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Integrin-linked kinase (ILK) has emerged as a controversial pseudokinase protein that plays a crucial role in the signaling process initiated by integrin-mediated signaling. However, ILK also exhibits a scaffolding protein function inside cells, controlling cytoskeletal dynamics, and has been related to non-neoplastic diseases such as chronic kidney disease (CKD). Although this protein always acts as a heterotrimeric complex bound to PINCH and parvin adaptor proteins, the role of parvin proteins is currently not well understood. Using in silico approaches for the design, we have generated and prepared a set of new tripeptides mimicking an alpha-parvin segment. These derivatives exhibit activity in phenotypic assays in an ILK-dependent manner without altering kinase activity, thus allowing the generation of new chemical probes and drug candidates with interesting ILK-modulating activities.

BACKGROUND AND AIMS Type 2 diabetes and obesity are metabolic disorders related to kidney risk and associated with white adipose tissue (WAT) hypertrophy, which is based in a pro-adipogenic transdifferentiation of the adipocytes and their precursors’ stem cells present in WAT. Several transcription factors are key regulators during this transdifferentiation. The consequence is that adipocytes’ size increases due to the excessive storage of triglycerides (TG) inside the cells. Therapeutic strategies for reducing WAT size may be based in the capacity of increasing lipolysis and reducing adipogenesis. We have previously demonstrated the implication of the extracellular matrix (ECM) mediator integrin beta 1 (INTB1) and intracellular mediation of actin cytoskeleton during obesity (Hatem-Vaquero. J Endocrinol 2017; Cell Physiol Biochem 2020). Here, we demonstrate for the first time the antiobesogenic properties of a new highly biocompatible and bioactive graphene-based product named BioGraph (Llorens-Gámez M. Int J Biol Macromol 2020; Salesa B. Biomedicines 2021), which physicochemical interaction with INTB1 give a great potential as a diagnostic and bioactive therapeutic tool. METHOD Pluripotent c3h10t1/2 cells were differentiated into adipocytes using a specific culture medium. After 7 days of differentiation, the adipocytes were treated for 24 h with a suspension of the graphene derivative BioGraph, developed, registered and patented by Graphenano Medical Care S.L. To determine viability and toxicity on adipocytes, trypan blue and MTT labeling techniques were used. The levels of intracellular TG contained in the lipid droplets were determined by fluorescent labeling with AdipoRed. PPARgamma, CEBP and INTB1 mRNA levels were determined by RT-qPCR. Actin polymerization in the cytoskeleton was quantified by fluorescent phalloidin staining. Active INTB1 quantification (phosphorylated isoform at thr788/9) and total and active lipase HSL (phosphorylated isoform at ser660) were performed by western blot. RESULTS BioGraph is safe and non-toxic on adipocytes in a two-order concentration range. The TG content is reduced in a dose-dependent manner. Adipogenesis markers PPARgamma and CEBP and actin polymerization were reduced after treatment with an intermediate dose. The expression of INTB1 was not modified, but its phosphorylation was increased rapidly after treatment. Lipolysis-main enzyme HSL expression and activity were increased 24 h after treatment. CONCLUSION BioGraph is biocompatible and bioactive on adipocytes. A rapid modulation of INTB1 produces the depolymerization of the cytoskeleton, the reduction of intracellular TG content and the expression of adipogenesis markers, while HSL-mediated lipolysis is increased. We demonstrate for the first time the mechanism followed by a graphene-based material modulating the adipocyte phenotype and behavior. BioGraph may be considered a new anti-obesogenic agent.

espanolEl contexto actual de cambio global obliga a realizar una selvicultura adaptativa en todo tipo de masas forestales, pero la particular situacion de miles de hectareas de repoblaciones protectoras, en las que una gestion inadecuada compromete la estabilidad de las masas, las debe convertir en asunto prioritario. Su edad, aunque algo alejada de los turnos habituales, no es impedimiento para la aplicacion de cortas de regeneracion que, ademas, pueden potenciar la naturalizacion y resiliencia de estas repoblaciones. Sin embargo, esta opcion apenas se ha aplicado en las miles de hectareas de pino resinero (Pinus pinaster Ait.) situadas al sur del Sistema Central. Por ello, en este trabajo se ha estudiado un canton de repoblacion de pino resinero situado en Fuencaliente (Ciudad Real), con cierto grado de diversificacion en elsubpiso (distintas especies de Quercus mediterraneos y madrono), que fue cortado a hecho en dos tiempos en 2012. Previamente a las cortas, se distinguieron tres tipos de masa (pinar puro, y dos grados de mezcla con frondosas), instalandose 78 parcelas de regeneracion repartidas equilibradamente entre los trestipos. Estas parcelas se han inventariado anualmente durante 7 anos, clasificando el regenerado de las especies arboreas en cuatro clases de altura, y midiendose diferentes parametros ecologicos asociados. Laregeneracion del pino puede considerarse exitosa tanto en cantidad como en distribucion, asociandose positivamente con veranos frescos y humedos. No obstante, no se ha promovido una diversificacion especifica apreciable, pues los Quercus solo regeneran bien donde eran abundantes ya antes de las cortas EnglishThe actual context of global change necessary leads to the application of adaptive silviculture techniques in forests. Nevertheless, the particular situation of several protective reforestations, in which an inadequate management jeopardizes its stability, makes them a priority nowadays. Although its ages are still far from rotation ages, regeneration fellings may enhance naturalization and resilience of these reforestations. However, this solution has been scarcely developed in the maritime pine (Pinus pinaster Ait.) forests located at the south of the Central System mountain range. Thus, this work studied a reforested maritime pine stand with variable presence of broad-leaved species under cover located in Fuencaliente (Ciudad Real). This stand, cut following a seed-tree method in 2012, presented before the cuttings three types of forest structure: pure pine forest and two mixlevels. 78 circular plots were installed to monitor pine and Quercus regeneration (classified in four height classes) and several ecological parameters. Pine regeneration could be considered as successful in both density and spatial distribution, and was positively associated with humid and warm summer. Specific diversification, however, was not promoted enough,as Quercus regeneration was abundant only in plots with significant densities of adult trees of these species

La gran superficie ocupada en España por repoblaciones protectoras con edades cercanas al turno exige abordar urgentemente su regeneración. El temperamento de luz de Pinus pinaster Ait. obliga a aplicar cortas continuas, justificadas selvícolamente pero que provocan impacto paisajístico y riesgo de erosión. Buscando conseguir regeneración suficiente y diversificación estructural y específica pero minimizando riesgos, se han instalado dos dispositivos experimentales en repoblaciones de Pinus pinaster de 50-65 años de edad situadas en Arbancón (Guadalajara) y Fuencaliente (Ciudad Real), sobre los que otoño de 2017 se cortó a hecho por bosquetes circulares (entresaca por bosquetes) de tamaños reducidos; en Fuencaliente también se ejecutaron cortas a hecho en dos tiempos sobre grandes superficies. Un año después de las cortas, los primeros resultados muestran que en Arbancón se ha conseguido regeneración suficiente en bosquetes con diámetros iguales a 1,5 y 2,5 veces la altura dominante de la masa: densidades medias en otoño de alrededor de 4500 pies ha-1 uniformemente repartidas, supervivencia estival del 67 % y nueva regeneración otoñal, con valores significativamente superiores a los de las zonas sin tratar). En Fuencaliente (bosquetes con diámetros entre 2 y 3,5 la altura dominante), la inadecuada ejecución de las cortas mecanizadas provocó remoción y compactación excesivas en el suelo, a pesar de lo cual en el otoño siguiente se alcanzan densidades de regeneración medias de 1500 pies ha-1 (en bosquetes) y de 2300 pies ha -1 (cortas a hecho en dos tiempos). En ningún caso se han apreciado signos de erosión.

