Biomedical application of graphitic carbon nitrides: tissue depositionin vivo, induction of reactive oxygen species (ROS) and cell viability in tumor cells.

The urgency for new materials in oncology is immediate. In this study we have developed the g-C3N4, a graphitic-like structure formed by periodically linked tris-s-triazine units. The g-C3N4has been synthesized by a simple and fast thermal process. XRD has shown the formation of the crystalline sheet with a compacted structure. The graphite-like structure and the functional groups have been shown by Raman and FTIR spectroscopy. TEM image and AFM revealed the porous composed of five or six C-N layers stacked. DRS and Photoluminescence analyses confirmed the structure with band gap of 2.87 eV and emission band at 448 nm in different wavelengths excitation conditions. The biological results showed inhibitory effect on cancer cell lines and non-toxic effect in normal cell lines. To the best of our knowledge, this is the first work demonstrating the cytotoxic effects of 2D g-C3N4in a cancer cell line, without any external or synergistic influence. The biodistribution/tissue accumulation showed that g-C3N4present a tendency to accumulation on the lung in the first 2 h, but after 24 h the profile of the biodistribution change and it is found mainly in the liver. Thus, 2D-g-C3N4showed great potential for the treatment of several cancer types.

Extracellular Matrix Derived from High Metastatic Human Breast Cancer Triggers Epithelial-Mesenchymal Transition in Epithelial Breast Cancer Cells through αvß3 Integrin.

Alterations in the composition and architecture of the extracellular matrix (ECM) can influence cancer growth and dissemination. During epithelial-mesenchymal transition (EMT), epithelial cells assume a mesenchymal cell phenotype, changing their adhesion profiles from cell-cell contacts to cell-matrix interactions, contributing to metastasis. Breast cancer cells present at different stages of differentiation, producing distinct ECMs in the same tumor mass. However, the contribution of ECM derived from metastatic tumor cells to EMT is unclear. Here, we showed the mechanisms involved in the interaction of MCF-7, a low-metastatic, epithelial breast cancer cell line, with the ECM produced by a high metastatic breast tumor cell, MDA-MB-231 (MDA-ECM). MDA-ECM induced morphological changes in MCF-7 cells, decreased the levels of E-cadherin, up-regulated mesenchymal markers, and augmented cell migration. These changes were accompanied by the activation of integrin-associated signaling, with increased phosphorylation of FAK, ERK, and AKT and activation canonical TGF-ß receptor signaling, enhancing phosphorylation of SMAD2 and SMAD4 nuclear translocation in MCF-7 cells. Treatment with Kistrin (Kr), a specific ligand of integrin αvß3 EMT induced by MDA-ECM, inhibited TGF-ß receptor signaling in treated MCF-7 cells. Our results revealed that after interaction with the ECM produced by a high metastatic breast cancer cell, MCF-7 cells lost their characteristic epithelial phenotype undergoing EMT, an effect modulated by integrin signaling in crosstalk with TGF-ß receptor signaling pathway. The data evidenced novel potential targets for antimetastatic breast cancer therapies.

Helicobacter pylori urease induces pro-inflammatory effects and differentiation of human endothelial cells: Cellular and molecular mechanism.

