El factor 2 de la Red de Comunicación Celular (CCN2) ejerce un papel esencial en el mantenimiento de la estructura y las respuestas vasculares de la aorta en ratones

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Medicina. Fecha de Lectura: 24-03-2023La prevalencia y la mortalidad de las enfermedades cardiovasculares (ECVs) han aumentado sustancialmente durante las últimas décadas. Un claro ejemplo de ello los aneurismas aórticos (AA), para los cuales no existen, actualmente, biomarcadores de fácil detección que permitan su diagnóstico precoz ni de dianas terapéuticas directas para su tratamiento. En este sentido, existen diversas alteraciones en genes esenciales para el mantenimiento funcional y estructural de los vasos sanguíneos descritas en patologías que cursan AA, como los síndromes de Marfan o Loeys-Dietz, que han cobrado una mayor importancia en los últimos años en el contexto aneurismático, siendo un claro ejemplo la vía molecular de TGF-β. Considerando que CCN2 es uno de los genes más sobrexpresados por TGF-β y su aumento en diversas patologías cardiovasculares, la primera parte del objetivo principal de esta tesis doctoral se centró en investigar los efectos a nivel aórtico inducidos por la deleción inducida de Ccn2 en ratones en condiciones fisiológicas y en un contexto de daño vascular. Para ello, se utilizaron ratones CCN2flox/floxROSA26-ERT/Cre (CCN2-KO), a los que se implantó una minibomba de infusión sistémica con angiotensina II (Ang II) o suero salino durante 15 días. Así, se demostró que aquellos ratones CCN2-KO infundidos con Ang II presentaban una elevada tasa de mortalidad debida a un desarrollo temprano de AA y un elevado índice de rotura. Además, la ausencia de CCN2 generó la disrupción de las fibras de elastina en la pared aórtica, variaciones en la función vascular y un incremento de la actividad metaloproteinasa, viéndose exacerbado con la infusión de Ang II. Curiosamente, la aproximación experimental para mantener los niveles de CCN2 elevados en los ratones CCN2-KO mediante la infusión sistémica del fragmento C terminal de CCN2 (CCN2(IV)) recombinante no modificó el desarrollo de AA ni los ratios de mortalidad tras la infusión de Ang II. Posteriormente, se realizó una secuenciación masiva del ARN mensajero del tejido aórtico que reflejó la existencia de un patrón de expresión génica diferencial entre los ratones CCN2-KO y los ratones control. El subsecuente análisis ontológico de los resultados de la secuenciación masiva reflejó que la biosíntesis de aldosterona era uno de los procesos bilógicos más enriquecidos, por lo que se realizó un nuevo modelo experimental en el que un grupo de ratones CCN2-KO fue tratado de manera preventiva con un bloqueante del receptor de mineralocorticoides, espironolactona. Los resultados obtenidos determinaron que el bloqueo de la vía de aldosterona inducía una bajada en el número de aneurismas aórticos generados en estos ratones, así como una reducción de la mortalidad asociada a ellos, debido, en parte, a la prevención del incremento de la actividad metaloproteinasa y a una mejora de la función vascular. La segunda parte del objetivo principal consistió en investigar el efecto de CCN2 en la regulación de los niveles de los receptores de TGF-β (TGFβR1 y TGFβR2) en una línea de células de músculo liso vascular de aorta de ratón. De esta manera, se observó que el tratamiento durante 48 horas con el fragmento CCN2(IV) aumentaba los niveles génicos y proteicos de TGFβR2, mientras que no generaba diferencias en los niveles de TGFβR1. Además, se comprobó que este incremento en los niveles de TGFβR2 estaba mediado directamente por la activación del receptor de EGF y de la ruta SMAD inducida directamente por CCN2 de manera independiente de TGF-β. Los datos obtenidos en esta tesis demuestran, por tanto, el papel crítico que desempeña CCN2 en el mantenimiento de la homeostasis vascular, tanto funcional como estructural, y su papel directo en la activación de la ruta de TGF-β, lo cual supone un cambio en el paradigma de la función que ejerce CCN2 en las patologías vasculare

Diseño de un software de intermediación de comunicación para sistemas distribuidos de tiempo real críticos en Java

Las facilidades e independencia de plataforma de Java han generado un gran interés en la comunidad de tiempo real. Dicho interés se ha reflejado en la especificación RTSJ (Real-Time Specification for Java), que extiende y adapta el lenguaje Java para permitir el desarrollo de sistemas de tiempo real. Adicionalmente, se han desarrollado perfiles de RTSJ para garantizar la predecibilidad en sistemas de tiempo real críticos. Sin embargo, RTSJ y sus perfiles no proporcionan facilidades para sistemas distribuidos. El objetivo de este trabajo es afrontar dicha limitación definiendo un nuevo modelo de RMI (Remote Method Invocation) basado en los principales perfiles de RTSJ para sistemas de tiempo real crítico. Este trabajo presenta el diseño y la implementación de RMI-HRT (RMI-Hard Real-Time) que está enfocado a sistemas de tiempo real crítico con requisitos de alta integridad

