Potencial terapéutico de la proteína reguladora SOCS1 en las complicaciones crónicas de la diabetes

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Farmacología. Fecha de lectura: 19-02-2016Esta tesis tiene embargado el acceso al texto completo hasta el 19-08-2017La diabetes mellitus es una enfermedad de una gran prevalencia entre la población mundial que desencadena una serie de complicaciones crónicas en los pacientes, lo que conduce a un deterioro de su calidad de vida. Entre estas complicaciones, la aterosclerosis y la nefropatía constituyen la principal causa de morbimortalidad y enfermedad renal crónica en los pacientes diabéticos, ya que actualmente no existen alternativas terapéuticas que frenen eficazmente la aparición de eventos cardiovasculares o la progresión a insuficiencia renal. La aterosclerosis y la nefropatía diabética comparten características similares desde el punto de vista de su patogenia, entre ellas la activación de mecanismos inflamatorios claves en el inicio y progresión del daño tisular. La vía de señalización intracelular JAK/STAT (Janus quinasa/Transductor de la señal y activador de la transcripción) regula un amplio rango de mediadores implicados en procesos de proliferación celular, inflamación y fibrosis, y es un importante mecanismo por el cual la hiperglicemia y la dislipemia contribuyen a la progresión de las complicaciones de la diabetes. Las proteínas SOCS (supresoras de la señal de citoquinas) constituyen uno de los principales mecanismos que regulan la magnitud y duración de la vía JAK/STAT. Este trabajo se ha centrado en el desarrollo de dos abordajes preclínicos basados en la proteína SOCS1, capaces de reducir la activación crónica de la vía JAK/STAT con el fin de prevenir el inicio y la progresión de la aterosclerosis y la nefropatía diabética. En primer lugar, se investigó el efecto ateroprotector y antiinflamatorio de la inhibición de JAK/STAT, mediante terapia génica con un adenovirus que codifica la proteína SOCS1, en un modelo experimental de aterosclerosis con ratones deficientes en apolipoproteína E (apoE-/-). La expresión del transgen SOCS1 atenuó la activación de STAT1 y STAT3 en aorta, redujo la expresión de sus genes dependientes (citoquinas, quimioquinas, moléculas de adhesión y receptores scavenger) y previno el desarrollo de aterosclerosis en fases tempranas, frenando también la progresión de las lesiones ateroscleróticas en fases avanzadas del modelo animal. Además, la terapia con SOCS1 promovió un fenotipo de placa más estable, caracterizado por un menor contenido de lípidos, linfocitos T y macrófagos M1 proinflamatorios y por un incremento de macrófagos M2 antiinflamatorios y colágeno. A nivel sistémico, la terapia génica con SOCS1 redujo la expresión de citoquinas de respuesta linfocitaria Th1 y Th17 y el estado de activación de los monocitos circulantes. En experimentos in vitro, SOCS1 inhibió la activación de STAT1 y STAT3 y la expresión de genes proinflamatorios en células vasculares y macrófagos estimulados con citoquinas y también redujo la proliferación y la migración celular. En segundo lugar, desarrollamos un péptido sintético con permeabilidad celular y que contiene la región inhibidora de quinasa (KIR) de la proteína SOCS1 para el tratamiento experimental de la aterosclerosis y la nefropatía asociadas a diabetes en un modelo de diabetes por inyección de estreptozotocina en ratones apoE-/-. La administración del péptido derivado de SOCS1 no afectó a la hiperglicemia ni al perfil lipídico de los animales diabéticos. Sin embargo, este tratamiento mostró un efecto ateroprotector en los ratones diabéticos, de manera que la supresión de la activación de STAT1 y STAT3 en la aorta redujo de forma efectiva el tamaño y extensión de las placas de ateroma tanto en fases iniciales como en fases avanzadas del proceso aterosclerótico. Además, las lesiones ateroscleróticas de los animales tratados con el péptido SOCS1 se caracterizaron por un menor contenido de lípidos, linfocitos T y macrófagos, y un mayor contenido de colágeno y células de musculo liso vascular, parámetros indicativos de estabilización de la placa. Asimismo, el tratamiento redujo la expresión de citoquinas proinflamatorias y el estrés oxidativo en el tejido aórtico. Por otra parte, los estudios en tejido renal demostraron el efecto protector del péptido derivado de SOCS1 en la nefropatía diabética experimental. El tratamiento mejoró significativamente la función renal y las lesiones renales asociadas a diabetes (expansión mesangial, infiltrado leucocitario y fibrosis) y redujo la activación de STAT1 y STAT3 y la expresión de genes proinflamatorios y profibróticos en el riñón diabético. Es de destacar que ninguno de estos efectos se observó en los animales tratados con el péptido mutante, utilizado en estos estudios como control estructural. In vitro, el péptido SOCS1 suprimió la activación de STAT y la