BACKGROUND: Helicobacter pylori urease (HPU) is a key virulence factor that enables bacteria to colonize and survive in the stomach. We early demonstrated that HPU, independent of its catalytic activity, induced inflammatory and angiogenic responses in vivo and directly activated human neutrophils to produce reactive oxygen species (ROS). We have investigated the effects of HPU on endothelial cells, focusing on the signaling mechanism involved. METHODS: Monolayers of human microvascular endothelial cells (HMEC-1) were stimulated with HPU (up to 10 nmol/L): Paracellular permeability was accessed through dextran-FITC passage. NO and ROS production was evaluated using intracellular probes. Proteins or mRNA expressions were detected by Western blotting and fluorescence microscopy or qPCR assays, respectively. RESULTS: Treatment with HPU enhanced paracellular permeability of HMEC-1, preceded by VE-cadherin phosphorylation and its dissociation from cell-cell junctions. This caused profound alterations in actin cytoskeleton dynamics and focal adhesion kinase (FAK) phosphorylation. HPU triggered ROS and nitric oxide (NO) production by endothelial cells. Increased intracellular ROS resulted in nuclear factor kappa B (NF-κB) activation and upregulated expression of cyclooxygenase-2 (COX-2), hemeoxygenase-1 (HO-1), interleukin-1ß (IL-1ß), and intercellular adhesion molecule-1 (ICAM-1). Higher ICAM-1 and E-selectin expression was associated with increased neutrophil adhesion on HPU-stimulated HMEC monolayers. The effects of HPU on endothelial cells were dependent on ROS production and lipoxygenase pathway activation, being inhibited by esculetin. Additionally, HPU improved vascular endothelial growth factor receptor 2 (VEGFR-2) expression. CONCLUSION: The data suggest that the pro-inflammatory properties of HPU drive endothelial cell to a ROS-dependent program of differentiation that contributes to the progression of H pylori infection.

Molecular and Cellular Risk Assessment of Healthy Human Cells and Cancer Human Cells Exposed to Nanoparticles.

: Nanodrugs have in recent years been a subject of great debate. In 2017 alone, almost 50 nanodrugs were approved for clinical use worldwide. Despite the advantages related to nanodrugs/nanomedicine, there is still a lack of information regarding the biological safety, as the real behavior of these nanodrugs in the body. In order to better understand these aspects, in this study, we evaluated the effect of polylactic acid (PLA) nanoparticles (NPs) and magnetic core mesoporous silica nanoparticles (MMSN), of 1000 nm and 50 nm, respectively, on human cells. In this direction we evaluated the cell cycle, cytochemistry, proliferation and tubulogenesis on tumor cells lines: from melanoma (MV3), breast cancer (MCF-7, MDA-MB-213), glioma (U373MG), prostate (PC3), gastric (AGS) and colon adenocarcinoma (HT-29) and non-tumor cell lines: from human melanocyte (NGM), fibroblast (FGH) and endothelial (HUVEC), respectively. The data showed that an acute exposure to both, polymeric nanoparticles or MMSN, did not show any relevant toxic effects on neither tumor cells nor non-tumor cells, suggesting that although nanodrugs may present unrevealed aspects, under acute exposition to human cells they are harmless.

Microparticles derived from obese adipose tissue elicit a pro-inflammatory phenotype of CD16+, CCR5+ and TLR8+ monocytes.

Macrophage infiltration into adipose tissue (AT) is a hallmark of the chronic inflammatory response in obesity and is supported by an intense monocyte migration towards AT. Although it has been detected an increased proportion of circulating CD16+ monocyte subsets in obese subjects, the mechanisms underlying this effect and the contribution of these cells to the inflamed profile of obese AT are still poorly understood. We investigated whether factors secreted by human obese omental AT could polarize monocytes to CD16+ enriched phenotype, and how these changes could modify their migratory capacity towards adipose tissue itself. We show that explants of human obese omental AT, obtained during bariatric surgery, released higher levels of MIP1-α, TNFα, leptin and also VEGF, together with increasing amounts of microparticles (MP), when compared to explants of lean subcutaneous AT. A higher content of circulating MP derived from preadipocytes and leukocytes was also detected in plasma of obese subjects. Conditioned media or MP released from obese omental AT increased CD16 and CCR5 expression on CD14+CD16- monocytes and augmented their migratory capacity towards the conditioned media from obese omental AT, itself. This effect was inhibited when MIP1-α was neutralized. Additionally, we demonstrate that MP derived from obese omental AT carry and transfer TLR8 to monocytes, thus triggering an increase in CD16 expression in those cells. Our data shows a positive feedback loop between blood monocytes and obese omental AT, which releases chemotactic mediators and TLR8-enriched MP, thus inducing an up-regulation of CD16+ monocytes, favoring leukocyte infiltration in the obese omental AT.

Circulating cell membrane microparticles transfer heme to endothelial cells and trigger vasoocclusions in sickle cell disease.

