Semaglutide modulates prothrombotic and atherosclerotic mechanisms, associated with epicardial fat, neutrophils and endothelial cells network.

BACKGROUND: Obesity has increased in recent years with consequences on diabetes and other comorbidities. Thus, 1 out of 3 diabetic patients suffers cardiovascular disease (CVD). The network among glucose, immune system, endothelium and epicardial fat has an important role on pro-inflammatory and thrombotic mechanisms of atherogenesis. Since semaglutide, long-acting glucagon like peptide 1- receptor agonist (GLP-1-RA), a glucose-lowering drug, reduces body weight, we aimed to study its effects on human epicardial fat (EAT), aortic endothelial cells and neutrophils as atherogenesis involved-cardiovascular cells. METHODS: EAT and subcutaneous fat (SAT) were collected from patients undergoing cardiac surgery. Differential glucose consumption and protein cargo of fat-released exosomes, after semaglutide or/and insulin treatment were analyzed by enzymatic and TripleTOF, respectively. Human neutrophils phenotype and their adhesion to aortic endothelial cells (HAEC) or angiogenesis were analyzed by flow cytometry and functional fluorescence analysis. Immune cells and plasma protein markers were determined by flow cytometry and Luminex-multiplex on patients before and after 6 months treatment with semaglutide. RESULTS: GLP-1 receptor was expressed on fat and neutrophils. Differential exosomes-protein cargo was identified on EAT explants after semaglutide treatment. This drug increased secretion of gelsolin, antithrombotic protein, by EAT, modulated CD11b on neutrophils, its migration and endothelial adhesion, induced by adiposity protein, FABP4, or a chemoattractant. Monocytes and neutrophils phenotype and plasma adiposity, stretch, mesothelial, fibrotic, and inflammatory markers on patients underwent semaglutide treatment for 6 months showed a 20% reduction with statistical significance on FABP4 levels and an 80% increase of neutrophils-CD88. CONCLUSION: Semaglutide increases endocrine activity of epicardial fat with antithrombotic properties. Moreover, this drug modulates the pro-inflammatory and atherogenic profile induced by the adiposity marker, FABP4, which is also reduced in patients after semaglutide treatment.

Synergism between obesity and HFpEF on neutrophils phenotype and its regulation by adipose tissue-molecules and SGLT2i dapagliflozin.

The adiposity invokes innate immune activity, coronary microvascular dysfunction and consequently heart failure preserved ejection fraction (HFpEF). Our aim was to study the neutrophils profile on obesity and cardiovascular disease and its regulation by adipose tissue-secretome and dapagliflozin. We have isolated neutrophils from patients undergoing open heart surgery (19 women and 51 men). Its migration activity was performed with culture-transwell, transcriptional studies of proteolytic enzymes, adhesion molecules or receptors were analysed by real-time PCR and proteomics (from 20 patients) analysis by TripleTOF mass spectrometer. Differentiated HL-60 (dHL-60) was used as a preclinical model on microfluidic for endothelial cells attaching assays and genes regulation with epicardial and subcutaneous fat secretomes from patients (3 women and 9 men) or dapagliflozin 1-10 µM treatments. The transcriptional and proteomics studies have determined higher levels of adhesion molecules in neutrophils from patients with obesity. The adhesion molecule CD11b levels were higher in those patients with the combined obesity and HFpEF factors (1.70 ± 0.06 a.u. without obesity, 1.72 ± 0.04 a.u. obesity or HFpEF without obesity and 1.79 ± 0.08 a.u. obesity and HFpEF; p < .01). While fat-secretome induces its upregulation, dapagliflozin can modulated it. Because CD11b upregulation is associated with higher neutrophils migration and adhesion into endothelial cells, dapagliflozin might modulate this mechanism on patients with obesity and HFpEF.

Naturally presented HLA class I-restricted epitopes from the neurotrophic factor S100-ß are targets of the autoimmune response in type 1 diabetes.

