RESUMEN
OBJECTIVE: Endothelial cells (EC) in obese adipose tissue (AT) are exposed to a chronic proinflammatory environment that may induce a mesenchymal-like phenotype and altered function. The objective of this study was to establish whether endothelial-to-mesenchymal transition (EndoMT) is present in human AT in obesity and to investigate the effect of such transition on endothelial function and the endothelial particulate secretome represented by extracellular vesicles (EV). Approach and Results: We identified EndoMT in obese human AT depots by immunohistochemical co-localization of CD31 or vWF and α-SMA (alpha-smooth muscle actin). We showed that AT EC exposed in vitro to TGF-ß (tumor growth factor-ß), TNF-α (tumor necrosis factor-α), and IFN-γ (interferon-γ) undergo EndoMT with progressive loss of endothelial markers. The phenotypic change results in failure to maintain a tight barrier in culture, increased migration, and reduced angiogenesis. EndoMT also reduced mitochondrial oxidative phosphorylation and glycolytic capacity of EC. EVs produced by EC that underwent EndoMT dramatically reduced angiogenic capacity of the recipient naïve ECs without affecting their migration or proliferation. Proteomic analysis of EV produced by EC in the proinflammatory conditions showed presence of several pro-inflammatory and immune proteins along with an enrichment in angiogenic receptors. CONCLUSIONS: We demonstrated the presence of EndoMT in human AT in obesity. EndoMT in vitro resulted in production of EV that transferred some of the functional and metabolic features to recipient naïve EC. This result suggests that functional and molecular features of EC that underwent EndoMT in vivo can be disseminated in a paracrine or endocrine fashion and may induce endothelial dysfunction in distant vascular beds.
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Tejido Adiposo/irrigación sanguínea , Transición Epitelial-Mesenquimal/genética , Neovascularización Patológica/genética , Obesidad/genética , Factor de Crecimiento Transformador beta1/farmacología , Tejido Adiposo/metabolismo , Análisis de Varianza , Biomarcadores/metabolismo , Estudios de Casos y Controles , Movimiento Celular/genética , Proliferación Celular/genética , Células Cultivadas , Células Endoteliales/metabolismo , Transición Epitelial-Mesenquimal/efectos de los fármacos , Citometría de Flujo/métodos , Humanos , Obesidad/fisiopatología , Proteómica/métodosRESUMEN
The metabolic syndrome and diabetic conditions support atherosclerosis, but the exact mechanisms for accelerated atherogenesis remain unclear. Although the proinflammatory role of STAT4 in atherosclerosis and diet-induced insulin resistance (IR) was recently established, the impact of STAT4 on atherogenesis in conditions of IR is not known. In this study, we generated Stat4-/-Ldlr-/- mice that were fed a diabetogenic diet with added cholesterol (DDC). DDC-fed Stat4-/-Ldlr-/- mice demonstrated improved glucose tolerance, insulin sensitivity, and a 36% reduction in atherosclerosis compared with Ldlr-/- controls. Interestingly, we detected a reduction in T follicular helper (Tfh) cells and plasma B cells but a sharp elevation in CD8+ regulatory T cells (Tregs) in spleens and aortas of Stat4-/-Ldlr-/- mice compared with Ldlr-/- mice. Similarly, STAT4 deficiency supported CD8+ Treg differentiation in vitro. STAT4-deficient CD8+ Tregs suppressed Tfh cell and germinal center B cell development upon immunization with keyhole limpet hemocyanin, indicating an important role for STAT4 in CD8+ Treg functions in vivo. Furthermore, adoptive transfer of Stat4-/-Ldlr-/- CD8+ Tregs versus Ldlr-/- CD8+ Tregs resulted in a significant reduction in plaque burden and suppression of Tfh cell and germinal center B cells in DDC-fed Ldlr-/- recipients. STAT4 expression in macrophages (MΦs) also affected the Tfh/CD8+ Treg axis, because conditioned media from Stat4-/-Ldlr-/- MΦs supported CD8+ Treg differentiation, but not Tfh cell differentiation, in a TGF-ß-dependent manner. These findings suggest a novel mechanism by which STAT4 supports atherosclerosis in IR Ldlr-/- mice via STAT4-dependent MΦs, as well as cell-intrinsic suppression of CD8+ Treg generation and functions and maintenance of Tfh cell generation and the accompanying humoral immune response.