Background and Aims Insulin resistance (IR)-related metabolic disorders, such as diabetes type 2 and obesity, are linked to renal and cardiovascular risk. Unfortunately, they lack a better prognosis before their full establishment. An important mechanism associated to the IR development is the dysregulation of lipolysis in white adipose tissue (WAT), which is driven by the excessive lipid accumulation, together with inflammation and the excessive deposition of ECM components such as collagens and fibronectin, known as fibrosis. The intracellular lipid adjustment is affected by changes in the expression and activity of the hormone-sensitive lipase (HSL) and the intracellular and circulating balance of the lipolysis products glycerol and free fatty acids. We published that transgenic depletion of the scaffold intracellular protein Integrin-linked kinase (ILK), one of the adhesome translators of the ECM signals, modulates IR in WAT [Hatem-Vaquero et al. J Endocrinol. 2017]. We also published that ILK downregulation in WAT is an early event in a short-term high fat diet (HFD) mice model that correlates with a faster insulin sensitivity loss, accompanied with an early increase of body weight and the erratic expression of WAT adipokines and metabolites transporters. [Hatem-Vaquero et al. Cell Physiol Biochem 2020]. Here we used in vivo and in vitro approaches to decode the role of ILK downregulation during WAT expansion and altered lipolysis. Method Adult mice with 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. We determined the weight changes on body and WAT depots, epidydimal (eWAT) and subcutaneous (scWAT), and the circulating lypolisis product glycerol. The expression of ECM component fibronectin (FN), fibrosis marker TGF-beta and the glycerol transporter AQP3 were determined in eWAT by RT-qPCR or Western blot. Comparative studies were performed in cultured C3H10t1/2-based adipocytes where ILK expression was deliberately downregulated by the transfection of siRNAs against ILK or in control adipocytes (scramble siRNAs). We determined the expression of ILK, total and active HSL isoforms (HSL, p-HSL s660) by RT-qPCR or Western blot and the intracellular fat droplet content (by adipo-red dye) Results HFD increased progressively CT eWAT, scWAT and whole body weight gains. HFD-fed cKD-ILK have an earlier increase of body and eWAT weights. However, the scWAT weight ratio against eWAT was inverted in the same cKD-ILK animals. This early depot-specific response to HFD coincide with increase in IR [Wajchenberg BL. Endocr Rev 2000]. HFD-fed cKD-ILK WAT early expressed altered levels of fibrotic markers (TGF-beta and Fibronectin). HFD-fed cKD-ILK shown a temporary increase of circulating glycerol and its WAT transporter AQP3. In cultured adipocytes with downregulated expression of ILK, the intracellular fat droplet was increased and the HSL contain and its lipolysis activity (measured as levels of p-HSL isoform) were reduced. Conclusion We suggest ILK as a modulator during the obesity establishment. Considering the observed relation between ILK expression with the altered lipolysis and fibrosis patterns observed in visceral WAT, ILK downregulation may be a predictive value and probably its pharmacological upregulation may be a novel therapeutic strategy against obesity and IR.

Background and Aims Patients with CKD have a higher risk of developing cardiovascular diseases. Protein-bound uremic toxins, such as p-cresol (pc) and indoxyl-sulfate (IS), are retention solutes, poorly removed during hemodialysis. They lead to endothelial dysfunction inducing the expression of adhesion molecules and leucocyte adhesion to endothelium. Monocyte extravasation can be carried out by dynamic adhesion structures called podosomes. Integrin-linked kinase (ILK), a kinase and an intracellular scaffold protein, links the cell-adhesion receptors, integrins and growth factors to the actin cytoskeleton and to a range of signalling pathways. We demonstrate the role of ILK in the accumulation of integrin-associated proteins inside the podosomes. The aim of this study was to investigate if ILK is involved in uremic toxin-induced leucocyte podosome formation and adhesion under uremic conditions that simulate CKD. Method In vitro experiments were carried out in human cell line of leukemic monocytes, THP-1. We tested the effect of uremic toxins on cell viability and ILK expression levels or activation by performing dose and time-response experiments. Cells were exposed to IS (25-100 µg ml-1) plus pc (10-100 µg ml-1) both combined, for different times. ILK expression levels were determined by Western Blot and RT-qPCR and its kinase activity was tested by its downstream effector GSK-3β phosphorylated levels. Cell adhesion of THP-1 cells stained with cell tracker to a monolayer of human endothelial cells (EA.hy926) and podosome formation and cell adhesion of THP-1 cells stained with phalloidin to a fibronectin extracellular matrix, was determined by fluorescence confocal microscopy. ILK co-localization with podosome specific protein cortactin was assessed by fluorescence confocal microscopy. For ex vivo experiments in ILK conditional-knockdown mice (cKD-ILK), male CRE-LOX mice were injected with tamoxifen (cKD-ILK) or vehicle (wild-type, WT), to induce ILK deletion. Peripheral blood mononuclear cells (PBMCs) were obtained and treated with uremic toxins. Cell adhesion to a fibronectin extracellular matrix was determined by cell count of the percentage of attached cells against the total cells collected. Results Our data suggests that uremic toxins did not induce toxicity in THP-1 cells. Furthermore, ILK was activated by uremic toxins both at low and high concentrations, without changes in the protein expression. In the cell adhesion assay to the endothelium, uremic toxins induces an increase of THP-1 cells adhesion, which is completely abolished when ILK is knocked down by ILK siRNA, both at low and high toxins concentrations. Similar results were obtained from the cell adhesion assay to the fibronectin extracellular matrix. Moreover, uremic toxins induced podosome formation in THP-1 cells, even at low concentrations, compared to the control, while ILK knockdown abrogated almost completely fibronectin adhesion and podosome formation in uremic toxin-treated cells. Interestingly, we tested that ILK co-localize with cortactin in podosomes, which confirmed the implication of ILK in podosome structures formation induced by uremic toxins. In cKD-ILK PBMC mice the transgenic ILK depletion significantly decrease non-excised ILK mRNA levels. These cKD-ILK PBMC mice exhibited a lower adhesion to the fibronectin extracellular matrix compared to WT. By last, uremic toxins induced a significant increase of mice PBMCs adhesion in WT animals compared to the control that was significantly lower in PBMCs of cKD-ILK animals. Conclusion These data suggest that ILK plays a critical role in the required processes for leukocyte extravasation. ILK deletion may prevent podosome formation and adhesion of leucocytes, decreasing the endothelial and vascular damage caused by the accumulation of uremic toxins in CKD. Therefore, ILK could be a potential therapeutic target for the treatment of vascular damage associated with CKD.