Intravascular hemolysis describes the relocalization of heme and hemoglobin (Hb) from erythrocytes to plasma. We investigated the concept that erythrocyte membrane microparticles (MPs) concentrate cell-free heme in human hemolytic diseases, and that heme-laden MPs have a physiopathological impact. Up to one-third of cell-free heme in plasma from 47 patients with sickle cell disease (SCD) was sequestered in circulating MPs. Erythrocyte vesiculation in vitro produced MPs loaded with heme. In silico analysis predicted that externalized phosphatidylserine (PS) in MPs may associate with and help retain heme at the cell surface. Immunohistology identified Hb-laden MPs adherent to capillary endothelium in kidney biopsies from hyperalbuminuric SCD patients. In addition, heme-laden erythrocyte MPs adhered and transferred heme to cultured endothelial cells, inducing oxidative stress and apoptosis. In transgenic SAD mice, infusion of heme-laden MPs triggered rapid vasoocclusions in kidneys and compromised microvascular dilation ex vivo. These vascular effects were largely blocked by heme-scavenging hemopexin and by the PS antagonist annexin-a5, in vitro and in vivo. Adversely remodeled MPs carrying heme may thus be a source of oxidant stress for the endothelium, linking hemolysis to vascular injury. This pathway might provide new targets for the therapeutic preservation of vascular function in SCD.

Priming Endothelial Cells With a Melanoma-Derived Extracellular Matrix Triggers the Activation of αvß3/VEGFR2 Axis.

The unique composition of tumor-produced extracellular matrix (ECM) can be a determining factor in changing the profile of endothelial cells in the tumor microenvironment. As the main receptor for ECM proteins, integrins can activate a series of signaling pathways related to cell adhesion, migration, and differentiation of endothelial cells that interact with ECM proteins. We studied the direct impact of the decellularized ECM produced by a highly metastatic human melanoma cell line (MV3) on the activation of endothelial cells and identified the intracellular signaling pathways associated with cell differentiation. Our data show that compared to the ECM derived from a human melanocyte cell line (NGM-ECM), ECM produced by a melanoma cell line (MV3-ECM) is considerably different in ultrastructural organization and composition and possesses a higher content of tenascin-C and laminin and a lower expression of fibronectin. When cultured directly on MV3-ECM, endothelial cells change morphology and show increased adhesion, migration, proliferation, and tubulogenesis. Interaction of endothelial cells with MV3-ECM induces the activation of integrin signaling, increasing FAK phosphorylation and its association with Src, which activates VEGFR2, potentiating the receptor response to VEGF. The blockage of αvß3 integrin inhibited the FAK-Src association and VEGFR activation, thus reducing tubulogenesis. Together, our data suggest that the interaction of endothelial cells with the melanoma-ECM triggers integrin-dependent signaling, leading to Src pathway activation that may potentiate VEGFR2 activation and up-regulate angiogenesis. J. Cell. Physiol. 231: 2464-2473, 2016. © 2016 Wiley Periodicals, Inc.

Reactive oxygen species involved in apoptosis induction of human respiratory epithelial (A549) cells by Streptococcus agalactiae.

Streptococcus agalactiae (Group B Streptococcus; GBS) is an important pathogen and is associated with pneumonia, sepsis and meningitis in neonates and adults. GBS infections induce cytotoxicity of respiratory epithelial cells (A549) with generation of reactive oxygen species (ROS) and loss of mitochondrial membrane potential (ψm). The apoptosis of A549 cells by GBS was dependent on the activation of caspase-3 and caspase-9 with increased pro-apoptotic Bim and Bax molecules and decreased Bcl-2 pro-survival protein. Treatment of infected A549 cells with ROS inhibitors (diphenyleniodonium chloride or apocynin) prevented intracellular ROS production and apoptosis. Consequently, oxidative stress is included among the cellular events leading to apoptosis during GBS human invasive infections.

ExoU-induced redox imbalance and oxidative stress in airway epithelial cells during Pseudomonas aeruginosa pneumosepsis.