Type 1 diabetes (T1D) results from the destruction of pancreatic ß-cells by the immune system, and CD8+ T lymphocytes are critical actors in this autoimmune response. Pancreatic islets are surrounded by a mesh of nervous cells, the peri-insular Schwann cells, which are also targeted by autoreactive T lymphocytes and express specific antigens, such as the neurotrophic factor S100-ß. Previous work has shown increased proliferative responses to whole S100-ß in both human T1D patients and the nonobese diabetic (NOD) mouse model. We describe for the first time naturally processed and presented epitopes (NPPEs) presented by class I human leukocyte antigen-A*02:01 (A2.1) molecules derived from S100-ß. These NPPEs triggered IFN-γ responses more frequently in both newly diagnosed and long-term T1D patients compared with healthy donors. Furthermore, the same NPPEs are recognized during the autoimmune response leading to diabetes in A2.1-transgenic NOD mice as early as 4 wk of age. Interestingly, when these NPPEs are used to prevent diabetes in this animal model, an acceleration of the disease is observed together with an exacerbation in insulitis and an increase in S100-ß-specific cytotoxicity in vaccinated animals. Whether these can be used in diabetes prevention needs to be carefully evaluated in animal models before use in future clinical assays.-Calviño-Sampedro, C., Gomez-Tourino, I., Cordero, O. J., Reche, P. A., Gómez-Perosanz, M., Sánchez-Trincado, J. L., Rodríguez, M. Á., Sueiro, A. M., Viñuela, J. E., Calviño, R. V. Naturally presented HLA class I-restricted epitopes from the neurotrophic factor S100-ß are targets of the autoimmune response in type 1 diabetes.

Action of obestatin in skeletal muscle repair: stem cell expansion, muscle growth, and microenvironment remodeling.

The development of therapeutic strategies for skeletal muscle diseases, such as physical injuries and myopathies, depends on the knowledge of regulatory signals that control the myogenic process. The obestatin/GPR39 system operates as an autocrine signal in the regulation of skeletal myogenesis. Using a mouse model of skeletal muscle regeneration after injury and several cellular strategies, we explored the potential use of obestatin as a therapeutic agent for the treatment of trauma-induced muscle injuries. Our results evidenced that the overexpression of the preproghrelin, and thus obestatin, and GPR39 in skeletal muscle increased regeneration after muscle injury. More importantly, the intramuscular injection of obestatin significantly enhanced muscle regeneration by simulating satellite stem cell expansion as well as myofiber hypertrophy through a kinase hierarchy. Added to the myogenic action, the obestatin administration resulted in an increased expression of vascular endothelial growth factor (VEGF)/vascular endothelial growth factor receptor 2 (VEGFR2) and the consequent microvascularization, with no effect on collagen deposition in skeletal muscle. Furthermore, the potential inhibition of myostatin during obestatin treatment might contribute to its myogenic action improving muscle growth and regeneration. Overall, our data demonstrate successful improvement of muscle regeneration, indicating obestatin is a potential therapeutic agent for skeletal muscle injury and would benefit other myopathies related to muscle regeneration.

Impaired adipogenesis and insulin resistance in epicardial fat-mesenchymal cells from patients with cardiovascular disease.

The thickness of epicardial adipose tissue (EAT), which is an inflammatory source for coronary artery disease (CAD), correlates with insulin resistance. One trigger factor is impaired adipogenesis. Here, our aim was to clarify the underlying mechanisms of insulin resistance on EAT-mesenchymal cells (MC). EAT and subcutaneous adipose tissue (SAT) were collected from 19 patients who were undergoing heart surgery. Their dedifferentiated adipocytes (DAs) and/or MCs were cultured. After the induction of adipogenesis or stimulation with insulin, the expression of adipokines was analyzed using real-time polymerase chain reaction (PCR). Colorimetric assays were performed to measure glucose levels and proliferation rate. Proteins modifications were detected via the proteomic approach and Western blot. Our results showed lower adipogenic ability in EAT-MCs than in SAT-MCs. Maximum adiponectin levels were reached within 28-35 days of exposure to adipogenic inducers. Moreover, the adipogenesis profile in EAT-MCs was dependent on the patients' clinical characteristics. The low adipogenic ability of EAT-MCs might be associated with an insulin-resistant state because chronic insulin treatment reduced the inflammatory cytokine expression levels, improved the glucose consumption, and increased the post-translational modifications (PTMs) of the glycolytic enzyme phosphoglycerate mutase 1 (PGAM1). We found lower adipogenic ability in EAT-MCs than in SAT-MCs. This lower ability level was dependent on gender and the presence of diabetes, obesity, and CAD. Low adipogenesis ability and insulin resistance in EAT-MCs might shed light on the association between EAT dysfunction and cardiovascular disease.