Background and Aims Renal ILK transgenic deletion on mice (cKD-ILK) address basal polyuria compatible with nephrogenic diabetes insipidus (NDI), due to disrupted tubular water channel aquaporin 2 (AQP2) AQP2 expression and vesicular trafficking to the apical membrane [Cano-Peñalver JL et al. FASEB J. 2014; Mamuya FA et al. Am J Physiol Renal Physiol. 2016]. An early symptom of chronic renal diseases (CKD) is NDI. ILK depletion also have a nephron-protective effect in renal damage in mice due to the increase in cGMP [Cano-Peñalver JL et al. Mol Med. 2016; de Frutos S et al. Biochim Biophys Acta Mol Basis Dis. 2019], the latter an alternative anti-NDI mediator. Here we demonstrate that ILK-mediated regulation of AQP2, under basal or CKD contexts, does not depend on cGMP. Method cKD-ILK and controls (WT) were treated orally with a NO donor, as precursor of cGMP formation (IDN, 300mg / Kg / day) for 24 hours. Urine volume was determined as a measure of tubular functionality. Fresh kidneys from non-treated mice were cultured ex vivo with precursors of cGMP (NO donor SNP, 1 µM or cGMP analogue 8Br-cGMP, 0.2 mM) for 30 minutes. AQP2 in the apical membrane of the tubules was quantified by immunohistochemistry. More cKD-ILK and WT were subjected for 6 weeks to a diet supplemented with 0.2% adenine (A) as CKD inducer or baseline standard diet. Urine volume and total ranal AQP2 levels were determined. Results Compared to WT, cGMP axis activation in vivo did not compensate the basal NDI present in cKD-ILK and the activation ex vivo did not improve the apical presence of AQP2 in cKD-ILK. Both WT and cKD-ILK subjected to CKD showed exacerbated polyuria and decreased levels of renal AQP2, but no differences were observed between groups. Conclusion ILK regulates AQP2, both in a basal and pathological context (CKD) independent of cGMP which otherwise is nephron-protective. Our results probably explain the co-existence of cGMP increase together with AQP2 decrease observed in the cKD-ILK kidneys.

Background and Aims Important mechanism associated to the development of Insulin resistance (IR) are dysregulation of lipid adjustment and excessive deposition of extracellular matrix (ECM) in the visceral white adipose tissue (WAT). The transgenic depletion of the ECM-to-cell scaffold intracellular protein Integrin-linked kinase (ILK) modulates IR in WAT [Hatem-Vaquero et al. J Endocrinol. 2017] and body weight gain and adipose malfunction correlates with early downregulation of ILK expression in WAT during a short-term high fat diet (HFD) mice model [Hatem-Vaquero et al. Cell Physiol Biochem 2020]. Thus, we demonstrated that transcriptional regulation of ILK might govern the expansion of adipocytes. Between several transcriptional factors implicated in lipid metabolism, the activation of the transcription factor peroxisome proliferator activated receptor (PPAR) family member PPARβ/δ ameliorates IR. Taking in consideration that it modulates the expression of ILK in other contexts aside metabolism [Di-Poï N et al. J Steroid Biochem Mol Biol. 2003, Zhu B et al. Oncogene. 2014], here we activated PPARβ/δ in vivo and in vitro to upregulate adipose ILK expression and prevent WAT expansion. Method Adult mice were fed with either standard (STD) or HFD diets during 4 weeks and divided into groups treated with PPARβ/δ activator L-165041 (i.p. 2 mg/kg b.w per day) or vehicle for 2 additional weeks with maintained diet challenges [Lim HJ et al. Eur J Pharmacol. 2009]. Body and epidydimal WAT depot (eWAT) weights changes were compared and ILK expression was determined by RT-qPCR. Comparative studies were performed in cultured C3H10t1/2-based adipocytes where ILK expression was deliberately downregulated by the transfection of siRNAs against ILK or in control adipocytes (scramble siRNAs). After cells were treated with L-165041 (1 microM, 24h), we determined the intracellular triglyceride content (by adipo-red dye), the expression of ILK and its downstream substrate AKT (total and active p-AKT in ser473) by RT-qPCR or Western blot. Results HFD increased progressively eWAT and whole body weight gains, which coincide with increase in IR. PPARβ/δ-activated mice under STD or HFD have preventive reduction of weight gains and increased expression of ILK in eWAT. Downregulated ILK expression in culture adipocytes coincide with increased triglyceride content, whereas the activation of PPARβ/δ reduced it at the same time that increased ILK expression and activity. Conclusion We suggest ILK modulation as a novel therapeutic strategy during the obesity establishment. The pharmacological activation of PPARβ/δ increases ILK expression and activity, which correlates with a weight loss in IR-based models in vivo. Although the increased presence of ILK induces the phosphorylation of AKT, in turn one of the main mediators during insulin signalling, we cannot conclude the role of ILK-mediated AKT modulation during the reduction of the adipocyte expansion observed

