT or NGT. At variance, SAT samples obtained from SO individuals with IGT displayed levels of TNFA which were threefold higher compared to those with NGT, but not different from those with T2D. Elevated levels of TNFα were also found in differentiated adipocytes, isolated from the SAT specimens of individuals with IGT and T2D, compared to those with NGT. Consistent with the pro-inflammatory milieu, IL-1ß and IP-10 secretion was significantly higher in adipocytes from individuals with IGT and T2D. Moreover, increased levels of TNFα, both mRNA and secreted protein were detected in MSCs obtained from IGT and T2D, compared to NGT SO individuals. Exposure of T2D and IGT-derived MSCs to the anti-inflammatory flavonoid quercetin reduced TNFα levels and was paralleled by a significant decrease of the secretion of inflammatory cytokines. CONCLUSION: In severe obesity, enhanced SAT-derived inflammatory phenotype is an early step in the progression toward T2D and maybe, at least in part, attenuated by quercetin.

Lanthionine, a Novel Uremic Toxin, in the Vascular Calcification of Chronic Kidney Disease: The Role of Proinflammatory Cytokines.

Vascular calcification (VC) is a risk factor for cardiovascular events and mortality in chronic kidney disease (CKD). Several components influence the occurrence of VC, among which inflammation. A novel uremic toxin, lanthionine, was shown to increase intracellular calcium in endothelial cells and may have a role in VC. A group of CKD patients was selected and divided into patients with a glomerular filtration rate (GFR) of <45 mL/min/1.73 m2 and ≥45 mL/min/1.73 m2. Total Calcium Score (TCS), based on the Agatston score, was assessed as circulating lanthionine and a panel of different cytokines. A hemodialysis patient group was also considered. Lanthionine was elevated in CKD patients, and levels increased significantly in hemodialysis patients with respect to the two CKD groups; in addition, lanthionine increased along with the increase in TCS, starting from one up to three. Interleukin IL-6, IL-8, and Eotaxin were significantly increased in patients with GFR < 45 mL/min/1.73 m2 with respect to those with GFR ≥ 45 mL/min/1.73 m2. IL-1b, IL-7, IL-8, IL-12, Eotaxin, and VEGF increased in calcified patients with respect to the non-calcified. IL-8 and Eotaxin were elevated both in the low GFR group and in the calcified group. We propose that lanthionine, but also IL-8 and Eotaxin, in particular, are a key feature of VC of CKD, with possible marker significance.

Prep1 regulates angiogenesis through a PGC-1α-mediated mechanism.

Angiogenesis depends on a delicate balance between the different transcription factors, and their control should be considered necessary for preventing or treating diseases. Pre-B-cell leukemia transcription factor regulating protein 1 (Prep1) is a homeodomain transcription factor that plays a primary role in organogenesis and metabolism. Observations performed in a Prep1 hypomorphic mouse model, expressing 3-5% of the protein, show an increase of embryonic lethality due, in part, to defects in angiogenesis. In this study, we provide evidence that overexpression of Prep1 in mouse aortic endothelial cells (MAECs) stimulates migration, proliferation, and tube formation. These effects are paralleled by an increase of several proangiogenic factors and by a decrease of the antiangiogenic gene neurogenic locus notch homolog protein 1 (Notch1). Prep1-mediated angiogenesis involves the activation of the p160 Myb-binding protein (p160)/peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) pathway. Indeed, Prep1 overexpression increases its binding with p160 and induces a 4-fold increase of p160 and 70% reduction of PGC-1α compared with control cells. Incubation of MAECs with a synthetic Prep1(54-72) peptide, mimicking the Prep1 region involved in the interaction with p160, reverts the proangiogenic effects mediated by Prep1. In addition, Prep1 levels increase by 3.2-fold during the fibroblast growth factor ß (bFGF)-mediated endothelial colony-forming cells' activation, whereas Prep1(54-72) peptide reduces the capability of these cells to generate tubular-like structures in response to bFGF, suggesting a possible role of Prep1 both in angiogenesis from preexisting vessels and in postnatal vasculogenesis. Finally, Prep1 hypomorphic heterozygous mice, expressing low levels of Prep1, show attenuated placental angiogenesis and vessel formation within Matrigel plugs. All of these observations indicate that Prep1, complexing with p160, decreases PGC-1α and stimulates angiogenesis.-Cimmino, I., Margheri, F., Prisco, F., Perruolo, G., D'Esposito, V., Laurenzana, A., Fibbi, G., Paciello, O., Doti, N., Ruvo, M., Miele, C., Beguinot, F., Formisano, P., Oriente, F. Prep1 regulates angiogenesis through a PGC-1α-mediated mechanism.