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Aterosclerosis/inmunología , Receptores de LDL/metabolismo , Factor de Transcripción STAT4/metabolismo , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Reguladores/inmunología , Animales , Antígenos CD8/metabolismo , Células Cultivadas , Colesterol/metabolismo , Dieta Aterogénica , Centro Germinal/inmunología , Humanos , Resistencia a la Insulina , Activación de Linfocitos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de LDL/genética , Factor de Transcripción STAT4/genéticaRESUMEN
The 12-lipoxygenase (12LO) pathway is a promising target to reduce islet dysfunction, adipose tissue (AT) inflammation and insulin resistance. Optimal pre-clinical models for the investigation of selective12LO inhibitors in this context have not yet been identified. The objective of this study was to characterize the time course of 12LO isoform expression and metabolite production in pancreatic islets and AT of C57BLKS/J-db/db obese diabetic mouse in a pre-diabetic state in order to establish a suitable therapeutic window for intervention with selective lipoxygenase inhibitors. Mice have 2 major 12LO isoforms -the leukocyte type (12/15LO) and the platelet type (p12LO) and both are expressed in islets and AT. We found a sharp increase in protein expression of 12/15LO in the pancreatic islets of 10-week old db-/- mice compared to 8- week old counterparts. Immunohistochemistry showed that the increase in islet 12/15LO parallels a decline in islet number. Analysis of 12- and 15-hydroperoxytetraeicosanoid acids (HETE)s showed a 2-3 fold increase especially in 12(S)-HETE that mirrored the increase in 12/15LO expression in islets. Analysis of AT and stromal vascular fraction (SVF) showed a significant increase of platelet 12LO gene expression along with 12- and 15- HETEs. The data demonstrate that the db/db mouse is a suitable model for investigation of 12/15LO inhibitors in the development of inflammatory mediated type 2 diabetes, with a narrow window of therapeutic intervention prior to 8 weeks of age.
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Araquidonato 12-Lipooxigenasa/metabolismo , Araquidonato 15-Lipooxigenasa/metabolismo , Diabetes Mellitus Tipo 2/enzimología , Células Secretoras de Insulina/enzimología , Inhibidores de la Lipooxigenasa/farmacología , Estado Prediabético/enzimología , Animales , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/patología , Activación Enzimática/efectos de los fármacos , Células Secretoras de Insulina/patología , Isoenzimas/antagonistas & inhibidores , Isoenzimas/metabolismo , Masculino , Ratones , Ratones Obesos , Estado Prediabético/tratamiento farmacológico , Estado Prediabético/patologíaRESUMEN
Visceral adipose tissue (AT) inflammation is linked to the complications of obesity, including insulin resistance (IR) and type 2 diabetes. Recent data from our lab showed that germline deficiency in STAT4 reduces inflammation and improves IR in obese mice. The objective of this study was to determine the contribution of selective STAT4 deficiency in subsets of hematopoietic cells to IR and AT inflammation. To determine the contribution of hematopoietic lineage, we sublethally irradiated Stat4-/-C57Bl6 mice and reconstituted them with bone marrow cells (BMC) from Stat4+/+C57Bl6 congenic donors. We also established the contribution of selective STAT4 deficiency in CD4+ or CD8+ T cells using adoptive transfer in Rag1-/- mice. All mice received a HFD for 15 weeks (n = 7-12 mice/group). BMC that expressed STAT4 induced increases in glucose intolerance and IR compared to STAT4-deficient cells. Also, AT inflammation was increased and the numbers of CD8+ cells infiltrating AT were higher in mice with STAT4 expressing BMC. Studies in Rag1-/- mice further confirmed the prominent role of CD8+ cells expressing STAT4 in insulin resistance and AT and islet inflammation. Collectively our results show specific and dominant contribution of STAT4 in the hematopoietic compartment to metabolic health and inflammation in diet-induced obesity.
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Tejido Adiposo/metabolismo , Inflamación/metabolismo , Factor de Transcripción STAT4/metabolismo , Adipocitos/metabolismo , Animales , Western Blotting , Citometría de Flujo , Hematopoyesis/genética , Hematopoyesis/fisiología , Inflamación/genética , Insulina/metabolismo , Resistencia a la Insulina/genética , Resistencia a la Insulina/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Reacción en Cadena en Tiempo Real de la Polimerasa , Factor de Transcripción STAT4/genéticaRESUMEN
Islet inflammation contributes to beta cell demise in both type 1 and type 2 diabetes. 12-Lipoxygenase (12-LO, gene expressed as ALOX12 in humans and 12-Lo in rodents in this manuscript) produces proinflammatory metabolites such as 12(S)-hydroxyeicosatetraenoic acids through dioxygenation of polyunsaturated fatty acids. 12-LO was first implicated in diabetes when the increase in 12-Lo expression and 12(S)-hydroxyeicosatetraenoic acid was noted in rodent models of diabetes. Subsequently, germline 12-Lo (-/-) was shown to prevent the development of hyperglycemia in mouse models of type 1 diabetes and in high-fat fed mice. More recently, beta cell-specific 12-Lo (-/-) was shown to protect mice against hyperglycaemia after streptozotocin and a high-fat diet. In humans, 12-LO expression is increased in pancreatic islets of autoantibody-positive, type 1 diabetic and type 2 diabetic organ donors. Interestingly, the high expression of ALOX12 is associated with the alteration in first-phase glucose-stimulated insulin secretion in human type 2 diabetic islets. To further clarify the role of islet 12-LO in diabetes and to validate 12-LO as a therapeutic target of diabetes, we have studied selective pharmacological inhibitors for 12-LO. The compounds we have identified show promise: they protect beta cell lines and human islets from apoptosis and preserve insulin secretion when challenged by proinflammatory cytokine mixture. Currently studies are underway to test the compounds in mouse models of diabetes. This review summarises a presentation given at the 'Islet inflammation in type 2 diabetes' symposium at the 2015 annual meeting of the EASD. It is accompanied two other mini-reviews on topics from this symposium (by Simone Baltrusch, DOI: 10.1007/s00125-016-3891-x and Marc Donath, DOI: 10.1007/s00125-016-3873-z ) and a commentary by the Session Chair, Piero Marchetti (DOI: 10.1007/s00125-016-3875-x ).
