Regulation of endothelin synthesis by extracellular matrix in human endothelial cells

Regulation of endothelin synthesis by extracellular matrix in human endothelial cells.BackgroundVascular diseases are characterized by the presence of structural changes and the progressive loss of endothelial function. Although the biochemical basis of these structural changes have started to be outlined, it seems that accumulation of normal extracellular matrix proteins as well as the appearance of interstitial collagens, mainly collagen type I, characterize this process. On the other hand, a role for endothelial vasoactive factors has been proposed in the genesis of endothelial dysfunction, and it is generally accepted that changes in extracellular matrix composition may modify cell behavior.MethodsExperiments were designed to test the influence of the supporting matrix on endothelin-1 (ET-1) synthesis by endothelial cells. Northern blot experiments were performed to analyze the prepro-endothelin-1 (prepro-ET-1) mRNA expression. ET-1 production was measured by ELISA.ResultsCells grown on collagen type I (Col I) showed an increase of prepro-ET-1 mRNA level when compared with cells cultured on collagen type IV (Col IV). According to these results, the release of ET-1 to culture medium was also higher in Col I-grown cells than in those cultured on Col IV. Treatment of cells with a peptide that interferes with Col I integrins (D6Y), or with protein tyrosine kinase inhibitors such as genistein and herbimycin, completely abolished the effect of Col I. Moreover, experiments with antibodies against integrins suggest that these cell surface receptors could be involved in the modulation of ET-1 system by extracellular matrix.ConclusionsThese results suggest that the presence of an abnormal extracellular matrix could stimulate endothelin synthesis by human endothelial cells, through integrin activation

Integrin Linked Kinase (ILK) Downregulation as an Early Event During the Development of Metabolic Alterations in a Short-Term High Fat Diet Mice Model.

Background/Aims: Diabetes type 2, metabolic syndrome or non-alcoholic fatty liver disease are insulin resistance-related metabolic disorders, which lack a better prognosis before their full establishment. We studied the importance of the intracellular scaffold protein integrin linked kinaes (ILK) as a key modulator in the initial pathogenesis and the early progression of those insulin resistance- related disorders. Methods: Adult mice with a global transgenic downregulation of ILK expression (cKD-ILK) and littermates without that depletion (CT) were fed with either standard (STD) or high fat (HFD) diets during 2 and 6 weeks. Weights, blood glucose and other systemic biochemical parameters were determined in animals under fasting conditions and after glucose or pyruvate intraperitoneal injections to test their tolerance. In RNA or proteins extracted from insulin-sensitive tissues, we determined by reverse transcription?quantitative PCR and western blot the expression of ILK, metabolites transporters and other metabolism and inflammatory markers. Glucose uptake capacity was studied in freshly isolated tissues. Results: HFD feeding was able to early and progressively increase glycaemia, insulinemia, circulating glycerol, body weight gain, liver-mediated gluconeogenesis along this time lapse, but cKD-ILK have all these systemic misbalances exacerbated compared to CT in the same HFD time lapse. Interestingly, the tisular expression of ILK in HFD-fed CT was dramatically downregulated in white adipose tissue (WAT), skeletal muscle and liver at the same extent of the original ILK downregulation of cKD-ILK. We previously published that basal STD-fed cKD-ILK compared to basal STD-CT have different expression of glucose transporters GLUT4 in WAT and skeletal muscle. In the same STD-fed cKD-ILK, we observed here the increased expressions of hepatic GLUT2 and WAT pro-inflammatory cytokines TNF-? and MCP-1. The administration of HFD exacerbated the expression changes in cKD-ILK of these and other markers related to the imbalanced metabolism observed, such as WAT lipolysis (HSL), hepatic gluconeogenesis (PCK-1) and glycerol transport (AQP9). Conclusion: ILK expression may be taken as a predictive determinant of metabolic disorders establishment, because its downregulation seems to correlate with the early imbalance of glucose and glycerol transport and the subsequent loss of systemic homeostasis of these metabolites.Instituto de Salud Carlos III-ISCIIIComunidad de MadridFondo Europeo de Desarrollo Regional-FEDERInstituto Ramon y Cajal de Investigación Sanitária-IRYCISFundación Renal Iñigo Álvarez de Toledo-FRIA