Pathologic endoplasmic reticulum stress induced by glucotoxic insults inhibits adipocyte differentiation and induces an inflammatory phenotype.

Adipocyte differentiation is critical in obesity. By controlling new adipocyte recruitment, adipogenesis contrasts adipocyte hypertrophy and its adverse consequences, such as insulin resistance. Contrasting data are present in literature on the effect of endoplasmic reticulum (ER) stress and subsequent unfolded protein response (UPR) on adipocyte differentiation, being reported to be either necessary or inhibitory. In this study, we sought to clarify the effect of ER stress and UPR on adipocyte differentiation. We have used two different cell lines, the widely used pre-adipocyte 3T3-L1 cells and a murine multipotent mesenchymal cell line, W20-17 cells. A strong ER stress activator, thapsigargin, and a pathologically relevant inducer of ER stress, glucosamine (GlcN), induced ER stress and UPR above those occurring in the absence of perturbation and inhibited adipocyte differentiation. Very low concentrations of 4-phenyl butyric acid (PBA, a chemical chaperone) inhibited only the overactivation of ER stress and UPR elicited by GlcN, leaving unaltered the part physiologically activated during differentiation, and reversed the inhibitory effect of GlcN on differentiation. In addition, GlcN stimulated proinflammatory cytokine release and PBA prevented these effects. An inhibitor of NF-kB also reversed the effects of GlcN on cytokine release. These results indicate that while ER stress and UPR activation is "physiologically" activated during adipocyte differentiation, the "pathologic" part of ER stress activation, secondary to a glucotoxic insult, inhibits differentiation. In addition, such a metabolic insult, causes a shift of the preadipocyte/adipocyte population towards a proinflammatory phenotype.

Comparison between fibroblast wound healing and cell random migration assays in vitro.

Cell migration plays a key role in many biological processes, including cancer growth and invasion, embryogenesis, angiogenesis, inflammatory response, and tissue repair. In this work, we compare two well-established experimental approaches for the investigation of cell motility in vitro: the cell random migration (CRM) and the wound healing (WH) assay. In the former, extensive tracking of individual live cells trajectories by time-lapse microscopy and elaborate data processing are used to calculate two intrinsic motility parameters of the cell population under investigation, i.e. the diffusion coefficient and the persistence time. In the WH assay, a scratch is made in a confluent cell monolayer and the closure time of the exposed area is taken as an easy-to-measure, empirical estimate of cell migration. To compare WH and CRM we applied the two assays to investigate the motility of skin fibroblasts isolated from wild type and transgenic mice (TgPED) overexpressing the protein PED/PEA-15, which is highly expressed in patients with type 2 diabetes. Our main result is that the cell motility parameters derived from CRM can be also estimated from a time-resolved analysis of the WH assay, thus showing that the latter is also amenable to a quantitative analysis for the characterization of cell migration. To our knowledge this is the first quantitative comparison of these two widely used techniques.

Substrate-zymography: a still worthwhile method for gelatinases analysis in biological samples.