expresión de sus genes dependientes en células vasculares y renales estimuladas con citoquinas y redujo procesos de proliferación, migración y adhesión celular. En conclusión, estos estudios demuestran que la inhibición de la vía JAK/STAT basada en la acción de la proteína reguladora SOCS1 suprime la inflamación y retarda la progresión del daño vascular y renal en ratones diabéticos, proporcionando así las bases para el desarrollo de nuevas terapias frente a las complicaciones inflamatorias de la diabetes.Diabetes mellitus is a highly prevalent systemic disease worldwide that triggers a series of chronic complications which reduce the quality of life of patients. Among these complications, atherosclerosis and nephropathy are the leading cause of morbidity, mortality and chronic kidney disease in diabetic patients, given that current therapies are unable to effectively stop the occurrence of cardiovascular events or the progression to kidney failure. Furthermore, atherosclerosis and diabetic nephropathy share similar pathological features, including the activation of inflammatory mechanisms that play a crucial role in all stages of tissue damage. The JAK/STAT (Janus kinase/Signal transducer and activator of transcription) intracellular signaling pathway regulates a broad range of mediators implicated in cell proliferation, inflammation and fibrosis and is an important mechanism whereby hyperglycemia and dyslipidemia contribute to the progression of diabetic complications. Among the mechanisms controlling the activation and duration of JAK/STAT, the suppressors of cytokine signaling (SOCS) proteins are highlighted. This study addresses the preclinical development of two different SOCS1-based therapeutic approaches targeting chronic activation of JAK/STAT to prevent the initiation and progression of atherosclerosis and diabetic nephropathy. Firstly, we investigated the atheroprotective and antiinflammatory effect of JAK/STAT inhibition by gene therapy with a SOCS1-encoding adenovirus in an experimental model of atherosclerosis in apolipoprotein E deficient mice (apoE-/-). SOCS1 transgene expression in mouse aorta attenuated STAT1 and STAT3 activation and the expression of STAT-dependent genes (cytokines, chemokines, adhesion molecules and scavenger receptors), therefore preventing early development of atherosclerosis and further progression to advanced lesions. Furthermore, SOCS1 gene delivery promoted a more stable plaque phenotype characterized by lower content of lipids, T lymphocytes and proinflammatory M1 macrophages and higher content of antiinflammatory M2 macrophages and collagen. Systemically, SOCS1 gene therapy reduced the expression of Th1 and Th17 cytokines and the activation state of circulating monocytes. In vitro, SOCS1 prevented STAT1 and STAT3 activation and proinflammatory gene expression in cytokine-stimulated vascular cells and macrophages, and also reduced cell proliferation and migration. Secondly, we developed a synthetic cell-permeable peptide containing the kinase inhibitory region (KIR) of SOCS1 as an experimental therapy for diabetes-associated atherosclerosis and nephropathy in streptozotocin-induced diabetic apoE-/- mice. Administration of SOCS1-derived peptide did not affect hyperglycemia and lipid profile in diabetic mice but resulted in atheroprotection. Indeed, SOCS1 peptide therapy suppressed STAT1 and STAT3 activity in mouse aorta and effectively reduced the size and extension of atheroma plaques at both early and advanced stages of atherosclerosis. Furthermore, atherosclerotic lesions of peptide-treated mice exhibited a reduced content of lipids, T lymphocytes and macrophages, and increased collagen and smooth muscle cell content, indicating a more stable plaque phenotype. Likewise, SOC1 peptide treatment reduced proinflammatory cytokine expression and oxidative stress in the aortic tissue. On the other hand, studies in kidney tissue revealed a protective effect of SOCS1 peptide in experimental diabetic nephropathy, as evidenced by a significant improvement of renal function and renal lesions (mesangial expansion, leukocyte infiltration and fibrosis). Treatment also attenuated STAT1 and STAT3 activation and reduced the expression of proinflammatory and profibrotic genes in diabetic kidneys. It is noteworthy that no significant effects were observed in mice treated with mutant peptide, the structural control. In vitro, SOCS1 peptide suppressed STAT activation and STAT-target gene expression in cytokine-stimulated vascular and renal cells, therefore preventing cell proliferation, migration and adhesion. Collectively, our studies demonstrate that SOCS1-based inhibition of JAK/STAT signaling pathway suppresses inflammation and retards the progression of vascular and renal injury in diabetic mice, thus providing the basis for the development of new therapies against diabetes inflammatory complications