The Integrin Beta1 Modulator Tirofiban Prevents Adipogenesis and Obesity by the Overexpression of Integrin-Linked Kinase: a Pre-Clinical Approach In Vitro and In Vivo

de Frutos, S., Griera, M., Hatem-Vaquero, M. et al. The integrin beta1 modulator Tirofiban prevents adipogenesis and obesity by the overexpression of integrin-linked kinase: a pre-clinical approach in vitro and in vivo. Cell Biosci 12, 10 (2022)Background: Obesity is caused by the enlargement of the white adipose tissue (WAT) depots, characterized by the hypertrophic enlargement of malfunctioning adipocytes within WAT which increases the storage of triglycerides (TG) in the lipid droplets (LD). Adipogenesis pathways as well as the expression and activity of some extracellular matrix receptors integrins are upregulated. Integrin?1 (INTB1) is the main isoform involved in WAT remodeling during obesity and insulin resistance-related diseases. We recently described Integrin Linked Kinase (ILK), a scafold protein recruited by INTB1, as an important mediator of WAT remodeling and insulin resistance. As the few approved drugs to fght obesity have brought long-term cardiovascular side efects and given that the consideration of INTB1 and/or ILK modulation as anti-obesogenic strategies remains unexplored, we aimed to evaluate the anti-obesogenic capacity of the clinically approved anticoagulant Tirofban (TF), stated in preclinical studies as a cardiovascular protector. Methods: Fully diferentiated adipocytes originating from C3H10T1/2 were exposed to TF and were co-treated with specifc INTB1 blockers or with siRNA-based knockdown ILK expression. Lipid-specifc dyes were used to determine the TG content in LD. The genetic expression pattern of ILK, pro-infammatory cytokines (MCP1, IL6), adipogenesis (PPAR?, Leptin), thermogenesis (UCP1), proliferation (PCNA), lipid metabolism (FASN, HSL, ATGL), and metabolite trans porters (FABP4, FAT, AQP7) were detected using quantitative PCR. Cytoskeletal actin polymerization was detected by confocal microscopy. Immunoblotting was performed to detect INTB1 phosphorylation at Thr788/9 and ILK activity as phosphorylation levels of protein kinase B (AKT) in Ser473 and glycogen synthase kinase 3? (GSK3?) at Ser9. TF was intraperitoneally administered once per day to wildtype and ILK knockdown mice (cKDILK) challenged with a high-fat diet (HFD) or control diet (STD) for 2 weeks. Body and WAT weight gains were compared. The expression of ILK and other markers was determined in the visceral epididymal (epi) and inguinal subcutaneous (sc) WAT. Results: TF reduced TG content and the expression of adipogenesis markers and transporters in adipocytes, while UCP-1 expression was increased and the expression of lipases, cytokines or PCNA was not afected. Mechanistically, TF rapidly increased and faded the intracellular phosphorylation of INTB1 but not AKT or GSK3?. F-actin levels were rapidly decreased, and INTB1 blockade avoided the TF efect. After 24 h, ILK expression and phosphorylation rates of AKT and GSK3? were upregulated, while ILK silencing increased TG content. INTB1 blockade and ILK silencing avoided TF efects on the TG content and the transcriptional expression of PPAR? and UCP1. In HFD-challenged mice, the systemic administration of TF for several days reduced the weight gain on WAT depots. TF reduced adipogenesis and pro-infammatory biomarkers and increased lipolysis markers HSL and FAT in epiWAT from HFD, while increased UCP1 in scWAT. In both WATs, TF upregulated ILK expression and activity, while no changes were observed in other tissues. In HFD-fed cKDILK, the blunted ILK in epiWAT worsened weight gain and avoided the anti-obesogenic efect of in vivo TF administration. Conclusions: ILK downregulation in WAT can be considered a biomarker of obesity establishment. Via an INTB1-ILK axis, TF restores malfunctioning hypertrophied WAT by changing the expression of adipocyte-related genes, increas ing ILK expression and activity, and reducing TG storage. TF prevents obesity, a property to be added to its anticoagu lant and cardiovascular protective advantages.Instituto de Salud Carlos IIIComunidad de MadridFondo Europeo de Desarrollo Regional-FEDE

H- ras deletion protects against angiotensin II-induced arterial hypertension and cardiac remodeling through protein kinase G-Ibeta pathway activation