Background and Aims Calpains are intracellular cysteine proteases that play a critical role in cell remodeling, being involved in multiple biological processes linked to tissue damage and repair mechanisms. In addition, they are released into the circulation, being able to carry out systemic actions with pathological consequences. The aim of this study was to investigate the role of calpains in the progression of chronic kidney disease (CKD) in an experimental model of chronic renal damage induced by adenine. Method We induced an experimental model of CKD, in mice fed for 2 weeks with an adenine-supplemented diet (0.2% adenine) (A). Animals receiving this diet develop a tubulointerstitial damage resembling that is observed in human CKD. Mice with standard diet were used as controls (C). Renal function was assessed by measuring serum blood urea nitrogen (BUN) and creatinine (mg/dl). Fibrosis markers (collagen type I and fibronectin) were determined by RT-qPCR. Changes in the renal content of calpains 1 and 2 were analyzed by western blot (protein content), and RT-qPCR (mRNA expression). Results Our results show functional and structural changes at renal level in the adenine-fed mice, with increased BUN (A: 72 mg/dl, C: 28 mg/dl, p < 0.05), creatinine (A: 0.58 mg/dl, C: 0.25 mg/dl, p < 0.05), collagen type I mRNA expression (A: 12.9 units, C: 1.2 units, p < 0.05) and fibronectin mRNA expression (A: 3.46 units, C: 1.3 units, p < 0.05). Furthermore, protein content of calpains 1 (A: 1.27 units, C: 0.78 units, p < 0.05) and 2 (A: 1.30 units, C: 0.66 units, p < 0.05) was significantly higher in adenine-fed mice when compared to control. At the same time, we observed a significant increase in gene expression of both calpain 1 (A: 4.21 units, C: 0.51 units, p < 0.05) and 2 (A: 4.93 units, C: 0.56 units, p < 0.05) in the adenine model regarding to mice with standard diet. Our results demonstrate that calpain 1 and 2 expression in renal tissue increases as CKD progresses. Interestingly, we found statistically significant correlations between renal calpains 1 and 2 protein and mRNA content and plasma BUN and creatinine (p < 0.05, r between 0.79 and 0.92), as well as protein expression of calpain 2 and mRNA expression of collagen type I (p < 0.05, r = 0.76). These data suggest a potential direct relationship between renal calpain 1 and 2 content and loss of renal function, in part due probably to the modulation of the fibrotic changes, in adenine fed mice. Conclusion We suggest an implication of calpains 1 and 2 in the development of CKD. Thus, effective calpain blockade or downregulation could be useful as a therapeutic strategy to prevent CKD. Further experiments will be necessary to establish the relationship between these factors.

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-ISCIII, Comunidad de Madrid, Fondo Europeo de Desarrollo Regional-FEDER, Instituto Ramon y Cajal de Investigación Sanitária-IRYCIS, Fundación Renal Iñigo Álvarez de Toledo-FRIAT

Resumen: Antecedentes: Los pacientes con enfermedad renal crónica presentan una acumulación de toxinas urémicas, las cuales han sido identificadas como agentes patogénicos asociados con la mortalidad cardiovascular, muy elevada en este grupo de enfermos. Un fenómeno común a la disfunción renal progresiva y al daño vascular asociado es la acumulación anormal de proteínas de la matriz extracelular (MEC) en las estructuras renales o vasculares. Objetivo: Estudiar la contribución de la uremia o las toxinas urémicas a la producción de citocinas y MEC en aortas de animales urémicos o células de músculo liso de aorta humana (HAOSMC). Materiales y métodos: Se utilizaron ratones con uremia inducida por una dieta rica en adenina (0,2%) durante 2, 4 o 6 semanas. Se evaluó la función renal mediante la diuresis, los niveles plasmáticos de creatinina y nitrógeno ureico plasmático, y la excreción fraccional de sodio y el daño vascular mediante histología y expresión proteica por RT-qPCR. In vitro, las HAOSMC se incubaron con toxinas urémicas: p-cresol 10-100 (μg/ml) e indoxil-sulfato 25-100 (μg/ml), solas o simultáneamente. La expresión proteica se evaluó por Western blot y microscopia confocal. Resultados: La administración de adenina produjo un progresivo daño renal en los ratones, un engrosamiento de la pared aórtica y un incremento de la expresión de TGF-β1 y proteínas de MEC. Las toxinas a dosis altas y combinadas también indujeron expresión de TGF-β1 y proteínas de MEC por las células HAOSMC. Conclusiones: La uremia producida por una dieta rica en adenina o las dosis altas de toxinas urémicas indujeron el depósito anormal de proteínas de MEC en las paredes vasculares o su producción por HAOSMC. La comprensión de los mecanismos que subyacen a este proceso fisiopatológico puede resultar de utilidad en la prevención del daño cardiovascular asociado a la progresión de la enfermedad renal crónica, una dolencia, de momento, irreversible y, en ocasiones, silenciosa hasta su diagnóstico en etapas avanzadas. Abstract: Background: Patients with chronic kidney disease present with an accumulation of uraemic toxins, which have been identified as pathogenic agents associated with cardiovascular mortality, which is very high is this patient group. A phenomenon common to the progressive renal dysfunction and associated vascular damage, is the abnormal accumulation of extracellular matrix (ECM) proteins in the renal or vascular structures. Objective: To determine the contribution of uraemia or the uraemic toxins to the production of cytokinins and ECM in aortas of uraemic animals or human aortic smooth muscle cells (HASMCs). Materials and methods: Mice were used with uraemia induced by a diet rich in adenine (0.2%) for 2, 4 or 6 weeks. Kidney function was evaluated by means of urine volume, plasma levels of creatinine, urea, fractional excretion of sodium, and vascular damage using histology, as well as protein expression using RT-qPCR. The HASMCs were incubated in vitro with uraemic toxins: p-cresol 10-100 (μg/ml) and indoxyl-sulphate25-100 (μg/ml) alone or simultaneously. The protein expression was evaluated using Western blot and confocal microscopy. Results: The administration of adenine produced progressive kidney damage in the mice, thickening of the aortic wall, and increasing the expression of TGF-β1 and ECM proteins. The toxins at high doses and combined also induced the expression of TGF-β1 and ECM proteins by the HASMCs. Conclusions: The uraemia produced by an adenine rich diet or high doses of uraemic toxins induced the abnormal deposit of ECM proteins in the vascular wall or its production by HASMCs. The understanding of the mechanisms that underlie this pathophysiological process may be useful in the prevention of cardiovascular damage associated with the progress of chronic kidney disease, a disease, at the moment that is irreversible and occasional silent until its diagnosis in advanced stages. Palabras clave: Enfermedad renal crónica, Daño vascular, Toxinas urémicas, Adenina, Fibrosis, Transforming Growth factor beta-1, Keywords: Chronic kidney disease, Vascular damage, Uraemic toxins, Adenine, Fibrosis, Transforming Growth factor beta-1

Two processes are associated with progressive loss of renal function: 1) decreased aquaporin-2 (AQP2) expression and urinary concentrating capacity (Nephrogenic Diabetes Insipidus, NDI); and 2) changes in extracellular matrix (ECM) composition, e.g. increased collagen I (Col I) deposition, characteristic of tubule-interstitial fibrosis. AQP2 expression is regulated by both the ECM-to-intracellular scaffold protein integrin-linked kinase (ILK) by NFATc/AP1 and other transcription factors. In the present work, we used in vivo and in vitro approaches to examine ILK participation in NFATc3/AP-1-mediated increases in AQP2 gene expression. Both NFATc3 knock-out mice and ILK conditional-knockdown mice (cKD-ILK) display symptoms of NDI (polyuria and reduced AQP2 expression). NFATc3 is upregulated in the renal medulla tubular cells of cKD-ILK mice but with reduced nuclear localization. Inner medullary collecting duct mIMCD3 cells were subjected to ILK depletion and transfected with reporter plasmids. Pharmacological activators or inhibitors determined the effect of ILK activity on NFATc/AP-1-dependent increases in transcription of AQP2. Finally, mIMCD3 cultured on Col I showed reduced activity of the ILK/GSK3β/NFATc/AQP2 axis, suggesting this pathway is a potential target for therapeutic treatment of NDI.