ExoU is a potent proinflammatory toxin produced by Pseudomonas aeruginosa, a major agent of severe lung infection and sepsis. Because inflammation is usually associated with oxidative stress, we investigated the effect of ExoU on free radical production and antioxidant defense mechanisms during the course of P. aeruginosa infection. In an experimental model of acute pneumonia, ExoU accounted for increased lipid peroxidation in mice lungs as soon as 3 h after intratracheal instillation of PA103 P. aeruginosa strain. The contribution of airway cells to the generation of a redox imbalance was assessed by in vitro tests carried out with A549 airway epithelial cells. Cultures infected with the ExoU-producing PA103 P. aeruginosa strain produced significantly increased concentrations of lipid hydroperoxides, 8-isoprostane, reactive oxygen intermediates, peroxynitrite and nitric oxide (NO), when compared to cells infected with exoU-deficient mutants. Overproduction of NO by PA103-infected cells likely resulted from overexpression of both inducible and endothelial NO synthase isoforms. PA103 infection was also associated with a significantly increased activity of superoxide dismutase (SOD) and decreased levels of reduced glutathione (GSH), a major antioxidant compound. Our findings unveil another potential mechanism of tissue damage during infection by ExoU-producing P. aeruginosa strains.

Rapid NOS-1-derived nitric oxide and peroxynitrite formation act as signaling agents for inducible NOS-2 expression in vascular smooth muscle cells.

Septic vascular dysfunction is characterized by hypotension and hyporeactivity to vasoconstrictors and nitric oxide (NO), reactive oxygen species and peroxynitrite have a prominent role in this condition. However, the mechanism whereby the vascular dysfunction is initiated is poorly understood. Based on previous studies of our group and the literature,we hypothesize that constitutive nitric oxide synthases (c-NOS) and peroxynitrite may play a role in the development of septic vascular dysfunction. Bacterial lipopolysaccharide (LPS) and interferon-γ (IFN) were used to stimulate rat aorta smooth muscle cells (A7r5) and rat aorta slices. This stimulation led to a rapid (within minutes) production of NO and superoxide anion, which led to peroxynitrite formation. When this rapid initial burst was reduced, through the inhibition of c-NOS and NADPH oxidases (NOX) or the scavenging of NO and superoxide the NF-κB activation, NOS-2 expression and nitrite production were significantly attenuated. Although vascular smooth muscle cells express both c-NOS isoforms, gene knockdown revealed that only NOS-1-dependent NO and peroxynitrite formation are important for the later NOS-2 expression. Similar findings were obtained by knockdown NOX-1 gene, one source of superoxide for peroxynitrite formation. Taking together, we show that smooth muscle cell activation by LPS/IFN leads to a rapid formation of NOS-1-derived NO and NOX-1-derived superoxide, forming peroxynitrite; and that this species act as a trigger for NOS-2 expression through NF-κB activation. Therefore, our findings suggest a critical role for NOS-1 and NOX-1 in the initiation of the vascular dysfunction associated with sepsis and septic shock.

Unlocking the Secrets of Adipose Tissue: How an Obesity-Associated Secretome Promotes Osteoblast Dedifferentiation via TGF-ß1 Signaling, Paving the Path to an Adipogenic Phenotype.

BACKGROUND: Obesity poses a significant global health challenge, given its association with the excessive accumulation of adipose tissue (AT) and various systemic disruptions. Within the adipose microenvironment, expansion and enrichment with immune cells trigger the release of inflammatory mediators and growth factors, which can disrupt tissues, including bones. While obesity's contribution to bone loss is well established, the direct impact of obese AT on osteoblast maturation remains uncertain. This study aimed to explore the influence of the secretomes from obese and lean AT on osteoblast differentiation and activity. METHODS: SAOS-2 cells were exposed to the secretomes obtained by culturing human subcutaneous AT from individuals with obesity (OATS) or lean patients, and their effects on osteoblasts were evaluated. RESULTS: In the presence of the OATS, mature osteoblasts underwent dedifferentiation, showing an increased proliferation accompanied by a morphological shift towards a mesenchymal phenotype, with detrimental effects on osteogenic markers and the calcification capacity. Concurrently, the OATS promoted the expression of mesenchymal and adipogenic markers, inducing the formation of cytoplasmic lipid droplets in SAOS-2 cells exposed to an adipogenic differentiation medium. Additionally, TGF-ß1 emerged as a key mediator of these effects, as the OATS was enriched with this growth factor. CONCLUSIONS: Our findings demonstrate that obese subcutaneous AT promotes the dedifferentiation of osteoblasts and increases the adipogenic profile in these cells.