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Inflamación/metabolismo , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patología , Animales , Araquidonato 12-Lipooxigenasa/metabolismo , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patología , Diabetes Mellitus Tipo 2/inmunología , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Humanos , Ácidos Hidroxieicosatetraenoicos/metabolismo , Inflamación/inmunología , Inflamación/patología , Células Secretoras de Insulina/inmunología , LípidosRESUMEN
OBJECTIVE: Prediabetic states are associated with accelerated atherosclerosis, but the availability of mouse models to study connections between these diseases has been limited. The aim of this study was to test the selective role of impaired insulin receptor/insulin receptor substrate-1 signaling on atherogenesis. METHODS AND RESULTS: To address the effects of impaired insulin signaling associated with hyperinsulinemia on atherosclerosis in the absence of obesity and hyperglycemia, we generated insulin receptor (Insr)/insulin receptor substrate-1 (Insr1) double heterozygous apolipoprotein (Apoe)-knockout mice (Insr(+/-)Irs1(+/-)Apoe(-/-)) mice. Insr(+/-)Irs1(+/-)Apoe(-/-) mice fed a Western diet for 15 weeks showed elevated levels of fasting insulin compared to Insr(+/+)Irs1(+/+)Apoe(-/-) mice. There were no significant differences in glucose, triglyceride, HDL, VLDL, cholesterol levels or free fatty acid in the plasma of Insr(+/-)Irs1(+/-)Apoe(-/-) and Insr(+/+)Irs1(+/+)Apoe(-/-) mice. Atherosclerotic lesions were increased in male (brachiocephalic artery) and female (aortic tree) Insr(+/-)Irs1(+/-)Apoe(-/-) compared to Insr(+/+)Irs1(+/+)Apoe(-/-) mice. Bone marrow transfer experiments demonstrated that nonhematopoietic cells have to be Insr(+/-)Irs1(+/-) to accelerate atherosclerosis. Impaired insulin signaling resulted in decreased levels of vascular phospho-eNOS, attenuated endothelium-dependent vasorelaxation and elevated VCAM-1 expression in aortas of Insr(+/-)Irs1(+/-)Apoe(-/-) mice. In addition, phospho-ERK and vascular smooth muscle cell proliferation were significantly elevated in aortas of Insr(+/-)Irs1(+/-)Apoe(-/-) mice. CONCLUSIONS: These results demonstrate that defective insulin signaling is involved in accelerated atherosclerosis in Insr(+/-)Irs1(+/-)Apoe(-/-) mice by promoting vascular dysfunction and inflammation.
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Apolipoproteínas E/deficiencia , Aterosclerosis/genética , Aterosclerosis/fisiopatología , Heterocigoto , Proteínas Sustrato del Receptor de Insulina/genética , Receptor de Insulina/genética , Transducción de Señal/fisiología , Animales , Apolipoproteínas E/genética , Aterosclerosis/patología , Proliferación Celular , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Proteínas Sustrato del Receptor de Insulina/fisiología , Sistema de Señalización de MAP Quinasas/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/patología , Óxido Nítrico Sintasa de Tipo III/metabolismo , Receptor de Insulina/fisiologíaRESUMEN
The lipoxygenases (LOs) are principal enzymes involved in the oxidative metabolism of polyunsaturated fatty acids, including arachidonic acid. 12- and 15-LO and their lipid metabolites have been implicated in the development of insulin resistance and diabetes. Adipose tissue, and in particular visceral adipose tissue, plays a primary role in the development of the inflammation seen in these conditions. 12- and 15-LO and their lipid metabolites act as upstream regulators of many of the cytokines involved in the inflammatory response in adipose tissue. While the role that 12- and 15-LO play in chronically inflamed adipose tissue is becoming clearer, there are still many questions that remain unanswered regarding their activation, signaling pathways, and roles in healthy fat. 12- and 15-LO also generate products with anti-inflammatory properties that are under investigation. Therefore, 12- and 15-LO have the potential to be very important targets for therapeutics aimed at reducing insulin resistance and the comorbid conditions associated with obesity.
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Araquidonato 12-Lipooxigenasa/metabolismo , Araquidonato 15-Lipooxigenasa/metabolismo , Ácido Araquidónico/metabolismo , Grasa Intraabdominal/enzimología , Obesidad/enzimología , Adipogénesis , Animales , Citocinas/metabolismo , Humanos , Inflamación/enzimología , Inflamación/patología , Grasa Intraabdominal/patología , Ratones , Obesidad/patología , Transducción de SeñalRESUMEN
Chronic kidney disease is associated with increased levels of assymetric N(G),N(G)-dimethylarginine (ADMA), which is predictive of increased mortality and cardiovascular disease. ADMA induces endothelial dysfunction through competitive inhibition of the endothelial nitric oxide (eNOS) substrate L-arginine. Kajimoto et al. show that ADMA may also reduce nitric oxide production via decreased eNOS phosphorylation; this effect is mediated by the MAPK pathway and can be reversed in vivo by increased catabolism of ADMA through dimethylarginine dimethylaminohydrolase-1 overexpression.