Control of Liver Gene Expression by Sex Steroids and Growth Hormone Interplay

Sex steroids have important physiological actions, which are not limited to reproductive organs, in both females and males. They exert important physiological roles, including the regulation of somatotropic-liver axis, intermediate metabolism, or gender dimorphism. This is in part because the liver is a sex steroid-responsive organ where sex steroid- and growth hormone (GH)-dependent signaling pathways connect to regulate complex gene expression networks. Sex steroids can impact liver gene expression by a direct, through hepatic estrogen receptor (ER)α and androgen receptor (AR), or indirect mechanisms, by modulation of pituitary GH secretion and/or interaction with the GHR-STAT5b signaling pathway. Therefore, deficiency of sex steroid- and GH-dependent signaling pathways might cause a dramatic impact on mammalian liver physiology. In this chapter, we will focus our attention on main concepts and paradigms involved in the role and interplay between sex steroid- and GH-dependent signaling to regulate gene expression networks in the mammalian liver. A better understanding of how sex steroids and interactions with GH-STAT5b signaling pathway influence physiological and pathological states in the liver will contribute to improve clinical management of patients with disorders in body growth, development, and metabolism

Cannabinoid Receptor 2 Modulates Neutrophil Recruitment in a Murine Model of Endotoxemia

The endocannabinoid system consists of endogenous lipid mediators and cannabinoid receptors (CB) 1 and 2. It has previously been demonstrated that activation of the leukocyte-expressed CB2 has anti-inflammatory effects in vivo. Here, we report its role under baseline conditions and in a model of low-dose endotoxemia by comparing CB2 knockout to littermate control mice. CB2-deficient mice displayed significantly more neutrophils and fewer monocytes in the bone marrow under steady state. In initial validation experiments, administration of 1 mg/kg LPS to male C57BL/6J mice was shown to transiently upregulate systemic proinflammatory mediators (peaked at 2 hours) and mobilise bone marrow neutrophils and monocytes into circulation. In CB2 knockout mice, the level of the metalloproteinase MMP-9 was significantly elevated by 2 hours and we also observed augmented recruitment of neutrophils to the spleen in addition to increased levels of Ccl2, Ccl3, Cxcl10, and Il6. Collectively, our data show that the absence of CB2 receptor increases the levels of innate immune cell populations in the bone marrow under steady state. Furthermore, during an acute systemic inflammatory insult, we observe a highly reproducible and site-specific increase in neutrophil recruitment and proinflammatory chemokine expression in the spleen of CB2 knockout mice