Heme modulates intestinal epithelial cell activation: involvement of NADPHox-derived ROS signaling.

In many gut chronic inflammatory conditions, intestinal epithelium (IE) is deprived of the protection of the mucus secreted by IE-specialized cells. In these events, bleeding and subsequent lysis of erythrocytes are common. This may lead to the release of high amounts of heme in the intestinal lumen, which interacts with IE. Previous works from our group have shown that heme itself is a proinflammatory molecule, activating a number of phlogistic signaling events in a nicotinamide adenine dinucleotide phosphate oxidase (NADPHox)-dependent manner. In this study, we aim to evaluate the effects of heme upon a well-established nontransformed small intestine epithelial cell lineage (IEC 6). Our results show that free heme evokes intracellular reactive oxygen species (ROS) production by IEC 6 cells, which is inhibited both by pharmacological inhibition with diphenyleneiodonium (10 µM), a NADPHox inhibitor, and small interfering RNA-mediated suppression of NOX1, a constitutive NADPHox isoform present in intestinal epithelial cells. Focal adhesion kinase phosphorylation and actin cytoskeleton polymerization are also induced by heme in a NADPHox-dependent manner. Heme increases monolayer permeability and redistributes key modulators of cell-cell adhesion as zona occludens-1 and E-cadherin proteins via NADPHox signaling. Heme promotes IEC 6 cell migration and proliferation, phenomena also regulated by NADPHox-derived ROS. Heme, in NADPHox-activating concentrations, is able to induce mRNA expression of IL-6, a cytokine implicated in inflammatory and tumorigenic responses. These data indicate a prominent role for heme-derived signaling in the pathophysiology of intestinal mucosa dysfunction and address an important role of NADPHox activity on the pathogenesis of intestinal inflammatory conditions.

Leukotriene B(4) inhibits neutrophil apoptosis via NADPH oxidase activity: redox control of NF-κB pathway and mitochondrial stability.

Leukotriene B(4), an arachidonic acid-derived lipid mediator, is a known proinflammatory agent that has a direct effect upon neutrophil physiology, inducing reactive oxygen species generation by the NADPH oxidase complex and impairing neutrophil spontaneous apoptosis, which in turn may corroborate to the onset of chronic inflammation. Despite those facts, a direct link between inhibition of neutrophil spontaneous apoptosis and NADPH oxidase activation by leukotriene B(4) has not been addressed so far. In this study, we aim to elucidate the putative role of NADPH oxidase-derived reactive oxygen species in leukotriene B(4)-induced anti-apoptotic effect. Our results indicate that NADPH oxidase-derived reactive oxygen species are critical to leukotriene B(4) pro-survival effect on neutrophils. This effect also relies on redox modulation of nuclear factor kappaB signaling pathway. We have also observed that LTB(4)-induced Bad degradation and mitochondrial stability require NADPH oxidase activity. All together, our results strongly suggest that LTB(4)-induced anti-apoptotic effect in neutrophils occurs in a reactive oxygen species-dependent manner. We do believe that a better knowledge of the molecular mechanisms underlying neutrophil spontaneous apoptosis may contribute to the development of more successful strategies to control chronic inflammatory conditions such as rheumatoid arthritis.

Bone marrow mononuclear cell transplantation increases metalloproteinase-9 and 13 and decreases tissue inhibitors of metalloproteinase-1 and 2 expression in the liver of cholestatic rats.