Distinctive CD26 Expression on CD4 T-Cell Subsets.

Immune system CD4 T-cells with high cell-surface CD26 expression show anti-tumoral properties. When engineered with a chimeric antigen receptor (CAR), they incite strong responses against solid cancers. This subset was originally associated to human CD4 T helper cells bearing the CD45R0 effector/memory phenotype and later to Th17 cells. CD26 is also found in soluble form (sCD26) in several biological fluids, and its serum levels correlate with specific T cell subsets. However, the relationship between glycoprotein sCD26 and its dipeptidyl peptidase 4 (DPP4) enzymatic activity, and cell-surface CD26 expression is not well understood. We have studied ex vivo cell-surface CD26 and in vitro surface and intracellular CD26 expression and secretome's sCD26 in cultured CD4 T cells under different polarization conditions. We show that most human CD26negative CD4 T cells in circulating lymphocytes are central memory (TCM) cells while CD26high expression is present in effector Th1, Th2, Th17, and TEM (effector memory) cells. However, there are significant percentages of Th1, Th2, Th17, and Th22 CD26 negative cells. This information may help to refine the research on CAR-Ts. The cell surface CD45R0 and CD26 levels in the different T helper subsets after in vitro polarization resemble those found ex vivo. In the secretomes of these cultures there was a significant amount of sCD26. However, in all polarizations, including Th1, the levels of sCD26 were lower (although not significantly) compared to the Th0 condition (activation without polarization). These differences could have an impact on the various physiological functions proposed for sCD26/DPP4.

Evaluation of Virola oleifera activity in musculoskeletal pathologies: Inhibition of human multiple myeloma cells proliferation and combination therapy with dexamethasone or bortezomib.

ETHNOPHARMACOLOGICAL RELEVANCE: Virola oleifera (Schott) A.C. Smith, Myristicaceae, has been widely used in traditional medicine in Brazil to treat rheumatic pain, joint tumours, skin diseases, halitosis, bronchial asthma, haemorrhoids, and intestinal worms. Recently, research data showed the antioxidant properties in several oxidative stress-related models. However, there is no experimental evidence supporting its potential use in managing rheumatic diseases and bone malignancies. AIMS OF THE STUDY: To evaluate the therapeutic potential of the resin from Virola oleifera in joint and bone diseases, namely arthritis, osteosarcoma, chondrosarcoma, and multiple myeloma. MATERIALS AND METHODS: To determine Virola oleifera resin (VO) effects on arthritis-associated inflammation and cartilage degradation, the LPS-induced NO production, and mRNA and protein expression of ADAMTS5, MMP13, COL2, and ACAN, were evaluated in chondrocytes (ATDC5 and TC28 cell lines). The cytotoxic effects of VO (0.05-50 µg/ml) on multiple myeloma (ARH-77), osteosarcoma (SAOS-2), and chondrosarcoma (SW-1353) cell lines were analysed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The VO effects, combined with dexamethasone or bortezomib, were evaluated in a multiple myeloma cell line. The mechanisms of VO, alone or in combination with bortezomib, were determined by cell cycle analysis through flow cytometry, while expression levels of p-Akt/Akt, p-ERK/ERK, p-p38/p38 MAPK, Bax, Bcl-2, and cleaved-caspase-3/caspase-3 proteins by Western blot. RESULTS: VO had no significant effect on LPS-induced NO production in chondrocytes at non-cytotoxic concentrations. VO treatment diminished the mRNA levels of metalloproteinases and ECM components; however, any significant effect was observed on the protein expression levels. The cell viability of a multiple myeloma cell line was strongly reduced by VO treatment in a dose- and time-dependent manner, while osteosarcoma and chondrosarcoma cell lines viability was significantly affected only by the highest dose assessed. In multiple myeloma cells, VO leads to G2/M cell cycle arrest. Furthermore, it synergizes with dexamethasone by increasing cell toxicity. Finally, VO reverts bortezomib activity by counteracting ERK1/2, Bax, and caspase-3 activation. CONCLUSIONS: The current work supports the ethnopharmacological use of Virola oleifera (Schott) A.C. Smith in bone and joint diseases, but there is no evidence for the amelioration of arthritis-associated inflammatory or catabolic processes. Our data also supports the potential use of Virola oleifera as adjuvant therapy to optimize the pharmacologic effects of current chemotherapeutic drugs. However, possible herb-drug interactions should be considered before clinical application.