Matrix metallo-proteinases (MMPs) are a family of zinc-dependent endopeptidases, capable of degrading all the molecular components of extracellular matrix. A class of MMPs is gelatinases which includes gelatinase A or MMP-2 (72 kDa) and gelatinase B or MMP-9 (92 kDa), which have been shown to play critical roles in pathophysiology of many human disease and, in particular, cancer progression. For these reasons they obtained a great interest as potential non-invasive biomarker in providing useful clinical information in cancer diagnosis and therapy. A sensitive and unexpensive method for analysis of gelatinases is the gelatine zymography, which allows to measure the relative amounts of active and inactive enzymes in body fluids and tissue extracts. The procedure involves the electrophoretic separation of proteins under denaturing but non reducing conditions through a polyacrylamide gel containing a synthetic substrate (gelatin). The aim of this mini-review has been to describe the general principles of gelatine zymography technique, underling the main advantages and disadvantages. Even though an improvement of this method is necessary for a better applicability in laboratory medicine, gelatine zymography represents the most convenient method to detect the activity of the different gelatinases from a wide range of biological samples.

A targeted secretome profiling by multiplexed immunoassay revealed that secreted chemokine ligand 2 (MCP-1/CCL2) affects neural differentiation in mesencephalic neural progenitor cells.

Chemokines and cytokines, primarily known for their roles in the immune and inflammatory response, have also been identified as key components of the neurogenic niche where they are involved in the modulation of neural stem cell proliferation and differentiation. However, a complete understanding of the functional role played in neural differentiation and a comprehensive profiling of these secreted molecules are lacking. By exploiting the multiplexing capability of magnetic bead-based immunoassays, we have investigated the changes of the expression levels of a set of chemokines and cytokines released from the pluripotent neural cell line mes-c-myc A1 following its differentiation from a proliferating phenotype (A1P) toward a neural (A1D) phenotype. We found a subset of molecules exclusively released from A1P, whereas others were differentially detected in A1P and A1D conditioned media. Among them, we identified monocyte chemoattractant protein-1/chemokine ligand 2 (MCP-1/CCL2) as a proneurogenic factor able to affect neuronal differentiation of A1 cells as well as of neuroblasts from primary cultures and to induce the elongation and/or formation of neuritic processes. Altogether, data are suggestive of a main role played by the CCL2/CCR2 signaling pathway and in general of the network of secreted cytokines/chemokines in the differentiation of neural progenitor cells toward a neural fate.

Platelet-Rich Plasma Increases Growth and Motility of Adipose Tissue-Derived Mesenchymal Stem Cells and Controls Adipocyte Secretory Function.

Adipose tissue-derived mesenchymal stem cells (Ad-MSC) and platelet derivatives have been used alone or in combination to achieve regeneration of injured tissues. We have tested the effect of platelet-rich plasma (PRP) on Ad-MSC and adipocyte function. PRP increased Ad-MSC viability, proliferation rate and G1-S cell cycle progression, by at least 7-, 2-, and 2.2-fold, respectively, and reduced caspase 3 cleavage. Higher PRP concentrations or PRPs derived from individuals with higher platelet counts were more effective in increasing Ad-MSC growth. PRP also accelerated cell migration by at least 1.5-fold. However, PRP did not significantly affect mature adipocyte viability, differentiation and expression levels of PPAR-γ and AP-2 mRNAs, while it increased leptin production by 3.5-fold. Interestingly, PRP treatment of mature adipocytes also enhanced the release of Interleukin (IL)-6, IL-8, IL-10, Interferon-γ, and Vascular Endothelial Growth Factor. Thus, data are consistent with a stimulatory effect of platelet derivatives on Ad-MSC growth and motility. Moreover, PRP did not reduce mature adipocyte survival and increased the release of pro-angiogenic factors, which may facilitate tissue regeneration processes.

Bisphenol-A plasma levels are related to inflammatory markers, visceral obesity and insulin-resistance: a cross-sectional study on adult male population.