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Arginina/análogos & derivados , Óxido Nítrico Sintasa de Tipo III/antagonistas & inhibidores , Insuficiencia Renal Crónica/fisiopatología , Animales , Arginina/sangre , Humanos , MasculinoRESUMEN
Cardiovascular disease (CVD) is the most prominent cause of death of adults in the United States with coronary artery disease being the most common type of CVD. Following a myocardial event, the coronary endothelium plays an important role in the recovery of the ischemic myocardium. Specifically, endothelial cells (EC) must be able to elicit a robust angiogenic response necessary for tissue revascularization and repair. However, local or distant cues may prevent effective revascularization. Extracellular vesicles (EV) are produced by all cells and endothelium is a rich source of EVs that have access to the main circulation thereby potentially impacting local and distant tissue function. Systemic inflammation associated with conditions such as obesity as well as the acute inflammatory response elicited by a cardiac event can significantly increase the EV release by endothelium and alter their miRNA, protein or lipid cargo. Our laboratory has previously shown that EVs released by adipose tissue endothelial cells exposed to chronic inflammation have angiostatic effects on naïve adipose tissue EC in vitro. Whether the observed effect is specific to EVs from adipose tissue endothelium or is a more general feature of the endothelial EVs exposed to pro-inflammatory cues is currently unclear. The objective of this study was to investigate the angiostatic effects of EVs produced by EC from the coronary artery and adipose microvasculature exposed to pro-inflammatory cytokines (PIC) on naïve coronary artery EC. We have found that EVs from both EC sources have angiostatic effects on the coronary endothelium. EVs produced by cells in a pro-inflammatory environment reduced proliferation and barrier function of EC without impacting cellular senescence. Some of these functional effects could be attributed to the miRNA cargo of EVs. Several miRNAs such as miR-451, let-7, or miR-23a impact on multiple pathways responsible for proliferation, cellular permeability and angiogenesis. Collectively, our data suggests that EVs may compete with pro-angiogenic cues in the ischemic myocardium therefore slowing down the repair response. Acute treatments with inhibitors that prevent endogenous EV release immediately after an ischemic event may contribute to better efficacy of therapeutic approaches using functionalized exogenous EVs or other pro-angiogenic approaches.
RESUMEN
Central obesity is associated with low-grade inflammation that promotes type 2 diabetes and cardiovascular disease in obese individuals. The 12- and 5-lipoxygenase (12-LO and 5-LO) enzymes have been linked to inflammatory changes, leading to the development of atherosclerosis. 12-LO has also been linked recently to inflammation and insulin resistance in adipocytes. We analyzed the expression of LO and proinflammatory cytokines in adipose tissue and adipocytes in obese Zucker rats, a widely studied genetic model of obesity, insulin resistance, and the metabolic syndrome. mRNA expression of 12-LO, 5-LO, and 5-LO-activating protein (FLAP) was upregulated in adipocytes and adipose tissue from obese Zucker rats compared with those from lean rats. Concomitant with increased LO gene expression, the 12-LO product 12-HETE and the 5-LO products 5-HETE and leukotriene B4 (LTB4) were also increased in adipocytes. Furthermore, upregulation of key proinflammatory markers interleukin (IL)-6, TNFα, and monocyte chemoattractant protein-1 were observed in adipocytes isolated from obese Zucker rats. Immunohistochemistry indicated that the positive 12-LO staining in adipose tissue represents cells in addition to adipocytes. This was confirmed by Western blotting in stromal vascular fractions. These changes were in part reversed by the novel anti-inflammatory drug lisofylline (LSF). LSF also reduced p-STAT4 in visceral adipose tissue from obese Zucker rats and improved the metabolic profile, reducing fasting plasma glucose and increasing insulin sensitivity in obese Zucker rats. In 3T3-L1 adipocytes, LSF abrogated the inflammatory response induced by LO products. Thus, therapeutic agents reducing LO or STAT4 activation may provide novel tools to reduce obesity-induced inflammation.