15 p.Ras proteins regulate cell survival, growth, differentiation, blood pressure, and fibrosis in some organs.We have demonstrated that H-rasgene deletion produces mice hypotensionviaa soluble guanylate cyclase-proteinkinase G (PKG)–dependent mechanism. In this study, we analyzed the consequences of H-rasdeletiononcardiacremodeling induced by continuous angiotensin II (AngII) infusion and the molecular mechanisms implied. Leftventricular posterior wall thickness and mass and cardiomyocyte cross-sectional area were similar between AngII-treated H-Ras knockout (H-ras2/2) and control wild-type (H-ras+/+) mice, as were extracellular matrix proteinexpression. Increased cardiac PKG-Ibprotein expression in H-ras2/2mice suggests the involvement of this proteinin heart protection.Ex vivoexperiments on cardiac explants could support this mechanism, as PKG blockadeblunted protection against AngII-induced cardiac hypertrophy and fibrosis markers in H-ras2/2mice. Geneticmodulation studies in cardiomyocytes and cardiac and embryonic fibroblasts revealed that the lack of H-Ras down-regulates the B-RAF/MEK/ERK pathway, which induces the glycogen synthase kinase-3b-dependent activation ofthe transcription factor, cAMP response element-binding protein, which is responsible for PKG-Iboverexpressionin H-ras2/2mouse embryonic fibroblasts. This study demonstrates that H-rasdeletion protects against AngII-induced cardiac remodeling, possiblyviaa mechanism in which PKG-Iboverexpression could play a partial role, andpoints to H-Ras and/or downstream proteins as potential therapeutic targets in cardiovascular disease.Instituto de Salud Carlos IIIUniversidad de AlcaláFundación SenefroFEDE

Comparison of commercial kits to measure cytokine responses to Plasmodium falciparum by multiplex microsphere suspension array technology

<p>Abstract</p> <p>Background</p> <p>Multiplex cytokine profiling systems are useful tools for investigating correlates of protective immunity. Several Luminex and flow cytometry methods are commercially available but there is limited information on the relative performance of different kits. A series of comparison experiments were carried out to determine the most appropriate method for our subsequent studies.</p> <p>Methods</p> <p>Two Luminex methods were compared, the Bio-Rad human 17-plex panel and the Invitrogen (formerly BioSource) human cytokine 10-plex kit, and two flow cytometry methods, the Becton Dickinson Human Th1/Th2 Cytokine Kit (CBA) and the Bender MedSystems Human Th1/Th2 11plex FlowCytomix Multiplex Kit. All kits were tested for the measurement of cytokines in supernatants collected from human leukocytes stimulated with viable <it>Plasmodium falciparum </it>infected red blood cells (iRBC) or <it>P. falciparum </it>schizont lysates.</p> <p>Results</p> <p>Data indicated that the kits differed in sensitivity and reproducibility depending on the cytokine, and detected different quantities of some cytokines. The Bio-Rad 17-plex kit was able to detect more positive responses than the Invitrogen 10-plex kit. However, only when detecting IL-1, IL-6 or TNF did the two Luminex based methods correlate with one another. In this study, the flow cytometry based techniques were less variable and correlated better with one another. The two flow cytometry based kits showed significant correlation when detecting IFN-γ, IL-2, TNF, IL-10 and IL-6, but overall the BD kit detected more positive responses than the Bender MedSystems kit.</p> <p>Conclusions</p> <p>The microsphere suspension array technologies tested differed in reproducibility and the absolute quantity of cytokine detected. Sample volume, the number of cytokines measured, and the time and cost of the assays also differed. These data provide an accurate assessment of the four techniques, which will allow individual researchers to select the tool most suited for their study population.</p

Cytokine profiling in immigrants with clinical malaria after extended periods of interrupted exposure to Plasmodium falciparum.

Immunity to malaria is believed to wane with time in the absence of exposure to Plasmodium falciparum infection, but immunoepidemiological data on longevity of immunity remain controversial. We quantified serum cytokines and chemokines by suspension array technology as potential biomarkers for durability of immunity in immigrants with clinical malaria after years without parasite exposure. These were compared to serum/plasma profiles in naïve adults (travelers) and semi-immune adults under continuous exposure, with malaria, along with immigrant and traveler patients without malaria. Immigrants had higher levels of IL-2, IL-5 and IL-8 compared to semi-immune adults with malaria (P≤0.0200). Time since immigration correlated with increased IL-2 (rho=0.2738P=0.0495) and IFN-γ (rho=0.3044P=0.0282). However, immigrants did not show as high IFN-γ concentrations as travelers during a first malaria episode (P<0.0001). Immigrants and travelers with malaria had higher levels of IFN-γ, IL-6, and IL-10 (P<0.0100) than patients with other diseases, and IL-8 and IL-1β were elevated in immigrants with malaria (P<0.0500). Therefore, malaria patients had a characteristic strong pro-inflammatory/Th1 signature. Upon loss of exposure, control of pro-inflammatory responses and tolerance to P. falciparum appeared to be reduced. Understanding the mechanisms to maintain non-pathogenic effector responses is important to develop new malaria control strategies

Hyperphosphatemia Promotes Senescence of Myoblasts by Impairing Autophagy Through Ilk Overexpression, A Possible Mechanism Involved in Sarcopenia