BACKGROUND: The current increase of obesity and metabolic syndrome (MS) focuses attention on bisphenol-A (BPA), "obesogen" endocrine disruptor, main plastic component. Aim was to verify the role of BPA in metabolic alterations, insulin resistance, low grade inflammation and visceral obesity. METHODS: A cross-sectional study was performed in 76 out of 139 environmentally exposed adult males, unselected Caucasian subjects, enrolled by routine health survey at the "Federico II" University of Naples outpatient facilities. BPA plasma levels (ELISA), metabolic risk factors, homeostasis model assessment of insulin resistance index, plasma monocyte chemoattractant protein 1, interleukin-6 (IL-6) and tumor necrosis factor-alpha were performed. Clinical and biochemical parameters have been compared with BPA and pro-inflammatory cytokines levels. RESULTS: In total 24 subjects out of 76 (32%) presented with waist circumference (WC) >102 cm, 36 (47%) had impaired fasting glucose and 24 (32%) subjects had insulin resistance [11 out 52 (21%) with WC ≤102 cm and 13 out of 24 with WC >102 cm (54%), χ(2) 6.825, p = 0.009]. BPA and pro-inflammatory cytokine levels were significantly higher in subjects with visceral adiposity (WC > 102 cm). BPA correlated with WC, triglycerides, glucose homeostasis and inflammatory markers. At the multivariate analysis WC and IL-6 remained the main predictors of BPA. CONCLUSIONS: Detectable BPA plasma levels have been found also in our population. The strictly association between BPA and WC, components of MS, and inflammatory markers, further supports the BPA role in visceral obesity-related low grade chronic inflammation.

Methylglyoxal impairs endothelial insulin sensitivity both in vitro and in vivo.

AIMS/HYPOTHESIS: Insulin exerts a direct action on vascular cells, thereby affecting the outcome and progression of diabetic vascular complications. However, the mechanism through which insulin signalling is impaired in the endothelium of diabetic individuals remains unclear. In this work, we have evaluated the role of the AGE precursor methylglyoxal (MGO) in generating endothelial insulin resistance both in cells and in animal models. METHODS: Time course experiments were performed on mouse aortic endothelial cells (MAECs) incubated with 500 µmol/l MGO. The glyoxalase-1 inhibitor S-p-bromobenzylglutathione-cyclopentyl-diester (SpBrBzGSHCp2) was used to increase the endogenous levels of MGO. For the in vivo study, an MGO solution was administrated i.p. to C57BL/6 mice for 7 weeks. RESULTS: MGO prevented the insulin-dependent activation of the IRS1/protein kinase Akt/endothelial nitric oxide synthase (eNOS) pathway, thereby blunting nitric oxide (NO) production, while extracellular signal-regulated kinase (ERK1/2) activation and endothelin-1 (ET-1) release were increased by MGO in MAECs. Similar results were obtained in MAECs treated with SpBrBzGSHCp2. In MGO- and SpBrBzGSHCp2-exposed cells, inhibition of ERK1/2 decreased IRS1 phosphorylation on S616 and rescued insulin-dependent Akt activation and NO generation, indicating that MGO inhibition of the IRS1/Akt/eNOS pathway is mediated, at least in part, by ERK1/2. Chronic administration of MGO to C57BL/6 mice impaired whole-body insulin sensitivity and induced endothelial insulin resistance. CONCLUSIONS/INTERPRETATION: MGO impairs the action of insulin on the endothelium both in vitro and in vivo, at least in part through an ERK1/2-mediated mechanism. These findings may be instrumental in developing novel strategies for preserving endothelial function in diabetes.

Bisphenol A in polycystic ovary syndrome and its association with liver-spleen axis.