The development of insulin resistance is characterized by the impairment of glucose uptake mediated by glucose transporter 4 (GLUT4). Extracellular matrix changes are induced when the metabolic dysregulation is sustained. The present work was devoted to analyze the possible link between the extracellular-to-intracellular mediator integrin-linked kinase (ILK) and the peripheral tissue modification that leads to glucose homeostasis impairment. Mice with general depletion of ILK in adulthood (cKD-ILK) maintained in a chow diet exhibited increased glycemia and insulinemia concurrently with a reduction of the expression and membrane presence of GLUT4 in the insulin-sensitive peripheral tissues compared with their wild-type littermates (WT). Tolerance tests and insulin sensitivity indexes confirmed the insulin resistance in cKD-ILK, suggesting a similar stage to prediabetes in humans. Under randomly fed conditions, no differences between cKD-ILK and WT were observed in the expression of insulin receptor (IR-B) and its substrate IRS-1 expressions. The IR-B isoform phosphorylated at tyrosines 1150/1151 was increased, but the AKT phosphorylation in serine 473 was reduced in cKD-ILK tissues. Similarly, ILK-blocked myotubes reduced their GLUT4 promoter activity and GLUT4 expression levels. On the other hand, the glucose uptake capacity in response to exogenous insulin was impaired when ILK was blockedin vivoandin vitro, although IR/IRS/AKT phosphorylation states were increased but not different between groups. We conclude that ILK depletion modifies the transcription of GLUT4, which results in reduced peripheral insulin sensitivity and glucose uptake, suggesting ILK as a molecular target and a prognostic biomarker of insulin resistance.

Kidney fibrosis is one of the main pathological findings of progressive chronic kidney disease (CKD) although the pathogenesis of renal scar formation remains incompletely explained. Integrin-linked kinase (ILK), a major scaffold protein between the extracellular matrix (ECM) and intracellular signaling pathways, is involved in several pathophysiological processes during renal damage. However, ILK contribution in the CKD progress remains to be fully elucidated. In the present work, we studied 1) the renal functional and structural consequences of CKD genesis and progression when ILK is depleted and 2) the potential of ILK depletion as a therapeutic approach to delay CKD progression. We induced an experimental CKD model, based on an adenine-supplemented diet on adult wild-type (WT) and ILK-depleted mice, with a tubulointerstitial damage profile resembling that is observed in human CKD. The adenine diet induced in WT mice a progressive increase in plasma creatinine and urea concentrations. In the renal cortex it was also observed tubular damage, interstitial fibrosis and progressive increased ECM components, pro-inflammatory and chemo-attractant cytokines, EMT markers and TGF-β1 expressions. These observations were highly correlated to a simultaneous increase of ILK expression and activity. In adenine-fed transgenic ILK-depleted mice, all these changes were prevented. Additionally, we evaluated the potential role of ILK depletion to be applied after the disease induction, as an effective approach to interventions in human CKD subjects. In this scenario, two weeks after the establishment of adenine-induced CKD, ILK was abrogated in WT mice and stabilized renal damage, avoiding CKD progression. We propose ILK to be a potential target to delay renal disease progression.

The pathogenesis of diabetic retinopathy (DR) remains unclear but hyperglycemia is an established risk factor. Endothelial dysfunction and changes in Ca2+signaling have been shown to precede the onset of DR. We recently demonstrated that high extracellular glucose activates the Ca2+/calcineurin-dependent transcription factor NFAT in cerebral arteries and aorta, promoting the expression of inflammatory markers. Here we show, using confocal immunofluorescence, that NFAT is expressed in the endothelium of retinal microvessels and is readily activated by high glucose. This was inhibited by the NFAT blocker A-285222 as well as by the ectonucleotidase apyrase, suggesting a mechanism involving the release of extracellular nucleotides. Acute hyperglycemia induced by an IP-GTT (intraperitoneal glucose tolerance test) resulted in increased NFATc3 nuclear accumulation and NFAT-dependent transcriptional activity in retinal vessels of NFAT-luciferase reporter mice. In both Akita (Ins2+/−) and streptozotocin- (STZ-) induced diabetic mice, NFAT transcriptional activity was elevated in retinal vessels.In vivoinhibition of NFAT with A-285222 decreased the expression ofOPNandICAM-1mRNA in retinal vessels, prevented a diabetes driven downregulation of anti-inflammatory IL-10 in retina, and abrogated the increased vascular permeability observed in diabetic mice. Results identify NFAT signaling as a putative target for treatment of microvascular complications in diabetes.

Vesicular stomatitis virus G glycoprotein (VSVg) is extensively used for retroviral and lentiviral vector (LV) pseudotyping. However, VSVg pseudotyped vectors are serum inactivated, blocking the in vivo gene delivery. Several strategies have been employed to prevent complement inactivation, including chemical and genetic envelope modifications. This study employed the streptococcal albumin-binding domain (ABD) to generate a construct to express ABD as a glycosylphosphatidylinositol-anchored protein. LV particles bearing ABD are able to bind bovine and human serum albumin in vitro. Neither the lentiviral vector production titer nor the in vitro transduction was affected by the ABD display. The study demonstrated that ABD-bearing LVs are protected from human complement inactivation. More importantly, intravenous administration demonstrated that the presence of ABD significantly reduces lentivector sequestration in liver and bone-marrow cells. Therefore, the use of ABD represents an improvement for in vivo gene therapy applications. The results strongly point to ABD display as a universal strategy to increase the in vivo efficacy of different viral vectors.

One of the clinical alterations observed in chronic renal disease (CRD) is the impaired urine concentration, known as diabetes insipidus (DI). Tubulointerstitial fibrosis of the kidney is also a pathological finding observed in CRD and involves composition of extracellular matrix (ECM). However, an association between these two events has not been elucidated. In this study, we showed that the extracellular-to-intracellular scaffold protein integrin-linked kinase (ILK) regulates expression of tubular water channel aquaporin-2 (AQP2) and its apical membrane presence in the renal tubule. Basally, polyuria and decreased urine osmolality were present in ILK conditional-knockdown (cKD-ILK) adult mice compared with nondepleted ILK littermates. No changes were observed in arginine-vasopressin (AVP) blood levels, renal receptor (V2R), or AQP3 expression. However, tubular AQP2 was decreased in expression and apical membrane presence in cKD-ILK mice, where the canonical V2R/cAMP axis activation is still functional, but independent of the absence of ILK. Thus, cKD-ILK constitutes a nephrogenic diabetes insipidus (NDI) model. AQP2 and ILK colocalize in cultured inner medullary collecting duct (mIMCD3) cells. Specific ILK siRNAs and collagen I (Col) decrease ILK and AQP2 levels and AQP2 presence on the membrane of tubular mIMCD3 cells, which impairs the capacity of the cells to transport water under hypotonic stress. The present work points to ILK as a therapeutic target in NDI.