JAK, an Oncokinase in Hematological Cancer

Janus kinases (JAKs) play an essential role in the regulation of cytokine signaling. They control cell survival, proliferation, differentiation, immune response, and hematopoiesis. Deregulation of JAK signaling has been associated to the pathogenesis of numerous immune-inflammatory diseases, hematological malignancies, and solid tumors. Thus, JAK proteins have emerged as attractive therapeutic targets in the last decade. The discovery of the gain-of-function JAK2 mutation (JAK2 V617F) as the main cause of polycythemia vera—a chronic myeloproliferative syndrome—led to the development of the JAK inhibitor ruxolitinib. This key finding opened the door to the search for new therapeutic agents able to suppress the constitutive activation of JAK signaling in hematological cancers and other tumors. However, given the conserved nature of the kinase domain among JAK family members, and the interrelated roles of JAK kinases in many physiological processes, including hematopoiesis and immunity, the broad usage of JAK inhibitors in hematology is challenged by their narrow therapeutic window. Novel therapies are, therefore, needed. This chapter focuses on the understanding of the complex signaling of JAK proteins in cancerous cells, the various JAK aberrations implicated in myeloproliferative neoplasms, leukemia, and lymphoma, and the clinically available JAK inhibitors in cancer therapy

Absence of the non-signalling chemerin receptor CCRL2 exacerbates acute inflammatory responses in vivo

Chemerin is a chemotactic protein that induces migration of several immune cells including macrophages, immature dendritic cells, and NK cells. Chemerin binds to three G protein-coupled receptors (GPCRs), including CCRL2. The exact function of CCRL2 remains unclear. CCRL2 expression is rapidly upregulated during inflammation, but it lacks the intracellular DRYLAIV motif required for classical GPCR downstream signalling pathways, and it has not been reported to internalise chemerin upon binding. The aim of this study was to investigate what role if any CCRL2 plays during acute inflammation. Using the zymosan- and thioglycollate-induced murine models of acute inflammation, we report that mice deficient in the Ccrl2 gene display exaggerated local and systemic inflammatory responses, characterised by increased myeloid cell recruitment. This amplified myeloid cell recruitment was associated with increased chemerin and CXCL1 levels. Furthermore, we report that the inflammatory phenotype observed in these mice is dependent upon elevated levels of endogenous chemerin. Antibody neutralisation of chemerin activity in Ccrl2−/− mice abrogated the amplified inflammatory responses. Importantly, chemerin did not directly recruit myeloid cells but rather increased the production of other chemotactic proteins such as CXCL1. Administration of recombinant chemerin to wild-type mice before inflammatory challenge recapitulated the increased myeloid cell recruitment and inflammatory mediator production observed in Ccrl2−/− mice. We have demonstrated that the absence of CCRL2 results in increased levels of local and systemic chemerin levels and exacerbated inflammatory responses during acute inflammatory challenge. These results further highlight the importance of chemerin as a therapeutic target in inflammatory diseases

JKST6, a novel multikinase modulator of the BCR-ABL1/STAT5 signaling pathway that potentiates direct BCR-ABL1 inhibition and overcomes imatinib resistance in chronic myelogenous leukemia