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Araquidonato 12-Lipooxigenasa/metabolismo , Araquidonato 5-Lipooxigenasa/metabolismo , Citocinas/metabolismo , Regulación de la Expresión Génica , Mediadores de Inflamación/metabolismo , Grasa Intraabdominal/metabolismo , Obesidad/metabolismo , Células 3T3-L1 , Proteínas Activadoras de la 5-Lipooxigenasa/genética , Proteínas Activadoras de la 5-Lipooxigenasa/metabolismo , Animales , Antiinflamatorios no Esteroideos/farmacología , Araquidonato 12-Lipooxigenasa/genética , Araquidonato 5-Lipooxigenasa/genética , Ácidos Araquidónicos/metabolismo , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Grasa Intraabdominal/citología , Grasa Intraabdominal/efectos de los fármacos , Grasa Intraabdominal/patología , Ratones , Obesidad/tratamiento farmacológico , Obesidad/patología , Obesidad/fisiopatología , Pentoxifilina/análogos & derivados , Pentoxifilina/farmacología , Fosforilación/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas , Ratas Zucker , Factor de Transcripción STAT4/metabolismoRESUMEN
Adipose tissue inflammation in obesity is a major factor leading to cardiovascular disease and type 2 diabetes.12/15 lipoxygenases (ALOX) play an important role in the generation of inflammatory mediators, insulin resistance and downstream immune activation in animal models of obesity. However, the expression and roles of 12/15ALOX isoforms, and their cellular sources in human subcutaneous (sc) and omental (om) fat in obesity is unknown. The objective of this study was to examine the gene expression and localization of ALOX isoforms and relevant downstream cytokines in subcutaneous (sc) and omental (om) adipose tissue in obese humans. Paired biopsies of sc and om fat were obtained during bariatric surgeries from 24 morbidly obese patients. Gene and protein expression for ALOX15a, ALOX15b and ALOX 12 were measured by real-time PCR and western blotting in adipocytes and stromal vascular fractions (SVF) from om and sc adipose tissue along with the mRNA expression of the downstream cytokines IL-12a, IL-12b, IL-6, IFNγ and the chemokine CXCL10. In a paired analysis, all ALOX isoforms, IL-6, IL-12a and CXCL10 were significantly higher in om vs. sc fat. ALOX15a mRNA and protein expression was found exclusively in om fat. All of the ALOX isoforms were expressed solely in the SVF. Further fractionation of the SVF in CD34+ and CD34- cells indicated that ALOX15a is predominantly expressed in the CD34+ fraction including vascular and progenitor cells, while ALOX15B is mostly expressed in the CD34- cells containing various leucocytes and myeloid cells. This result was confirmed by immunohistochemistry showing exclusive localization of ALOX15a in the om fat and predominantly in the vasculature and non-adipocyte cells. Our finding is identifying selective expression of ALOX15a in human om but not sc fat. This is a study showing a major inflammatory gene exclusively expressed in visceral fat in humans.
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Tejido Adiposo/enzimología , Araquidonato 12-Lipooxigenasa/metabolismo , Araquidonato 15-Lipooxigenasa/metabolismo , Obesidad/enzimología , Adulto , Araquidonato 12-Lipooxigenasa/genética , Araquidonato 15-Lipooxigenasa/genética , Citocinas/metabolismo , Femenino , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Persona de Mediana EdadRESUMEN
Introduction: Evidence-based medicine (EBM) is pivotal in shaping patient care, yet it is challenging to incorporate into undergraduate medical education (UME) due to a lack of dedicated resources within the preclinical curriculum. To address this challenge, we used a peer-led approach to explain difficult concepts through language that students can understand at their shared level of understanding. Methods: Four second-year medical students trained in EBM over 18 months by facilitating monthly journal clubs, ultimately leading to their involvement as peer-instructors. With input from a faculty expert, peer-instructors designed integrative PowerPoint modules and interactive problem sets on basic biostatistics and EBM principles. Assessment included formative quizzes with multiple attempts to achieve at least 80% to demonstrate mastery of core learning objectives. Afterwards, students were invited to provide feedback using a 5-point Likert scale survey. Results: Of second-year students who participated, all 151 demonstrated 80% competency on each quiz. Eighty-seven (58%) students completed the survey on which, 77% agreed/strongly agreed that their level of understanding of EBM improved after the peer-led sessions, 76% agreed/strongly agreed that the sessions were more conducive to learning compared to traditional lectures, and 94% agreed/strongly agreed that the material covered was relevant to the USMLE Step 1. Discussion: This peer-led approach has been rated as effective by learners, improving their ability to critically appraise and apply clinical evidence. To promote integration of EBM into UME, we have prepared modules, problem sets, quizzes, and an outline of the problem-solving sessions for universal adoption.
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Educación de Pregrado en Medicina , Estudiantes de Medicina , Bioestadística , Curriculum , Medicina Basada en la Evidencia/educación , HumanosRESUMEN
Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor response to insulin, local hypoxia, and inflammation. By stimulating the differentiation of functional white adipocytes from progenitors, radical hypertrophy of the adipocyte population can be prevented and, consequently, the metabolic health of adipose tissue can be improved along with a reduction of inflammation. Also, by stimulating a differentiation of beige/brown adipocytes, the total body energy expenditure can be increased, resulting in weight loss. This approach could prevent the development of obesity co-morbidities such as type 2 diabetes and cardiovascular disease. This paper describes the isolation, expansion, and differentiation of white and beige adipocytes from a subset of human adipose tissue endothelial cells that co-express the CD31 and CD34 markers. The method is relatively cheap and is not labor-intensive. It requires access to human adipose tissue and the subcutaneous depot is suitable for sampling. For this protocol, fresh adipose tissue samples from morbidly obese subjects [body mass index (BMI) >35] are collected during bariatric surgery procedures. Using a sequential immunoseparation from the stromal vascular fraction, enough cells are produced from as little as 2-3 g of fat. These cells can be expanded in culture over 10-14 days, can be cryopreserved, and retain their adipogenic properties with passaging up to passage 5-6. The cells are treated for 14 days with an adipogenic cocktail using a combination of human insulin and the PPARγ agonist-rosiglitazone. This methodology can be used for obtaining proof of concept experiments on molecular mechanisms that drive adipogenic responses in adipose endothelial cells, or for screening new drugs that can enhance the adipogenic response directed either towards white or beige/brown adipocyte differentiation. Using small subcutaneous biopsies, this methodology can be used to screen out non-responder subjects for clinical trials aimed to stimulate beige/brown and white adipocytes for the treatment of obesity and co-morbidities.