Aims Integrin-linked kinase (ILK) regulates proliferation, differentiation, cell adhesion, and motility in many cell types and has been related to cancer progression, fibrosis, and vascular diseases. We designed the present study to directly explore the effect of ILK deletion on the regulation of vascular tone through the soluble guanylate cyclase (sGC) /protein kinase G (PKG) pathway in healthy adult mice. Methods and results Experiments were carried out using a tamoxifen-inducible CRE-LOX system to conditionally delete the ILK gene in adult mice. Mice lacking ILK expression (cKO) presented increased vascular content and increased activity of sGC and PKG, resulting in a more intense vasodilatory response to a single dose of a nitric oxide (NO) donor [sodium nitroprusside (SNP)] or PKG agonist [8-bromoguanosine 3′,5′-cyclic monophosphate sodium salt (8-Br)]. Five minutes after SNP or 8-Br administration the reduction in the systolic arterial pressure was enhanced in cKO mice (SNP WT: −7.4 ± 1.2 mmHG; SNP cKO: −14.0 ± 2.5; 8-Br WT: −2.9 ± 1.5 mmHG; 8-Br cKO: −10.0 ± 3.4 mmHG). ILK deletion restored the vascular response to SNP after chronic oral nitrite administration. In addition, ILK deletion also increased hypotensive SNP effect in angiotensin II-treated animals, suggesting a role for ILK in basal and pathological states. Conclusion Deletion of ILK in adult animals increased the vascular response to NO. These findings show, for the first time, a requirement for ILK in regulating sGC–PKG expression in vivo .

This paper represents research in progress in Simultaneous Localization and Mapping (SLAM) for Micro Aerial Vehicles (MAVs) in the context of rescue and/or recognition navigation tasks in indoor environments. In this kind of applications, the MAV must rely on its own onboard sensors to autonomously navigate in unknown, hostile and GPS denied environments, such as ruined or semi-demolished buildings. This article aims to investigate a SLAM technique that fuses visual information and measurements from the inertial measurement unit (IMU), to robustly obtain the 6DOF pose estimation of a MAV within a local map of the environment. The monocular visual SLAM algorithm along with the IMU calculate the pose estimation through an Extended Kalman Filter (EKF). The system consists of a low-cost commercial drone and a remote control unit to computationally afford the SLAM algorithms using a distributed node system based on ROS (Robot Operating System). Some experimental results show how sensor fusion improves the position estimation and the obtained map under different test conditions.

Soluble guanylyl cyclase (sGC) is activated by nitric oxide (NO) and produces cGMP, which activates cGMP-dependent protein kinases (PKG) and is hydrolyzed by specific phosphodiesterases (PDE). The vasodilatory and cytoprotective capacity of cGMP-axis activation results in a therapeutic strategy for several pathologies. Integrin-linked kinase (ILK), a major scaffold protein between the extracellular matrix and intracellular signaling pathways, may modulate the expression and functionality of the cGMP-axis–related proteins. We introduce ILK as a novel modulator in renal homeostasis as well as a potential target for cisplatin (CIS)-induced acute kidney injury (AKI) improvement. We used an adult mice model of depletion of ILK (cKD-ILK), which showed basal increase of sGC and PKG expressions and activities in renal cortex when compared with wildtype (WT) littermates. Twenty-four h activation of sGC activation with NO enhanced the filtration rate in cKD-ILK. During AKI, cKD-ILK maintained the cGMP-axis upregulation with consequent filtration rates enhancement and ameliorated CIS-dependent tubular epithelial-to-mesenchymal transition and inflammation and markers. To emphasize the role of cGMP-axis upregulation due to ILK depletion, we modulated the cGMP axis under AKI in vivo and in renal cultured cells. A suboptimal dose of the PDE inhibitor ZAP enhanced the beneficial effects of the ILK depletion in AKI mice. On the other hand, CIS increased contractility-related events in cultured glomerular mesangial cells and necrosis rates in cultured tubular cells; ILK depletion protected the cells while sGC blockade with ODQ fully recovered the damage.

Within the past decade tremendous efforts have been made to understand the mechanism behind aquaporin-2 (AQP2) water channel trafficking and recycling, to open a path toward effective diabetes insipidus therapeutics. A recent study has shown that integrin-linked kinase (ILK) conditional-knockdown mice developed polyuria along with decreased AQP2 expression. To understand whether ILK also regulates AQP2 trafficking in kidney tubular cells, we performed in vitro analysis using LLCPK1 cells stably expressing rat AQP2 (LLC-AQP2 cells). Upon treatment of LLC-AQP2 cells with ILK inhibitor cpd22 and ILK-siRNA, we observed increased accumulation of AQP2 in the perinuclear region, without any significant increase in the rate of endocytosis. This perinuclear accumulation did not occur in cells expressing a serine-256-aspartic acid mutation that retains AQP2 in the plasma membrane. We then examined clathrin-mediated endocytosis after ILK inhibition using rhodamine-conjugated transferrin. Despite no differences in overall transferrin endocytosis, the endocytosed transferrin also accumulated in the perinuclear region where it colocalized with AQP2. These accumulated vesicles also contained the recycling endosome marker Rab11. In parallel, the usual vasopressin-induced AQP2 membrane accumulation was prevented after ILK inhibition; however, ILK inhibition did not measurably affect AQP2 phosphorylation at serine-256 or its dephosphorylation at serine-261. Instead, we found that inhibition of ILK increased F-actin polymerization. When F-actin was depolymerized with latrunculin, the perinuclear located AQP2 dispersed. We conclude that ILK is important in orchestrating dynamic cytoskeletal architecture during recycling of AQP2, which is necessary for its subsequent entry into the exocytotic pathway.