Liver fibrosis results from chronic injury followed by activation of macrophages and fibrogenic cells like myofibroblasts and activated hepatic stellate cells. These fibrogenic cells express α-smooth muscle actin (α-SMA) and produce excessive extracellular matrix (ECM), with disorganization and loss of function of hepatic parenchyma. It is known that increased levels of metalloproteinases (MMPs) in liver fibrosis are associated with reduction of the pathologic ECM and fibrosis resolution. Recently, it has been shown that bone marrow mononuclear cells (BMMNCs) may reduce collagen and α-SMA expression, and ameliorate liver function in cholestatic rats. Therefore, this study aimed to analyze MMP-2, MMP-9 and MMP-13, and tissue inhibitors of MMPs (TIMPs)-1 and TIMP-2 in the liver of cholestatic rats transplanted with BMMNC. Animals were divided into normal rats, cholestatic rats obtained after 14 and 21 days of bile duct ligation (BDL), and rats obtained after 14 days of BDL that received BMMNCs and were killed after 7 days. MMP and TIMP expression was assessed by Western blotting, along with α-SMA, CD68 and CD11b expression by confocal microscopy. Western blotting analysis showed that 14-day BDL animals had significantly reduced amounts of MMP-2 and MMP-13, but increased amounts of MMP-9 compared to normal rats. After 21 days of BDL, overall MMP amounts were decreased and TIMPs were increased. BMMNC transplantation significantly increased MMP-9 and MMP-13, and decreased TIMP expression. Increased MMP activity was confirmed by zymography. MMP-9 and MMP-13 were expressed by macrophages near fibrotic septa, suggesting BMMNC may stimulate MMP production in fibrotic livers, contributing to ECM degradation and hepatic regeneration.

Leukotriene B4 mediates neutrophil migration induced by heme.

High concentrations of free heme found during hemolytic events or cell damage leads to inflammation, characterized by neutrophil recruitment and production of reactive oxygen species, through mechanisms not yet elucidated. In this study, we provide evidence that heme-induced neutrophilic inflammation depends on endogenous activity of the macrophage-derived lipid mediator leukotriene B(4) (LTB(4)). In vivo, heme-induced neutrophil recruitment into the peritoneal cavity of mice was attenuated by pretreatment with 5-lipoxygenase (5-LO) inhibitors and leukotriene B(4) receptor 1 (BLT1) receptor antagonists as well as in 5-LO knockout (5-LO(-/-)) mice. Heme administration in vivo increased peritoneal levels of LTB(4) prior to and during neutrophil recruitment. Evidence that LTB(4) was synthesized by resident macrophages, but not mast cells, included the following: 1) immuno-localization of heme-induced LTB(4) was compartmentalized exclusively within lipid bodies of resident macrophages; 2) an increase in the macrophage population enhanced heme-induced neutrophil migration; 3) depletion of resident mast cells did not affect heme-induced LTB(4) production or neutrophil influx; 4) increased levels of LTB(4) were found in heme-stimulated peritoneal cavities displaying increased macrophage numbers; and 5) in vitro, heme was able to activate directly macrophages to synthesize LTB(4). Our findings uncover a crucial role of LTB(4) in neutrophil migration induced by heme and suggest that beneficial therapeutic outcomes could be achieved by targeting the 5-LO pathway in the treatment of inflammation associated with hemolytic processes.

Nickel induces apoptosis in human neutrophils.

Nickel is an ubiquitous transition metal that is industrially applied in many forms, which inevitably leads to a high degree of occupational and environmental exposure. Over-exposure to nickel can produce a variety of adverse effects on human health, including allergy and lung and nasal cancers. In the present study, it is demonstrated, for the first time, that nickel [(Ni(II)] (as a nickel nitrate salt) at concentrations that may be attained in vivo, induces neutrophils' apoptosis by the intrinsic pathway. The use of diphenyleneiodonium, a NADPH oxidase inhibitor, delayed Ni(II)-induced apoptosis, suggesting that NADPH oxidase-derived reactive oxygen species and subsequent signaling could contribute to this event. This is an important finding since increased apoptosis mediated by nickel may disrupt the physiological activities of neutrophils, with potential impact in its immunological and antimicrobial role.

Proinflammatory responses of heme in alveolar macrophages: repercussion in lung hemorrhagic episodes.