Postnatal overfeeding during lactation induces endothelial dysfunction and cardiac insulin resistance in adult rats

Early overnutrition is associated with cardiometabolic alterations in adulthood, likely attributed to reduced insulin sensitivity due to its crucial role in the cardiovascular system. This study aimed to assess the long-term effects of early overnutrition on the development of cardiovascular insulin resistance. An experimental childhood obesity model was established using male Sprague Dawley rats. Rats were organized into litters of 12 pups/mother (L12-Controls) or 3 pups/mother (L3-Overfed) at birth. After weaning, animals from L12 and L3 were housed three per cage and provided ad libitum access to food for 6 months. L3 rats exhibited elevated body weight, along with increased visceral, subcutaneous, and perivascular fat accumulation. However, heart weight at sacrifice was reduced in L3 rats. Furthermore, L3 rats displayed elevated serum levels of glucose, leptin, adiponectin, total lipids, and triglycerides compared to control rats. In the myocardium, overfed rats showed decreased IL-10 mRNA levels and alterations in contractility and heart rate in response to insulin. Similarly, aortic tissue exhibited modified gene expression of TNFα, iNOS, and IL-6. Additionally, L3 aortas exhibited endothelial dysfunction in response to acetylcholine, although insulin-induced relaxation remained unchanged compared to controls. At the molecular level, L3 rats displayed reduced Akt phosphorylation in response to insulin, both in myocardial and aortic tissues, whereas MAPK phosphorylation was elevated solely in the myocardium. Overfeeding during lactation in rats induces endothelial dysfunction and cardiac insulin resistance in adulthood, potentially contributing to the cardiovascular alterations observed in this experimental mode

A mixture of algae and extra virgin olive oils attenuates the cardiometabolic alterations associated with aging in male wistar rats

Aging is one of the major risk factors for suffering cardiovascular and metabolic diseases. Due to the increase in life expectancy, there is a strong interest in the search for anti-aging strategies to treat and prevent these aging-induced disorders. Both omega 3 polyunsaturated fatty acids (ω-3 PUFA) and extra virgin olive oil (EVOO) exert numerous metabolic and cardiovascular benefits in the elderly. In addition, EVOO constitutes an interesting ingredient to stabilize ω-3 PUFA and decrease their oxidation process due to its high content in antioxidant compounds. ω-3 PUFA are commonly obtained from fish. However, more ecological and sustainable sources, such as algae oil (AO) can also be used. In this study, we aimed to study the possible beneficial effect of an oil mixture composed by EVOO (75%) and AO (25%) rich in ω-3 PUFA (35% docosahexaenoic acid (DHA) and 20% eicosapentaenoic acid (EPA)) on the cardiometabolic alterations associated with aging. For this purpose; young (three months old) and old (24 months old) male Wistar rats were treated with vehicle or with the oil mixture (2.5 mL/kg) for 21 days. Treatment with the oil mixture prevented the aging-induced increase in the serum levels of saturated fatty acids (SFA) and the aging-induced decrease in the serum concentrations of mono-unsaturated fatty acids (MUFA). Old treated rats showed increased serum concentrations of EPA and DHA and decreased HOMA-IR index and circulating levels of total cholesterol, insulin and IL-6. Treatment with the oil mixture increased the mRNA levels of antioxidant and insulin sensitivity-related enzymes, as well as reduced the gene expression of pro-inflammatory markers in the liver and in cardiac and aortic tissues. In addition, the treatment also prevented the aging-induced endothelial dysfunction and vascular insulin resistance through activation of the PI3K/Akt pathway. Moreover, aortic rings from old rats treated with the oil mixture showed a decreased response to the vasoconstrictor AngII. In conclusion, treatment with a mixture of EVOO and AO improves the lipid profile, insulin sensitivity and vascular function in aged rats and decreases aging-induced inflammation and oxidative stress in the liver, and in the cardiovascular system. Thus, it could be an interesting strategy to deal with cardiometabolic alterations associated with aging.This project was funded by the call “Doctorados Industriales 2017” (IND2017/BIO7701), a grant from Community of Madrid (Spain). This program aims to promote the effective collaboration between Universities and Companies and provides funding for the development of the research project at the University and, to hire a PhD student (Daniel González-Hedström) by the Company (Pharmactive Biotech Products S.L.) over a three-year period. Community of Madrid also funded the contract of María de la Fuente-Fernández through the Youth Employment Program (PEJ-2018-AI/SAL-11315