The nitric oxide/soluble guanylyl cyclase (sGC) signal transduction pathway plays an important role in smooth muscle relaxation and phenotypic regulation. However, the transcriptional regulation of sGC gene expression is largely unknown. It has been shown that sGC expression increases in pulmonary arteries from chronic hypoxia-induced pulmonary hypertensive animals. Since the transcription factor NFATc3 is required for the upregulation of the smooth muscle hypertrophic/differentiation marker alpha-actin in pulmonary artery smooth muscle cells from chronically hypoxic mice, we hypothesized that NFATc3 is required for the regulation of sGC-alpha1 expression during chronic hypoxia. Exposure to chronic hypoxia for 2 days induced a decrease in sGC-alpha1 expression in mouse pulmonary arteries. This reduction was independent of NFATc3 but mediated by nuclear accumulation of the mRNA-stabilizing protein human antigen R (HuR). Consistent with our hypothesis, chronic hypoxia (21 days) upregulated pulmonary artery sGC-alpha1 expression, bringing it back to the level of the normoxic controls. This response was prevented in NFATc3 knockout and cyclosporin (calcineurin/NFATc inhibitor)-treated mice. Furthermore, we identified effective binding sites for NFATc in the mouse sGC-alpha1 promoter. Activation of NFATc3 increased sGC-alpha1 promoter activity in human embryonic derived kidney cells, rat aortic-derived smooth muscle cells, and human pulmonary artery smooth muscle cells. Our results suggest that NFATc3 and HuR are important regulators of sGC-alpha1 expression in pulmonary vascular smooth muscle cells during chronic hypoxia-induced pulmonary hypertension.

Sleep apnea, defined as intermittent respiratory arrest during sleep, is associated with increased incidence of hypertension and peripheral vascular disease. Exposure of rodents to brief periods of intermittent hypercarbia/hypoxia (H-IH) during sleep mimics the cyclical hypoxia-normoxia of sleep apnea. Endothelin-1, an upstream activator of nuclear factor of activated T cells (NFAT), is increased during H-IH. Therefore, we hypothesized that NFATc3 is activated by H-IH and is required for H-IH-induced hypertension. Consistent with this hypothesis, we found that H-IH (20 brief exposures per hour to 5% O2-5% CO2for 7 h/day) induces systemic hypertension in mice [mean arterial pressure (MAP) = 97 ± 2 vs. 124 ± 2 mmHg, P < 0.05, n = 5] and increases NFATc3 transcriptional activity in aorta and mesenteric arteries. Cyclosporin A, an NFAT inhibitor, and genetic ablation of NFATc3 [NFATc3 knockout (KO)] prevented NFAT activation. More importantly, H-IH-induced hypertension was attenuated in cyclosporin A-treated mice and prevented in NFATc3 KO mice. MAP was significantly elevated in wild-type mice (Δ = 23.5 ± 6.1 mmHg), but not in KO mice (Δ = −3.9 ± 5.7). These results indicate that H-IH-induced increases in MAP require NFATc3 and that NFATc3 may contribute to the vascular changes associated with H-IH-induced hypertension.

Glomerular diseases are characterized by an abnormal synthesis of extracellular matrix proteins, such as collagen type I. Evidence that growth on collagen type I downregulates soluble guanylyl cyclase (sGC) expression and the responsiveness of human mesangial cells to nitric oxide (NO) by activating specific integrin signals involving integrin-linked kinase (ILK) is presented. Human mesangial cells were grown on collagen type I or IV for 24 to 72 h. Compared with collagen IV, growth on collagen I reduced the protein expression and NO-stimulated enzyme activity of sGC. This downregulation was effected at the level of transcription, because steady-state sGC mRNA expression was reduced on collagen I, but inhibition of transcription with Actinomycin D revealed no differences in transcript stability between the two culture conditions. Collagen I also reduced the capacity of cells to relax in response to NO after H2O2 challenge and inhibited NO-induced phosphorylation of vasodilator-activated phosphoprotein, a target of cyclic guanine monophosphate-dependent protein kinase. Examination of the surface expression of integrins, the receptors for extracellular matrix components, revealed that alpha1 and alpha2 integrin subunits were more abundant on cells that were grown on collagen IV and that surface expression of beta1 integrin did not vary with collagen type. However, growth on collagen I induced beta1 integrin to adopt its active conformation, and this activation of beta1 integrin was accompanied by increased activity of its downstream effector ILK. Dominant-negative suppression of ILK signaling relieved the suppression of sGC expression and NO-induced vasodilator-activated phosphoprotein phosphorylation induced by collagen I.

Natriuretic peptides (NP) activate particulate guanylate cyclase (pGC) and nitric oxide (NO) activates soluble guanylate cyclase (sGC). Both guanylate cyclases catalyse the formation of the same second messenger, cyclic guanosine 3',5'-monophosphate (cGMP), which activates the cGMP-dependent protein kinases (PKG). PKG then starts a signalling cascade that mediates many cardiovascular and renal effects, such as smooth muscle relaxation and diuresis. Many cell types possess both sGC and pGC. Because both GC-cGMP systems play complementary roles, an interaction between the two pathways might represent an important physiological control mechanism. In this report we demonstrate an interaction between the two pathways. C-type natriuretic peptide (CNP) decreased the beta-subunit of sGC (sGC-beta) steady-state protein levels and enzymatic activity in cultured human mesangial cells (HMC) in a time- and dose-dependent manner. This down-regulation was not dependent on changes in sGC-beta mRNA levels. Treatment of the cells with the stable cGMP analogue 8-Br-cGMP or the phosphodiesterase type-5 inhibitor Zaprinast produced the same down-regulatory effect. Inhibition of PKG or proteasome activity prevented the CNP-induced reduction of sGC-beta protein levels and activity. Taken together, these results demonstrate that pGC activation induces a post-transductional down-regulation of sGC by a mechanism involving PKG and the proteasome pathway.

One of the main challenges of aerial robots navigation in indoor or GPS-denied environments is position estimation using only the available onboard sensors. This paper presents a Simultaneous Localization and Mapping (SLAM) system that remotely calculates the pose and environment map of different low-cost commercial aerial platforms, whose onboard computing capacity is usually limited. The proposed system adapts to the sensory configuration of the aerial robot, by integrating different state-of-the art SLAM methods based on vision, laser and/or inertial measurements using an Extended Kalman Filter (EKF). To do this, a minimum onboard sensory configuration is supposed, consisting of a monocular camera, an Inertial Measurement Unit (IMU) and an altimeter. It allows to improve the results of well-known monocular visual SLAM methods (LSD-SLAM and ORB-SLAM are tested and compared in this work) by solving scale ambiguity and providing additional information to the EKF. When payload and computational capabilities permit, a 2D laser sensor can be easily incorporated to the SLAM system, obtaining a local 2.5D map and a footprint estimation of the robot position that improves the 6D pose estimation through the EKF. We present some experimental results with two different commercial platforms, and validate the system by applying it to their position control., Comunidad de Madrid, Universidad de Alcalá

The circulating levels of heat shock proteins (HSP) are increased in cardiovascular diseases; however, the implication of this for the fibrotic process typical of such diseases remains unclear. HSP70 can interact with the vascular smooth muscle cells (SMC), the major producer of extracellular matrix (ECM) proteins, through the Toll-like receptors 4 (TLR4). The transforming growth factor type-β1 (TGF-β1) is a well known vascular pro-fibrotic cytokine that is regulated in part by AP-1-dependent transcriptional mechanisms. We hypothesized that extracellular HSP70 could interact with SMCs, inducing TGF-β1 synthesis and subsequent changes in the vascular ECM. We demonstrate that extracellular HSP70 binds to human aorta SMC TLR4, which up-regulates the AP-1-dependent transcriptional activity of the TGF-β1 promoter. This is achieved through the mitogen activated protein kinases JNK and ERK, as demonstrated by the use of specific blockers and the knockdown of TLR4 with specific small interfering RNAs. The TGF-β1 upregulation increase the expression of the ECM proteins type I collagen and fibronectin. This novel observation may elucidate the mechanisms by which HSP70 contributes in the inflammation and fibrosis present in atherosclerosis and other fibrosis-related diseases.