CONTEXT: Bisphenol A, one of the highest-volume chemicals currently available, is known to act as endocrine disruptor and alters several metabolic functions, including inflammatory pathways. Elevated serum levels of bisphenol A have been found in women with polycystic ovary syndrome (PCOS) and a role of low-grade chronic inflammation has been recently reported in the pathogenesis of this syndrome. Increased spleen volume, a reliable and stable index of chronic inflammation, was strictly associated with the severity of hepatic steatosis (HS) in obese subjects, determining the so-called liver-spleen axis. OBJECTIVE: To evaluate the contribution of increased serum bisphenol A levels to low-grade chronic inflammation, HS and hyperandrogenism in women with PCOS. DESIGN, SETTING AND PARTICIPANTS: Forty lean and overweight/obese premenopausal women with PCOS and 20 healthy age-matched women were consecutively enrolled in a cross-sectional study from 2009 to 2011 at the Federico II University Hospital in Naples. MEASUREMENTS: Bisphenol A, homoeostasis model assessment of insulin resistance (HoMA-IR), laboratory liver tests, testosterone, sex hormone-binding globulin, free androgen index (FAI), C-reactive protein, interleukin-6, and the ultrasound quantification of HS and spleen longitudinal diameter. RESULTS: Independently of body weight, higher bisphenol A levels in PCOS women were associated with higher grades of insulin resistance, HS, FAI and inflammation, spleen size showing the best correlation. At multivariate analysis, spleen size and FAI were the best predictors of bisphenol A (ß coefficients 0.379, P = 0.007 and 0.343, P = 0.014, respectively). CONCLUSIONS: In premenopausal women with PCOS, we evidenced an association of serum bisphenol A levels with HS and markers of low-grade inflammation, in particular with spleen size, unravelling the presence of the liver-spleen axis in this syndrome.

Human Platelet-Rich Plasma Regulates Canine Mesenchymal Stem Cell Migration through Aquaporins.

Platelet products are commonly used in regenerative medicine due to their effects on the acceleration and promotion of wound healing, reduction of bleeding, synthesis of new connective tissue, and revascularization. Furthermore, a novel approach for the treatment of damaged tissues, following trauma or other pathological damages, is represented by the use of mesenchymal stem cells (MSCs). In dogs, both platelet-rich plasma (PRP) and MSCs have been suggested to be promising options for subacute skin wounds. However, the collection of canine PRP is not always feasible. In this study, we investigated the effect of human PRP (hPRP) on canine MSCs (cMSCs). We isolated cMSCs and observed that hPRP did not modify the expression levels of the primary class of major histocompatibility complex genes. However, hPRP was able to increase cMSC viability and migration by at least 1.5-fold. hPRP treatment enhanced both Aquaporin (AQP) 1 and AQP5 protein levels, and their inhibition by tetraethylammonium chloride led to a reduction of PRP-induced migration of cMSCs. In conclusion, we have provided evidence that hPRP supports cMSC survival and may promote cell migration, at least through AQP activation. Thus, hPRP may be useful in canine tissue regeneration and repair, placing as a promising tool for veterinary therapeutic approaches.

Correction: Bisphenol-A Impairs Insulin Action and Up-Regulates Inflammatory Pathways in Human Subcutaneous Adipocytes and 3T3-L1 Cells.

[This corrects the article DOI: 10.1371/journal.pone.0082099.].

Does Gut-breast Microbiota Axis Orchestrates Cancer Progression?

Breast cancer, even today, can cause death. Therefore, prevention and early detection are fundamental factors. The mechanisms that favour it are genetic and epigenetic, and seem to play a significant role; also, the microbiota can change estrogen levels and can induce chronic inflammation in the neoplastic site, alternating the balance between proliferation and cell death. Activated steroid hormone receptors induce transcription of genes that encode for proteins involved in cell proliferation and activate another transduction pathway, inducing cell cycle progression and cell migration. These important studies have allowed to develop therapies with selective modulators of estrogen receptors (SERMs), able to block their proliferative and pro-tumorigenic action. Of fundamental importance is also the role played by the microbiota in regulating the metabolism of estrogens and their levels in the blood. There are microbial populations that are able to promote the development of breast cancer, through the production of enzymes responsible for the deconjugation of estrogens, the increase of these in the intestine, subsequent circulation and migration to other locations, such as the udder. Other microbial populations are, instead, able to synthesize estrogen compounds or mimic estrogenic action, and interfere with the metabolism of drugs, affecting the outcome of therapies. The microbial composition of the intestine and hormonal metabolism depend largely on eating habits; the consumption of fats and proteins favours the increase of estrogen in the blood, unlike a diet rich in fiber. Therefore, in-depth knowledge of the microbiota present in the intestine-breast axis could, in the future, encourage the development of new diagnostic and therapeutic approaches to breast cancers.