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Antígenos CD34/metabolismo , Células Endoteliales/metabolismo , Obesidad Mórbida/fisiopatología , Epiplón/metabolismo , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Grasa Subcutánea/metabolismo , Animales , Diferenciación Celular , Humanos , Masculino , Grasa Subcutánea/patologíaRESUMEN
Diabetic non-healing wounds are a major clinical problem. The mechanisms leading to poor wound healing in diabetes are multifactorial but unresolved inflammation may be a major contributing factor. The complement system (CS) is the most potent inflammatory cascade in humans and contributes to poor wound healing in animal models. Signal transducer and activator of transcription 4 (STAT4) is a transcription factor expressed in immune and adipose cells and contributes to upregulation of some inflammatory chemokines and cytokines. Persistent CS and STAT4 expression in diabetic wounds may thus contribute to chronic inflammation and delayed healing. The purpose of this study was to characterize CS and STAT4 in early diabetic wounds using db/db mice as a diabetic skin wound model. The CS was found to be activated early in the diabetic wounds as demonstrated by increased anaphylatoxin C5a in wound fluid and C3-fragment deposition by immunostaining. These changes were associated with a 76% increase in nucleated cells in the wounds of db/db mice vs. CONTROLS: The novel classical CS inhibitor, Peptide Inhibitor of Complement C1 (PIC1) reduced inflammation when added directly or saturated in an acellular skin scaffold, as reflected by reduced CS components and leukocyte infiltration. A significant increase in expression of STAT4 and the downstream macrophage chemokine CCL2 and its receptor CCR2 were also found in the early wounds of db/db mice compared to non-diabetic controls. These studies provide evidence for two new promising targets to reduce unresolved inflammation and to improve healing of diabetic skin wounds.
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Activación de Complemento/fisiología , Complicaciones de la Diabetes/fisiopatología , Factor de Transcripción STAT4/fisiología , Heridas y Lesiones/fisiopatología , Animales , Complemento C5a/fisiología , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Inflamación/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal/fisiología , Cicatrización de Heridas/fisiología , Heridas y Lesiones/complicacionesRESUMEN
Aging leads to a proinflammatory state within the vasculature without disease, yet whether this inflammatory state occurs during atherogenesis remains unclear. Here, we examined how aging impacts atherosclerosis using Ldlr(-/-) mice, an established murine model of atherosclerosis. We found that aged atherosclerotic Ldlr(-/-) mice exhibited enhanced atherogenesis within the aorta. Aging also led to increased LDL levels, elevated blood pressure on a low-fat diet, and insulin resistance after a high-fat diet (HFD). On a HFD, aging increased a monocytosis in the peripheral blood and enhanced macrophage accumulation within the aorta. When we conducted bone marrow transplant experiments, we found that stromal factors contributed to age-enhanced atherosclerosis. To delineate these stromal factors, we determined that the vasculature exhibited an age-enhanced inflammatory response consisting of elevated production of CCL-2, osteopontin, and IL-6 during atherogenesis. In addition, in vitro cultures showed that aging enhanced the production of osteopontin by vascular smooth muscle cells. Functionally, aged atherosclerotic aortas displayed higher monocyte chemotaxis than young aortas. Hence, our study has revealed that aging induces metabolic dysfunction and enhances vascular inflammation to promote a peripheral monocytosis and macrophage accumulation within the atherosclerotic aorta.
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Envejecimiento/patología , Aterosclerosis/patología , Vasos Sanguíneos/patología , Inflamación/patología , Leucocitosis/patología , Monocitos/patología , Animales , Aorta/efectos de los fármacos , Aorta/patología , Aterosclerosis/complicaciones , Aterosclerosis/metabolismo , Recuento de Células , Quimiotaxis/efectos de los fármacos , Medios de Cultivo Condicionados/farmacología , Dieta Alta en Grasa , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Hematopoyesis/efectos de los fármacos , Hemodinámica/efectos de los fármacos , Inflamación/complicaciones , Inflamación/metabolismo , Mediadores de Inflamación/metabolismo , Resistencia a la Insulina , Interleucina-6/metabolismo , Leucocitosis/complicaciones , Leucocitosis/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Receptores de LDL/metabolismo , Células del Estroma/efectos de los fármacos , Células del Estroma/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
BACKGROUND: Normal pregnancy is characterized by sodium and water conservation and an increase in plasma volume that is required for an uncomplicated pregnancy. Renal interstitial hydrostatic pressure (RIHP) is significantly decreased in pregnant rats. This decrease in RIHP may play an important role in the sodium and water retention that characterizes normal pregnancy. Paradoxically this enhanced renal sodium and water reabsorption appear to conflict with the consistent findings of a general decrease in abundance of renal tubular sodium transporters during normal pregnancy. The objective of this review is to examine the apparent discrepancy between the increases in renal tubular sodium and water reabsorption, facilitated by decreases in RIHP, and the seemingly discordant decreases in abundance of renal tubular transporters during normal pregnancy in rats. METHODS: Western blots and immunohistochemistry were used to evaluate abundance and localization of renal tubular transporters. RIHP was measured directly and continuously via a polyethylene (PE) matrix that was implanted in the left kidney of rats at the age of 11 to 16 weeks. RESULTS: Average basal RIHP and fractional excretion of sodium (FENa) were found to be significantly lower (P < .05) in midterm pregnant (MP; n = 18) and late-term pregnant (LP; n = 20) rats compared with nonpregnant (NP; n = 16) rats (3.5 +/- 0.3 mm Hg and 1.46 +/- 0.24% for MP; 3.3 +/- 0.1 mm Hg and 1.41 +/- 0.21% for LP; and 7.6 +/- 0.6 mm Hg and 3.67 +/- 0.24% for NP). Cortical Na+-K+-ATPase and Na-Pi2a cotransporter (Na-Pi) protein expression tend to decline with pregnancy. Also cortical Na+-H+ exchanger-1 (NHE-1) protein expression declines steadily during the course of pregnancy from MP to LP compared with that in NP rats, and cortical Na+-H+ exchanger-3 (NHE-3) protein expression is significantly lower in MP and LP compared with NP rats. CONCLUSIONS: We propose that during normal uncomplicated pregnancy, simultaneous decreases in RIHP and in net abundance of renal tubular sodium transporters occur. The effects of decreased RIHP exceed those of the reduction in net abundance, and presumably activity, of renal tubular transporters resulting in an enhanced net sodium and water retention during pregnancy.