Chronic myelogenous leukemia (CML) is a hematological malignancy that highly depends on the BCR-ABL1/STAT5 signaling pathway for cell survival. First-line treatments for CML consist of tyrosine kinase inhibitors that efficiently target BCR-ABL1 activity. However, drug resistance and intolerance are still therapeutic limitations in Ph+ cells. Therefore, the development of new anti-CML drugs that exhibit alternative mechanisms to overcome these limitations is a desirable goal. In this work, the antitumoral activity of JKST6, a naphthoquinone-pyrone hybrid, was assessed in imatinib-sensitive and imatinib-resistant human CML cells. Live-cell imaging analysis revealed JKST6 potent antiproliferative activity in 2D and 3D CML cultures. JKST6 provoked cell increase in the subG1 phase along with a reduction in the G0/G1 phase and altered the expression of key proteins involved in the control of mitosis and DNA damage. Rapid increases in Annexin V staining and activation/cleavage of caspases 8, 9 and 3 were observed after JKST6 treatment in CML cells. Of interest, JKST6 inhibited BCR-ABL1/STAT5 signaling through oncokinase downregulation that was preceded by rapid polyubiquitination. In addition, JKST6 caused a transient increase in JNK and AKT phosphorylation, whereas the phosphorylation of P38-MAPK and Src was reduced. Combinatory treatment unveiled synergistic effects between imatinib and JKST6. Notably, JKST6 maintained its antitumor efficacy in BCR-ABL1-T315I-positive cells and CML cells that overexpress BCR-ABL and even restored imatinib efficacy after a short exposure time. These findings, together with the observed low toxicity of JKST6, reveal a novel multikinase modulator that might overcome the limitations of BCR-ABL1 inhibitors in CML therapy.This research has been funded by Spanish Ministry of Economy and Competitiveness - MINECO - (SAF 2015–65113-C2–1-R and RTI2018–094356-B-C21 to AEB, SAF2015–65113-C2–2 to LFP, SAF2017–88026-R to JL) with the co-funding of European Regional Development Fund (EU-ERDF), Canary Islands Government (CEI2018–23/ACIISI to BG, CEI2019–08/ACIISI to BG and LFP, ProID2021010037 to AEB, LFP and BG) and "Juan de la Cierva Incorporacion" Grant Program from the Ministry of Science, Innovation and Universities (IJC2018-035193-I to CR). This project has been also supported by Alfredo Martin-Reyes Foundation (Arehucas)-Canary Islands Foundation for Cancer Research (FICIC). HAT is recipient of a predoctoral program grant from ULPGC (2016). JCM was funded by the Instituto de Salud Carlos III through a Miguel Servet program (CPII17/ 00015)

Nrf2 Activation Provides Atheroprotection in Diabetic Mice Through Concerted Upregulation of Antioxidant, Anti-inflammatory, and Autophagy Mechanisms

Interactive relationships between metabolism, inflammation, oxidative stress, and autophagy in the vascular system play a key role in the pathogenesis of diabetic cardiovascular disease. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a stress-sensitive guarantor of cellular homeostasis, which cytoprotective contributions extend beyond the antioxidant defense. We investigated the beneficial effects and underlying mechanisms of the Nrf2 inducer tert-butyl hydroquinone (tBHQ) on diabetes-driven atherosclerosis. In the experimental model of streptozotocin-induced diabetes in apolipoprotein E-deficient mice, treatment with tBHQ increased Nrf2 activity in macrophages and vascular smooth muscle cells within atherosclerotic lesions. Moreover, tBHQ significantly decreased the size, extension and lipid content of atheroma plaques, and attenuated inflammation by reducing lesional macrophages (total number and M1/M2 phenotype balance), foam cell size and chemokine expression. Atheroprotection was accompanied by both systemic and local antioxidant effects, characterized by lower levels of superoxide anion and oxidative DNA marker 8-hydroxy-2′-deoxyguanosine, reduced expression of NADPH oxidase subunits, and increased antioxidant capacity. Interestingly, tBHQ treatment upregulated the gene and protein expression of autophagy-related molecules and also enhanced autophagic flux in diabetic mouse aorta. In vitro, Nrf2 activation by tBHQ suppressed cytokine-induced expression of pro-inflammatory and oxidative stress genes, altered macrophage phenotypes, and promoted autophagic activity. Our results reinforce pharmacological Nrf2 activation as a promising atheroprotective approach in diabetes, according to the plethora of cytoprotective mechanisms involved in the resolution of inflammation and oxidative stress, and restoring autophagy

SOCS2 protects against chemical-induced hepatocellular carcinoma progression by modulating inflammation and cell proliferation in the liver