Lifestyle and Dietary Habits Affect Plasma Levels of Specific Cytokines in Healthy Subjects.

Low-grade chronic inflammation (LGCI) is a common feature of non-communicable diseases. Cytokines play a crucial role in LGCI. This study aimed to assess how LGCI risk factors [e.g., age, body mass index (BMI), smoke, physical activity, and diet] may impact on specific cytokine levels in a healthy population. In total, 150 healthy volunteers were recruited and subjected to questionnaires about the last 7-day lifestyle, including smoking habit, physical activity, and food frequency. A panel of circulating cytokines, chemokines, and growth factors was analyzed by multiplex ELISA. BMI showed the heaviest impact on the correlation between LGCI-related risk factors and cytokines and was significantly associated with CRP levels. Aging was characterized by an increase in IL-1b, eotaxin, MCP-1, and MIP-1α. Smoking was related to higher levels of IL-1b and CCL5/RANTES, while physical activity was related to MIP-1α. Within the different eating habits, CRP levels were modulated by eggs, red meat, shelled fruits, and greens consumption; however, these associations were not confirmed in a multivariate model after adjusting for BMI. Nevertheless, red meat consumption was associated with an inflammatory pattern, characterized by an increase in IL-6 and IL-8. IL-8 levels were also increased with the frequent intake of sweets, while a higher intake of shelled fruits correlated with lower levels of IL-6. Moreover, IL-6 and IL-8 formed a cluster that also included IL-1b and TNF-α. In conclusion, age, BMI, smoke, physical activity, and dietary habits are associated with specific cytokines that may represent potential markers for LGCI.

ZMAT3 hypomethylation contributes to early senescence of preadipocytes from healthy first-degree relatives of type 2 diabetics.

Senescence of adipose precursor cells (APC) impairs adipogenesis, contributes to the age-related subcutaneous adipose tissue (SAT) dysfunction, and increases risk of type 2 diabetes (T2D). First-degree relatives of T2D individuals (FDR) feature restricted adipogenesis, reflecting the detrimental effects of APC senescence earlier in life and rendering FDR more vulnerable to T2D. Epigenetics may contribute to these abnormalities but the underlying mechanisms remain unclear. In previous methylome comparison in APC from FDR and individuals with no diabetes familiarity (CTRL), ZMAT3 emerged as one of the top-ranked senescence-related genes featuring hypomethylation in FDR and associated with T2D risk. Here, we investigated whether and how DNA methylation changes at ZMAT3 promote early APC senescence. APC from FDR individuals revealed increases in multiple senescence markers compared to CTRL. Senescence in these cells was accompanied by ZMAT3 hypomethylation, which caused ZMAT3 upregulation. Demethylation at this gene in CTRL APC led to increased ZMAT3 expression and premature senescence, which were reverted by ZMAT3 siRNA. Furthermore, ZMAT3 overexpression in APC determined senescence and activation of the p53/p21 pathway, as observed in FDR APC. Adipogenesis was also inhibited in ZMAT3-overexpressing APC. In FDR APC, rescue of ZMAT3 methylation through senolytic exposure simultaneously downregulated ZMAT3 expression and improved adipogenesis. Interestingly, in human SAT, aging and T2D were associated with significantly increased expression of both ZMAT3 and the P53 senescence marker. Thus, DNA hypomethylation causes ZMAT3 upregulation in FDR APC accompanied by acquisition of the senescence phenotype and impaired adipogenesis, which may contribute to FDR predisposition for T2D.