Hypoploidy defines patients with poor prognosis in breast cancer.

The clinicopathological features currently used in breast cancer prognosis often fail to characterize the clinical heterogeneity of the disease accurately. Our study is aimed to investigate the predictive value of DNA flow cytometry in breast cancer. Previously untreated breast carcinoma samples (584) were snap frozen for flow-cytometry. Tumors were classified into three DNA index (DI) categories: i) tumors showing a DI =0.96-1.15 (diploid and near-diploid); ii) tumors with a DI >or=1.16 (hyperdiploid, tetraploid, multiploid and/or those with more than one diploid population); and iii) tumors with a DI

The human VGF-derived bioactive peptide TLQP-21 binds heat shock 71 kDa protein 8 (HSPA8)on the surface of SH-SY5Y cells.

VGF (non-acronymic)is a secreted chromogranin/secretogranin that gives rise to a number of bioactive peptides by a complex proteolysis mechanism. VGF-derived peptides exert an extensive array of biological effects in energy metabolism, mood regulation, pain, gastric secretion function, reproduction and, perhaps, cancer. It is therefore surprising that very little is known about receptors and binding partners of VGF-derived peptides and their downstream molecular mechanisms of action. Here, using affinity chromatography and mass spectrometry-based protein identification, we have identified the heat shock cognate 71 kDa protein A8 (HSPA8)as a binding partner of human TLQP-21 on the surface of human neuroblastomaSH-SY5Y cells. Binding of TLQP-21 to membrane associated HSPA8 in live SH-SY5Y cells was further supported by cross-linking to live cells. Interaction between HSPA8 and TLQP-21 was confirmed in vitro by label-free Dynamic Mass Redistribution (DMR) studies. Furthermore, molecular modeling studies show that TLQP-21 can be docked into the HSPA8 peptide binding pocket. Identification of HSPA8 as a cell surface binding partner of TLQP-21 opens new avenues to explore the molecular mechanisms of its physiological actions, and of pharmacological modulation thereof.

Non classical Monocytes Levels, Increased by Subcutaneous Fat-Secretome, Are Associated with Less Rehospitalization after Heart Failure Admission.

Differential monocyte subsets are increased in obesity and heart failure (HF). We studied their role as predictors of rehospitalization for HF and their regulation by adipose tissue. Monocyte subsets and body fat composition were determined from 136 patients at the discharge after HF admission. Regulation of monocytes by SAT secretomes from obese/non-obese patients with HF was studied in a cell culture method. Proteomic analysis of secretome SAT was performed by LC-MALDI TOF/TOF. High CD14-CD16+ monocyte levels indicated less rehospitalization for HF (p = 0.018). SAT secretomes from obese patients increased the CD14-CD16+monocytes (11.8 ± 5.3 vs 3.9 ± 2.6%; p < 0.01). Differential proteins were determined between obese and non-obese patients with HF. High levels of CD14-CD16+ monocytes are associated with less rehospitalization for HF. This phenotype is upregulated by SAT secretome from obese patients with HF. This mechanism might help us to understand the obesity paradox in HF.

Orosomucoid as prognosis factor associated with inflammation in acute or nutritional status in chronic heart failure.