Clinical and experimental observations have supported the notion that free heme released during hemorrhagic and hemolytic episodes may have a major role in lung inflammation. With alveolar macrophages (AM) being the main line of defense in lung environments, the influence of free heme on AM activity and function was investigated. We observed that heme in a concentration range found during hemolytic episodes (3-30 µM) elicits AM to present a proinflammatory profile, stimulating reactive oxygen species (ROS) and nitric oxide (NO) generation and inducing IL-1ß, IL-6, and IL-10 secretion. ROS production is NADPH oxidase-dependent, being inhibited by DPI and apocynin, and involves p47 subunit phosphorylation. Furthermore, heme induces NF- κB nuclear translocation, iNOS, and also HO-1 expression. Moreover, AM stimulated with free heme show enhanced phagocytic and bactericidal activities. Taken together, the data support a dual role for heme in the inflammatory response associated with lung hemorrhage, acting as a proinflammatory molecule that can either act as both an adjuvant of the innate immunity and as an amplifier of the inflammatory response, leading tissue injury. The understanding of heme effects on pulmonary inflammatory processes can lead to the development of new strategies to ameliorate tissue damage associated with hemorrhagic episodes.

Cellular and Molecular Mechanisms Associating Obesity to Bone Loss.

Obesity is an alarming disease that favors the upset of other illnesses and enhances mortality. It is spreading fast worldwide may affect more than 1 billion people by 2030. The imbalance between excessive food ingestion and less energy expenditure leads to pathological adipose tissue expansion, characterized by increased production of proinflammatory mediators with harmful interferences in the whole organism. Bone tissue is one of those target tissues in obesity. Bone is a mineralized connective tissue that is constantly renewed to maintain its mechanical properties. Osteoblasts are responsible for extracellular matrix synthesis, while osteoclasts resorb damaged bone, and the osteocytes have a regulatory role in this process, releasing growth factors and other proteins. A balanced activity among these actors is necessary for healthy bone remodeling. In obesity, several mechanisms may trigger incorrect remodeling, increasing bone resorption to the detriment of bone formation rates. Thus, excessive weight gain may represent higher bone fragility and fracture risk. This review highlights recent insights on the central mechanisms related to obesity-associated abnormal bone. Publications from the last ten years have shown that the main molecular mechanisms associated with obesity and bone loss involve: proinflammatory adipokines and osteokines production, oxidative stress, non-coding RNA interference, insulin resistance, and changes in gut microbiota. The data collection unveils new targets for prevention and putative therapeutic tools against unbalancing bone metabolism during obesity.

Maxadilan, the Lutzomyia longipalpis vasodilator, drives plasma leakage via PAC1-CXCR1/2-pathway.

Experiments were designed to determine if the vasodilatory peptides maxadilan and pituitary adenylate cyclase-activating peptide (PACAP-38) may cause plasma leakage through activation of leukocytes and to what extent these effects could be due to PAC1 and CXCR1/2 receptor stimulation. Intravital microscopy of hamster cheek pouches utilizing FITC-dextran and rhodamine, respectively, as plasma and leukocyte markers was used to measure arteriolar diameter, plasma leakage and leukocyte accumulation in a selected area (5mm(2)) representative of the hamster cheek pouch microcirculation. Our studies showed that the sand fly vasodilator maxadilan and PACAP-38 induced arteriolar dilation, leukocyte accumulation and plasma leakage in postcapillary venules. The recombinant mutant of maxadilan M65 and an antagonist of CXCR1/2 receptors, reparixin, and an inhibitor of CD11b/CD18 up-regulation, ropivacaine, inhibited all these effects as induced by maxadilan. Dextran sulfate, a complement inhibitor with heparin-like anti-inflammatory effects, inhibited plasma leakage and leukocyte accumulation but not arteriolar dilation as induced by maxadilan and PACAP-38. In vitro studies with isolated human neutrophils showed that maxadilan is a potent stimulator of neutrophil migration comparable with fMLP and leukotriene B(4) and that M65 and reparixin inhibited such migration. The data suggest that leukocyte accumulation and plasma leakage induced by maxadilan involves a mechanism related to PAC1- and CXCR1/2-receptors on leukocytes and endothelial cells.