Asunto(s)
Riñón/irrigación sanguínea , Volumen Plasmático/fisiología , Proteínas Cotransportadoras de Sodio-Fosfato/metabolismo , Animales , Proteínas de Transporte de Catión/biosíntesis , Femenino , Presión Hidrostática , Inmunohistoquímica , Riñón/metabolismo , Túbulos Renales/irrigación sanguínea , Túbulos Renales/metabolismo , Proteínas de la Membrana/biosíntesis , Natriuresis/fisiología , Embarazo , Ratas , Ratas Sprague-Dawley , Circulación Renal/fisiología , Intercambiador 1 de Sodio-Hidrógeno , Intercambiador 3 de Sodio-Hidrógeno , Intercambiadores de Sodio-Hidrógeno/biosíntesis , Proteínas Cotransportadoras de Sodio-Fosfato/biosíntesis , ATPasa Intercambiadora de Sodio-Potasio/biosíntesisRESUMEN
Atherosclerosis is a chronic inflammatory process that leads to plaque formation in large and medium sized vessels. T helper 1 (Th1) cells constitute the majority of plaque infiltrating pro-atherogenic T cells and are induced via IFNγ-dependent activation of T-box (Tbet) and/or IL-12-dependent activation of signal transducer and activator of transcription 4 (STAT4). We thus aimed to define a role for STAT4 in atherosclerosis. STAT4-deficiency resulted in a â¼71% reduction (p < 0.001) in plaque burden in Stat4(-/-)Apoe(-/-) vs Apoe(-/-) mice fed chow diet and significantly attenuated atherosclerosis (â¼31%, p < 0.01) in western diet fed Stat4(-/-)Apoe(-/-) mice. Surprisingly, reduced atherogenesis in Stat4(-/-)Apoe(-/-) mice was not due to attenuated IFNγ production in vivo by Th1 cells, suggesting an at least partially IFNγ-independent pro-atherogenic role of STAT4. STAT4 is expressed in T cells, but also detected in macrophages (MΦs). Stat4(-/-)Apoe(-/-)in vitro differentiated M1 or M2 MΦs had reduced cytokine production compare to Apoe(-/-) M1 and M2 MΦs that was accompanied by reduced induction of CD69, I-A(b), and CD86 in response to LPS stimulation. Stat4(-/-)Apoe(-/-) MΦs expressed attenuated levels of CCR2 and demonstrated reduced migration toward CCL2 in a transwell assay. Importantly, the percentage of aortic CD11b(+)F4/80(+)Ly6C(hi) MΦs was reduced in Stat4(-/-)Apoe(-/-) vs Apoe(-/-) mice. Thus, this study identifies for the first time a pro-atherogenic role of STAT4 that is at least partially independent of Th1 cell-derived IFNγ, and primarily involving the modulation of MΦ responses.