Overfeeding during lactation in rats is associated with cardiovascular insulin resistance in the short-term

Childhood obesity is associated with metabolic and cardiovascular comorbidities. The development of these alterations may have its origin in early life stages such as the lactation period through metabolic programming. Insulin resistance is a common complication in obese patients and may be responsible for the cardiovascular alterations associated with this condition. This study analyzed the development of cardiovascular insulin resistance in a rat model of childhood overweight induced by overfeeding during the lactation period. On birth day, litters were divided into twelve (L12) or three pups per mother (L3). Overfed rats showed a lower increase in myocardial contractility in response to insulin perfusion and a reduced insulin-induced vasodilation, suggesting a state of cardiovascular insulin resistance. Vascular insulin resistance was due to decreased activation of phosphoinositide 3-kinase (PI3K)/Akt pathway, whereas cardiac insulin resistance was associated with mitogen-activated protein kinase (MAPK) hyperactivity. Early overfeeding was also associated with a proinflammatory and pro-oxidant state; endothelial dysfunction; decreased release of nitrites and nitrates; and decreased gene expression of insulin receptor (IR), glucose transporter-4 (GLUT-4), and endothelial nitric oxide synthase (eNOS) in response to insulin. In conclusion, overweight induced by lactational overnutrition in rat pups is associated with cardiovascular insulin resistance that could be related to the cardiovascular alterations associated with this conditionThis study has been funded by the grant to Precompetitive Projects of Universidad San Pablo CEU and Banco Santander 2016, and by a grant from the Community of Madrid (CAM) awarded to Daniel González-Hedström (IND2017/BIO7701, Spain

Supplementation with a carob (Ceratonia siliqua l.) fruit extract attenuates the cardiometabolic alterations associated with metabolic syndrome in mice

The incidence of metabolic syndrome (MetS) is increasing worldwide which makes necessary the finding of new strategies to treat and/or prevent it. The aim of this study was to analyze the possible beneficial effects of a carob fruit extract (CSAT+®) on the cardiometabolic alterations associated with MetS in mice. 16-week-old C57BL/6J male mice were fed for 26 weeks either with a standard diet (chow) or with a diet rich in fats and sugars (HFHS), supplemented or not with 4.8% of CSAT+®. CSAT+® supplementation reduced blood glucose, Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) and circulating levels of total cholesterol, low-density lipoprotein (LDL) cholesterol (LDL-c), insulin, and interleukin-6 (IL-6). In adipose tissue and skeletal muscle, CSAT+® prevented MetS-induced insulin resistance, reduced macrophage infiltration and the expression of pro-inflammatory markers, and up-regulated the mRNA levels of antioxidant markers. Supplementation with CSAT+® prevented MetS-induced hypertension and decreased the vascular response of aortic rings to angiotensin II (AngII). Moreover, treatment with CSAT+® attenuated endothelial dysfunction and increased vascular sensitivity to insulin. In the heart, CSAT+® supplementation reduced cardiomyocyte apoptosis and prevented ischemia-reperfusion-induced decrease in cardiac contractility. The beneficial effects at the cardiovascular level were associated with a lower expression of pro-inflammatory and pro-oxidant markers in aortic and cardiac tissues.This work has been funded by Pharmactive Biotech Products S.L. and by Grants from Community of Madrid awarded to Daniel González-Hedström (IND2017/BIO7701,) and María de la Fuente-Fernández (PEJ-2018-AI/SAL-11315

CCN2 Binds to Tubular Epithelial Cells in the Kidney

Cellular communication network-2 (CCN2), also called connective tissue growth factor (CTGF), is considered a fibrotic biomarker and has been suggested as a potential therapeutic target for kidney pathologies. CCN2 is a matricellular protein with four distinct structural modules that can exert a dual function as a matricellular protein and as a growth factor. Previous experiments using surface plasmon resonance and cultured renal cells have demonstrated that the C-terminal module of CCN2 (CCN2(IV)) interacts with the epidermal growth factor receptor (EGFR). Moreover, CCN2(IV) activates proinflammatory and profibrotic responses in the mouse kidney. The aim of this paper was to locate the in vivo cellular CCN2/EGFR binding sites in the kidney. To this aim, the C-terminal module CCN2(IV) was labeled with a fluorophore (Cy5), and two different administration routes were employed. Both intraperitoneal and direct intra-renal injection of Cy5-CCN2(IV) in mice demonstrated that CCN2(IV) preferentially binds to the tubular epithelial cells, while no signal was detected in glomeruli. Moreover, co-localization of Cy5-CCN2(IV) binding and activated EGFR was found in tubules. In cultured tubular epithelial cells, live-cell confocal microscopy experiments showed that EGFR gene silencing blocked Cy5-CCN2(IV) binding to tubuloepithelial cells. These data clearly show the existence of CCN2/EGFR binding sites in the kidney, mainly in tubular epithelial cells. In conclusion, these studies show that circulating CCN2(IV) can directly bind and activate tubular cells, supporting the role of CCN2 as a growth factor involved in kidney damage progression