BACKGROUND: Inflammation and nutritional state are involved in the pathogenesis of heart failure (HF). OBJECTIVE: To study the contribution of alpha-1-acid-glycoprotein (AGP) to these factors and its prognostic value in acute (AHF) or chronic HF (CHF). METHODS: The observational study has included 147 patients (mean age 70years, 62% men) admitted to a cardiology department for HF and followed-up for an average 326.6±140.8days. Blood AGP values were measured by Enzyme-Linked ImmunoSorbent Assay. Monocytes subsets were determined with CD14 and CD16 antibodies by flow cytometry and body composition was measured by dual-energy X-ray absorptiometry. The regulation of tumor necrosis factor (TNF-α) and leptin by AGP in epicardial adipose tissue (EAT) were analyzed by real time polymerase chain reaction. RESULTS: High AGP, that was associated with CD14+CD16+ monocytes, and proBNP levels at the discharge were indicators of rehospitalization for HF in AHF patients. However, low AGP levels determined a worse nutritional state in CHF patients. The leptin levels were downregulated by high AGP concentration in epicardial fat. CONCLUSION: AGP is a dual indicator in HF because high levels are predictors of adverse outcomes in AHF but low levels are related to the worse nutritional status in CHF. The regulation of leptin by AGP in epicardial fat might suggest a new pathway as protective mechanism in CHF.

CD26 Expression on T Helper Populations and sCD26 Serum Levels in Patients with Rheumatoid Arthritis.

We studied dipeptidyl peptidase IV (DPP-IV, CD26) expression in different T helper cells and serum soluble DPP-IV/sCD26 levels in rheumatoid arthritis (RA) patients, correlated these with disease activity score (DAS), and examined how they were affected by different therapies, conventional or biological (anti-TNF, anti-CD20 and anti-IL6R or Ig-CTLA4). The percentage of CD4+CD45R0+CD26- cells was greatly reduced in patients (up to 50%) when compared with healthy subjects. Three other subsets of CD4 cells, including a CD26high Th1-associated population, changed variably with therapies. Data from these subsets (frequency and staining density) significantly correlated with DAS28 or DAS28 components but different in each group of patients undergoing the different therapies. Th17 and Th22 subsets were implicated in RA as independent CCR4+ and CCR4- populations each, with distinct CD26 expression, and were targeted with varying efficiency by each therapy. Serum DPP-IV activity rather than sCD26 levels was lower in RA patients compared to healthy donors. DPP-IV and sCD26 serum levels were found related to specific T cell subsets but not to disease activity. We conclude that, according to their CD26 expression, different cell subsets could serve to monitor RA course, and an uncharacterized T helper CD26- subset, not targeted by therapies, should be monitored for early diagnosis.

Leptin does not induce hypertrophy, cell cycle alterations, or production of MCP-1 in cultured rat and mouse cardiomyocytes.

BACKGROUND: Leptin has been proposed as an important mediator in cardiovascular pathophysiology. Patients with congestive heart failure (CHF) present high plasma leptin levels. CHF is generally preceded by myocardial remodeling involving hypertrophy, necrosis, and apoptosis. AIM: To investigate whether leptin causes hypertrophy or cell cycle alterations, or induces MCP-1 synthesis in cardiomyocytes. METHODS: Primary cultures of neonatal rat cardiomyocytes (PC) and murine cell line HL-1 were used. RT-PCR was used to identify Ob-Rb gene expression. Metabolic activity and proliferation of cardiomyocytes was studied using MTT and BrdU uptake, while apoptosis was assayed with Hoechst dye vital staining and flow cytometry. Measurement of the cell surface area was used to determine hypertrophy. MCP-1 levels were measured by ELISA. RESULTS: RT-PCR showed Ob-Rb mRNA expression in HL-1 cells. Exposure to leptin induced no changes in metabolic activity, proliferation, and apoptotic rates, and did not alter cell cycle in cardiomyocytes. Leptin did not increase cell size of cardiomyocytes, and MCP-1 synthesis was not detected in PC and HL-1 cells treated with leptin. CONCLUSION: This work shows that leptin does not induce changes in viability, proliferation, size or apoptosis of rat neonatal and HL-1 cardiomyocytes, and it does not induce MCP-1 secretion in these cells.