Hepatocellular carcinoma (HCC) is one of the most prevalent and lethal cancers worldwide, but the precise intracellular mechanisms underlying the progression of this inflammation associated cancer are not well established. SOCS2 protein plays an important role in the carcinogenesis of different tumors by regulating cytokine signalling through the JAK/STAT axis. However, its role in HCC is unclear. Here, we investigate the role of SOCS2 in HCC progression and its potential as HCC biomarker. The effects of SOCS2 in HCC progression were evaluated in an experimental model of diethylnitrosamine (DEN)-induced HCC in C57BL/6 and SOCS2 deficient mice, in cultured hepatic cells, and in liver samples from HCC patients. Mice lacking SOCS2 showed higher liver tumor burden with increased malignancy grade, inflammation, fibrosis, and proliferation than their controls. Protein and gene expression analysis reported higher pSTAT5 and pSTAT3 activation, upregulation of different proteins involved in survival and proliferation, and increased levels of proinflammatory and pro-tumoral mediators in the absence of SOCS2. Clinically relevant, downregulated expression of SOCS2 was found in neoplasia from HCC patients compared to healthy liver tissue, correlating with the malignancy grade. In summary, our data show that lack of SOCS2 increases susceptibility to chemical-induced HCC and suggest the tumor suppressor role of this protein by regulating the oncogenic and inflammatory responses mediated by STAT5 and STAT3 in the liver. Hence, SOCS2 emerges as an attractive target molecule and potential biomarker to deepen in the study of HCC treatment.This research was supported by the “Premios Fundación DISA a la Investigación Biomédica 2019” funded by DISA Foundation, “Ayudas para la financiación de Proyectos de investigación, programa de ayudas ULPGC 2018” funded by the University of Las Palmas of Gran Canaria, and “Ayudas Juan de la Cierva Incorporación 2018” funded by the Ministry of Science and Innovation.Peer reviewe

Design and development of a digital intervention for workplace stress and mental health (EMPOWER)

Purpose: We describe the design and development of the European Platform to Promote health and wellbeing in the workplace (EMPOWER) digital intervention that provides an integrative user programme meeting the needs of employees and employers in addressing work stress. Results: A user-centred design process was followed from January 2020 until November 2021. A tailored algorithm was developed to provide support at the individual employee level and the company level. Each element of the digital intervention was developed in English and then translated in Spanish, English, Polish and Finnish. The digital intervention consists of a website and a mobile application (app) that provides algorithm-based personalised content after assessing a user's somatic and psychological symptoms, work functioning, and psychosocial risk factors for work stress. It has a public section and an employer portal that provides recommendations to reduce psychosocial risks in their company based upon clustered input from employees. Usability testing was conducted and showed high ease of use and completion of tasks by participants. Conclusion: The EMPOWER digital intervention is a tailored multimodal intervention addressing wellbeing, work stress, mental and physical health problems, and work productivity. This will be used in a planned RCT in four countries to evaluate its effectiveness

Spread of a SARS-CoV-2 variant through Europe in the summer of 2020

[EN] Following its emergence in late 2019, the spread of SARS-CoV-21,2 has been tracked by phylogenetic analysis of viral genome sequences in unprecedented detail3,4,5. Although the virus spread globally in early 2020 before borders closed, intercontinental travel has since been greatly reduced. However, travel within Europe resumed in the summer of 2020. Here we report on a SARS-CoV-2 variant, 20E (EU1), that was identified in Spain in early summer 2020 and subsequently spread across Europe. We find no evidence that this variant has increased transmissibility, but instead demonstrate how rising incidence in Spain, resumption of travel, and lack of effective screening and containment may explain the variant’s success. Despite travel restrictions, we estimate that 20E (EU1) was introduced hundreds of times to European countries by summertime travellers, which is likely to have undermined local efforts to minimize infection with SARS-CoV-2. Our results illustrate how a variant can rapidly become dominant even in the absence of a substantial transmission advantage in favourable epidemiological settings. Genomic surveillance is critical for understanding how travel can affect transmission of SARS-CoV-2, and thus for informing future containment strategies as travel resumes.S