In mammalians, advancing age is associated with sarcopenia, the progressive and involuntary loss of muscle mass and strength. Hyperphosphatemia is an aging-related condition involved in several pathologies. The aim of this work was to assess whether hyperphosphatemia plays a role in the age-related loss of mass muscle and strength by inducing cellular senescence in murine myoblasts and to explore the intracellular mechanism involved in this effect. Cultured mouse C2C12 cells were treated with 10 mM beta-glycerophosphate (BGP] at different periods of time to induce hyperphosphatemia. BGP promoted cellular senescence after 24 h of treatment, assessed by the increased expression of p53, acetylated-p53 and p21 and senescence associated beta-galactosidase activity. In parallel, BGP increased ILK expression and activity, followed by mTOR activation and autophagy reduction. Knocking-down ILK expression increased autophagy and protected cells from senescence induced by hyperphosphatemia. BGP also reduced the proliferative capacity of cultured myoblasts. Old mice (24months-old] presented higher serum phosphate concentration, lower forelimb strength, higher expression of p53 and ILK and less autophagy in vastus muscle than young mice (5-months-old]. In conclusion, we propose that hyperphosphatemia induces senescence in cultured myoblasts through ILK overexpression, reducing their proliferative capacity, which could be a mechanism involved in the development of sarcopenia, since old mice showed loss of muscular strength correlated with high serum phosphate concentration and increased levels of ILK and p53

Markers of endothelial damage in patients with chronic kidney disease on hemodialysis

Patients with Stage 5 chronic kidney disease who are on hemodialysis (HD) remain in a chronic inflammatory state, characterized by the accumulation of uremic toxins that induce endothelial damage and cardiovascular disease (CVD). Our aim was to examine microvesicles (MVs), monocyte subpopulations, and angiopoietins (Ang) to identify prognostic markers in HD patients with or without diabetes mellitus (DM). A total of 160 prevalent HD patients from 10 centers across Spain were obtained from the Biobank of the Nephrology Renal Network (Madrid, Spain): 80 patients with DM and 80 patients without DM who were matched for clinical and demographic criteria. MVs from plasma and several monocyte subpopulations (CD142+/CD16+, CD14+/CD162+) were analyzed by flow cytometry, and the plasma concentrations of Ang1 and Ang2 were quantified by ELISA. Data on CVD were gathered over the 5.5 yr after these samples were obtained. MV level, monocyte subpopulations (CD14+/CD162+ and CD142+/CD16+), and Ang2-to-Ang1 ratios increased in HD patients with DM compared with non-DM patients. Moreover, MV level above the median (264 MVs/µl) was associated independently with greater mortality. MVs, monocyte subpopulations, and Ang2-to-Ang1 ratio can be used as predictors for CVD. In addition, MV level has a potential predictive value in the prevention of CVD in HD patients. These parameters undergo more extensive changes in patients with DM.Support for this work was provided by Plan Nacional de IDi Proyectos de Investigación en Salud of Instituto de Salud Carlos III (ISCIII)–Subdirección General de Evaluación, Fondos de desarrollo regional (FEDER; PI11/01536, PI12/01489, PI14/00806, PI15/01785); Junta de Andalucía grants (P010-CTS-6337, P11-CTS-7352); and Fundación Nefrológica. P. Buendía, A. Carmona, and C. Luna-Ruiz are fellows from Consejería de Innovacion, Ciencia y Empresa, Junta de Andalucía

Renal tubule Cpt1a overexpression protects from kidney fibrosis by restoring mitochondrial homeostasis

Chronic kidney disease (CKD) remains a major epidemiological, clinical, and biomedical challenge. During CKD, renal tubular epithelial cells (TECs) present a persistent inflammatory and profibrotic response. Fatty acid oxidation (FAO), the main source of energy for TECs, is reduced in kidney fibrosis and contributes to its pathogenesis. To determine whether gain of function in FAO (FAO-GOF) could protect from fibrosis, we generated a conditional transgenic mouse model with overexpression of the fatty acid shuttling enzyme carnitine palmitoyl-transferase 1A (CPT1A) in TECs. Cpt1a-knockin (CPT1A-KI) mice subjected to 3 models of renal fibrosis (unilateral ureteral obstruction, folic acid nephropathy [FAN], and adenine-induced nephrotoxicity) exhibited decreased expression of fibrotic markers, a blunted proinflammatory response, and reduced epithelial cell damage and macrophage influx. Protection from fibrosis was also observed when Cpt1a overexpression was induced after FAN. FAO-GOF restored oxidative metabolism and mitochondrial number and enhanced bioenergetics, increasing palmitate oxidation and ATP levels, changes that were also recapitulated in TECs exposed to profibrotic stimuli. Studies in patients showed decreased CPT1 levels and increased accumulation of short- and middle-chain acylcarnitines, reflecting impaired FAO in human CKD. We propose that strategies based on FAO-GOF may constitute powerful alternatives to combat fibrosis inherent to CKD