Avian reoviruses cause apoptosis in cultured cells: viral uncoating, but not viral gene expression, is required for apoptosis induction.

The cytopathic effect evidenced by cells infected with avian reovirus S1133 suggests that this virus may induce apoptosis in primary cultures of chicken embryo fibroblasts. In this report we present evidence that avian reovirus infection of cultured cells causes activation of the intracellular apoptotic program and that this activation takes place during an early stage of the viral life cycle. The ability of avian reoviruses to induce apoptosis is not restricted to a particular virus strain or to a specific cell type, since different avian reovirus isolates were able to induce apoptosis in several avian and mammalian cell lines. Apoptosis was also provoked in ribavirin-treated avian reovirus-infected cells and in cells infected with UV-irradiated reovirions, indicating that viral mRNA synthesis and subsequent steps in viral replication are not needed for apoptosis induction in avian reovirus-infected cells and that the number of inoculated virus particles, not their infectivity, is the critical factor for apoptosis induction by avian reovirus. Our finding that apoptosis is no longer induced when intracellular viral uncoating is blocked indicates that intraendosomal virion disassembly is required for apoptosis induction and that attachment and uptake of parental reovirions are not sufficient to cause apoptosis. Taken together, our results suggest that apoptosis is triggered from within the infected cell by viral products generated after intraendosomal uncoating of parental reovirions.

Inhibition of Cdk4 activity enhances translation of p27kip1 in quiescent Rb-negative cells.

We show in this work that the inhibition of Cdk4 (6) in Rb(-/-) 3T3 cells enhances the accumulation of the p27(kip1) cyclin-dependent kinase inhibitor when these cells are induced into quiescence. Two different forms of inhibition of Cdk4 (6), namely overexpression of the Cdk4 (6) inhibitor p16 and overexpression of a dominant negative mutant of Cdk4 (Cdk4(N158)), result in this effect. This suggests that the relevant activity of Cdk4 (6) that has to be inactivated in this setting is its kinase activity. The accumulation of p27(kip1) is due to enhanced translation of the protein, mediated by the 3'-untranslated region of the p27(kip1) mRNA. Moreover, the cells that overexpress p16(ink4a) or Cdk4(N158) show a delay in G(1) when made quiescent and restimulated to proliferate. This delay is overcome by transfection of a plasmid expressing antisense p27(kip1), which shows that the accumulation of p27(kip1) in these cells is related to their G(1) delay. In summary, we report a new functional link between two important cell cycle regulators, Cdk4 and p27(kip1), and provide a mechanistic explanation to the previously reported epistatic relations between these two proteins.

DNA ploidy and cytonuclear area of peritumoral and paratumoral samples of mastectomy specimens: a useful prognostic marker?

OBJECTIVE: To investigate a possible relationship between DNA alterations in the "normal" residual mammary tissue of patients with breast cancer and survival. DESIGN: Prospective study. SETTING: University hospital, Spain. SUBJECTS: 162 patients operated on for breast cancer between 1991 and 1995. MAIN OUTCOME MEASURES: Cytology, histopathology, optic karyometry, DNA ploidy, and S-phase fraction measured by flow cytometry in peritumoral and paratumoral tissue. Mortality, and univariate analysis. RESULTS: DNA ploidy in peritumoral tissue was altered in 42 patients (26%), in 41 of whom the mean cytonuclear area was also altered. Of the 19 patients whose death within the study period was attributed to their cancer, 13 had peritumoral tissue in which both DNA ploidy and mean cytonuclear area were altered. On univariate analysis, there was significantly worse survival among the patients in whom both tumoral and peritumoral tissues were DNA aneuploid than among those in whom only tumoural tissue was DNA aneuploid (p < 0.0001). CONCLUSIONS: The presence of peritumoral or paratumoral tissue or both, with anomalous DNA content is associated with a reduced survival among women whose breast cancer has been treated by mastectomy. Additional studies including multivariate analysis are necessary to confirm our findings.