Asunto(s)
Aterosclerosis/genética , Interferón gamma/metabolismo , Macrófagos/citología , Placa Aterosclerótica/genética , Factor de Transcripción STAT4/genética , Células TH1/citología , Animales , Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos T/metabolismo , Aorta/patología , Antígeno B7-2/metabolismo , Células de la Médula Ósea/citología , Diferenciación Celular , Movimiento Celular , Citocinas/metabolismo , Femenino , Citometría de Flujo , Inflamación , Lectinas Tipo C/metabolismo , Lípidos/sangre , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
CONTEXT: Visceral adipose tissue (VAT) is a key contributor to chronic inflammation in obesity. The 12/15-lipoxygenase pathway (ALOX) is present in adipose tissue (AT) and leads to inflammatory cascades that are causal for the onset of insulin resistance in rodent models of obesity. OBJECTIVE: The pathophysiology of the ALOX 12/15 pathway in human AT is unknown. We characterized the ALOX pathway in different AT depots in obese humans with or without type 2 diabetes (T2D). DESIGN: This study includes a cross-sectional cohort of 46 morbidly obese (body mass index >39 kg/m(2)) nondiabetic (n = 25) and T2D (n = 21) subjects. SETTING: This study was conducted at Eastern Virginia Medical School (Norfolk, Virginia) in collaboration with Sentara Metabolic and Weight Loss Surgery Center (Sentara Medical Group, Norfolk, Virginia). PATIENTS: Twenty-five obese (body mass index 44.8 ± 4.4 kg/m(2)) nondiabetic (hemoglobin A1c 5.83% ± 0.27%) and 21 obese (43.4 ± 4.1 kg/m(2)) and T2D (hemoglobin A1c 7.66% ± 1.22%) subjects were included in the study. The subjects were age matched and both groups had a bias toward female gender. MAIN OUTCOMES AND MEASURES: Expression of ALOX isoforms along with fatty acid substrates and downstream lipid metabolites were measured. Correlations with depot-specific inflammatory markers were also established. RESULTS: ALOX 12 expression and its metabolite 12(S)-hydroxyeicosatetraenoic acid were significantly increased in the VAT of T2D subjects. ALOX 15A was exclusively expressed in VAT in both groups. ALOX 12 expression positively correlated with expression of inflammatory genes IL-6, IL-12a, CXCL10, and lipocalin-2. CONCLUSIONS: ALOX 12 may have a critical role in regulation of inflammation in VAT in obesity and T2D. Selective ALOX 12 inhibitors may constitute a new approach to limit AT inflammation in human obesity.
Asunto(s)
Araquidonato 12-Lipooxigenasa/metabolismo , Araquidonato 15-Lipooxigenasa/metabolismo , Diabetes Mellitus Tipo 2/enzimología , Grasa Intraabdominal/enzimología , Obesidad Mórbida/enzimología , Adolescente , Adulto , Anciano , Araquidonato 12-Lipooxigenasa/genética , Araquidonato 15-Lipooxigenasa/genética , Biomarcadores/metabolismo , Estudios Transversales , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/inmunología , Ácidos Grasos/metabolismo , Femenino , Regulación Enzimológica de la Expresión Génica/inmunología , Hemoglobina Glucada/metabolismo , Humanos , Inflamación/enzimología , Inflamación/genética , Inflamación/inmunología , Grasa Intraabdominal/inmunología , Metabolismo de los Lípidos/genética , Metabolismo de los Lípidos/inmunología , Masculino , Persona de Mediana Edad , Obesidad Mórbida/genética , Obesidad Mórbida/inmunología , Adulto JovenRESUMEN
In the past decade, islet inflammation has emerged as a contributor to the loss of functional ß cell mass in both type 1 (T1D) and type 2 diabetes (T2D). Evidence supports the idea that overnutrition and insulin resistance result in the production of proinflammatory mediators by ß cells. In addition to compromising ß cell function and survival, cytokines may recruit macrophages into islets, thus augmenting inflammation. Limited but intriguing data imply a role of adaptive immune response in islet dysfunction in T2D. Clinical trials have validated anti-inflammatory therapies in T2D, whereas immune therapy for T1D remains challenging. Further research is required to improve our understanding of islet inflammatory pathways and to identify more effective therapeutic targets for T1D and T2D.
Asunto(s)
Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 2/inmunología , Mediadores de Inflamación/metabolismo , Islotes Pancreáticos/inmunología , Animales , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Estrés del Retículo Endoplásmico/efectos de los fármacos , Humanos , Hipoglucemiantes/farmacología , Hipoglucemiantes/uso terapéutico , Resistencia a la Insulina , Polipéptido Amiloide de los Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Terapia Molecular DirigidaRESUMEN
Signal transducer and activator of transcription (STAT) 4 is one of the seven members of the STAT family. STAT4 has a prominent role in mediating interleukin-12-induced T-helper cell type 1 lineage differentiation. T cells are key players in the maintenance of adipose tissue (AT) inflammation. The role of STAT4 in obesity and AT inflammation is unknown. We sought to determine the role of STAT4 in AT inflammation in obesity-induced insulin resistance. We studied STAT4-null mice on the C57Bl6/J background. We have found that STAT4(-/-)C57Bl6/J mice develop high-fat diet-induced obesity (DIO) similar to wild-type controls, but that they have significantly improved insulin sensitivity and better glucose tolerance. Using flow cytometry and real-time PCR, we show that STAT4(-/-) mice with DIO produce significantly reduced numbers of inflammatory cytokines and chemokines in adipocytes, have reduced numbers of CD8(+) cells, and display increased alternative (M2) macrophage polarization. CD8(+) cells, but not CD4(+) cells, from STAT4(-/-) mice displayed reduced in vitro migration. Also, we found that adipocyte inflammation is reduced and insulin signaling is improved in STAT4(-/-) mice with DIO. We have identified STAT4 as a key contributor to insulin resistance and AT inflammation in DIO. Targeting STAT4 activation could be a novel approach to reducing AT inflammation and insulin resistance in obesity.