Oxidative Stress and Cellular Senescence Are Involved in the Aging Kidney

Chronic kidney disease (CKD) can be considered as a clinical model for premature aging. However, non‐invasive biomarkers to detect early kidney damage and the onset of a senescent phenotype are lacking. Most of the preclinical senescence studies in aging have been done in very old mice. Furthermore, the precise characterization and over-time development of age-related senes-cence in the kidney remain unclear. To address these limitations, the age-related activation of cellular senescence-associated mechanisms and their correlation with early structural changes in the kidney were investigated in 3- to 18-month-old C57BL6 mice. Inflammatory cell infiltration was ob-served by 12 months, whereas tubular damage and collagen accumulation occurred later. Early activation of cellular-senescence-associated mechanisms was found in 12-month-old mice, character-ized by activation of the DNA-damage-response (DDR), mainly in tubular cells; activation of the antioxidant NRF2 pathway; and klotho downregulation. However, induction of tubular-cell-cycle-arrest (CCA) and overexpression of renal senescent-associated secretory phenotype (SASP) components was only found in 18-month-old mice. In aging mice, both inflammation and oxidative stress (marked by elevated lipid peroxidation and NRF2 inactivation) remained increased. These findings support the hypothesis that prolonged DDR and CCA, loss of nephroprotective factors (klotho), and dysfunctional redox regulatory mechanisms (NRF2/antioxidant defense) can be early drivers of age-related kidney-damage progressionThis research was funded by grants from the Instituto de Salud Carlos III (ISCIII); Fondos FEDER European Union (PI17/00119, PI20/00140; and DTS20/00083 to M.R.-O.; PI18/01133 to A.M.R.); Sara Borrell’ program from Instituto de Salud Carlos III (ISCIII) (grant number CD20/00042 to R.R.R.-D.); Red de Investigación Renal REDINREN: RD16/0009/0003 and RICORS program to RICORS2040 496 (RD21/0005), to M.R.-O., Sociedad Española de Nefrología; “NOVELREN-CM: Enfermedad renal crónica: nuevas Estrategias para la prevención, Diagnóstico y tratamiento” (B2017/BMD3751 to M.R.-O.); “Convocatoria Dinamización Europa Investigación 2019” MINECO (EIN2019-103294 to M.R.-O.); Juan de la Cierva incorporacion grant: IJC2018-035187-I to S.R.-M.; innovation program under the Marie Skłodowska-Curie grant of the European Union’s Horizon 2020 (IMProvePD ID: 812699) to M.R.-O.; and Fundacion Conchita Rabago to L.T.-

Epigenetic Modulation of Gremlin-1/NOTCH Pathway in Experimental Crescentic Immune-Mediated Glomerulonephritis

Crescentic glomerulonephritis is a devastating autoimmune disease that without early and properly treatment may rapidly progress to end-stage renal disease and death. Current immunosuppressive treatment provides limited efficacy and an important burden of adverse events. Epigenetic drugs are a source of novel therapeutic tools. Among them, bromodomain and extraterminal domain (BET) inhibitors (iBETs) block the interaction between bromodomains and acetylated proteins, including histones and transcription factors. iBETs have demonstrated protective effects on malignancy, inflammatory disorders and experimental kidney disease. Recently, Gremlin-1 was proposed as a urinary biomarker of disease progression in human anti-neutrophil cytoplasmic antibody (ANCA)-associated crescentic glomerulonephritis. We have now evaluated whether iBETs could regulate Gremlin-1 in experimental anti-glomerular basement membrane nephritis induced by nephrotoxic serum (NTS) in mice, a model resembling human crescentic glomerulonephritis. In NTS-injected mice, the iBET JQ1 inhibited renal Gremlin-1 overexpression and diminished glomerular damage, restoring podocyte numbers. Chromatin immunoprecipitation assay demonstrated BRD4 enrichment of the Grem-1 gene promoter in injured kidneys, consistent with Gremlin-1 epigenetic regulation. Moreover, JQ1 blocked BRD4 binding and inhibited Grem-1 gene transcription. The beneficial effect of iBETs was also mediated by modulation of NOTCH pathway. JQ1 inhibited the gene expression of the NOTCH effectors Hes-1 and Hey-1 in NTS-injured kidneys. Our results further support the role for epigenetic drugs, such as iBETs, in the treatment of rapidly progressive crescentic glomerulonephritis