Membrane-anchored ephrinB2 and its receptor EphB4 are involved in the formation of blood and lymphatic vessels in normal and pathologic conditions. Eph/ephrin activation requires cell-cell interactions and leads to bidirectional signaling pathways in both ligand- and receptor-expressing cells. To investigate the functional consequences of blocking ephrinB2 activity, 2 highly specific human single-chain Fv (scFv) Ab fragments against ephrinB2 were generated and characterized. Both Ab fragments suppressed endothelial cell migration and tube formation in vitro in response to VEGF and provoked abnormal cell motility and actin cytoskeleton alterations in isolated endothelial cells. As only one of them (B11) competed for binding of ephrinB2 to EphB4, these data suggest an EphB-receptor–independent blocking mechanism. Anti-ephrinB2 therapy reduced VEGF-induced neovascularization in a mouse Matrigel plug assay. Moreover, systemic administration of ephrinB2-blocking Abs caused a drastic reduction in the number of blood and lymphatic vessels in xenografted mice and a concomitant reduction in tumor growth. Our results show for the first time that specific Ab-based ephrinB2 targeting may represent an effective therapeutic strategy to be used as an alternative or in combination with existing antiangiogenic drugs for treating patients with cancer and other angiogenesis-related diseases.

Background/aims: We have previously shown that aquaporin-2 (AQP2) is down-regulated in the renal medulla of rats made hypertensive by chronic inhibition of nitric oxide synthase (NOS). It has been shown that AQP2 expression is regulated by the calcineurin/ nuclear factor of activated T cells (NFATc). Nitric oxide (NO) regulates the activity of NFATc via c-Jun-N-terminal kinase 2 (JNK2). Therefore, we hypothesized that increases in NO enhance NFATc-mediated up-regulation of AQP2 promoter activity. Methods: AQP2 mRNA and protein expression were detected in mouse renal papilla. AQP2-promoter-luciferase reporter and NFAT-luciferase reporter transfected MDCK cells were used to determine AQP2 promoter activity and NFATc activity, respectively. Cells were incubated with classic activators and inhibitors of NFATc and NO pathway. Results: Our results demonstrate that both Ca2+ and NO have a synergistic effect in increasing AQP2 mRNA and protein in mouse papilla, and in activating the AQP2 promoter in kidney-derived cells. In addition, NO enhances Ca2+-induced NFATc activation. The underlying mechanism involves increased NFATc nuclear import and decreased export via PKG-mediated inhibition of JNK1/2. Conclusions: This is the first study defining novel regulatory roles for NO and NFATc in the control of AQP2 expression, which is an important renal protein. Fil: Albertoni Borghese, Maria Florencia. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Biología Celular y Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina Fil: Bettini, Layne M.. University Of New Mexico. School Of Medicine. Departament Of Cell Biology And Phisiology; Estados Unidos Fil: Nitta, Carlos H.. University Of New Mexico. School Of Medicine. Departament Of Cell Biology And Phisiology; Estados Unidos Fil: de Frutos, Sergio. University Of New Mexico. School Of Medicine. Departament Of Cell Biology And Phisiology; Estados Unidos Fil: Majowicz, Mónica. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Biología Celular y Molecular; Argentina Fil: González Bosc, Laura Veronica. University Of New Mexico. School Of Medicine. Departament Of Cell Biology And Phisiology; Estados Unidos

Chronic hypoxia (CH) activates the Ca2+-dependent transcription factor nuclear factor of activated T cells isoform c3 (NFATc3) in mouse pulmonary arteries. However, the mechanism of this response has not been explored. Since we have demonstrated that NFATc3 is required for CH-induced pulmonary arterial remodeling, establishing how CH activates NFATc3 is physiologically significant. The goal of this study was to test the hypothesis that endothelin-1 (ET-1) contributes to CH-induced NFATc3 activation. We propose that this mechanism requires increased pulmonary arterial smooth muscle cell (PASMC) intracellular Ca2+concentration ([Ca2+]i) and stimulation of RhoA/Rho kinase (ROK), leading to calcineurin activation and actin cytoskeleton polymerization, respectively. We found that: 1) CH increases pulmonary arterial pre-pro-ET-1 mRNA expression and lung RhoA activity; 2) inhibition of ET receptors, calcineurin, L-type Ca2+channels, and ROK blunts CH-induced NFATc3 activation in isolated intrapulmonary arteries from NFAT-luciferase reporter mice; and 3) both ET-1-induced NFATc3 activation in isolated mouse pulmonary arteries ex vivo and ET-1-induced NFATc3-green fluorescence protein nuclear import in human PASMC depend on ROK and actin polymerization. This study suggests that CH increases ET-1 expression, thereby elevating PASMC [Ca2+]iand RhoA/ROK activity. As previously demonstrated, elevated [Ca2+]iis required to activate calcineurin, which dephosphorylates NFATc3, allowing its nuclear import. Here, we demonstrate that ROK increases actin polymerization, thus providing structural support for NFATc3 nuclear transport.

Sleep apnea (SA) is defined as intermittent respiratory arrest during sleep and affects up to 20% of the adult population. SA is also associated with an increased incidence of hypertension and peripheral vascular disease. Exposing rodents to intermittent hypoxia during sleep mimics the cyclical hypoxia/normoxia of SA. We have previously shown that in mice and rats intermittent hypoxia induces ET-1 upregulation and systemic hypertension. Furthermore, intermittent hypoxia (IH) in mice increases nuclear factor of activated T cells isoform 3 (NFATc3) transcriptional activity in aorta and mesenteric arteries, whereas the calcineurin/NFAT inhibitor cyclosporin A prevents IH-induced hypertension. More importantly, NFATc3 knockout (KO) mice do not develop IH-induced hypertension. The goals of this study were to determine the role of NFATc3 in IH-induced arterial remodeling and whether IH-induced NFATc3 activation is mediated by ET-1. Oral administration of both a dual (bosentan) and a selective endothelin receptor type A antagonist (PD155080) during 2 days of IH exposure attenuated NFAT activation in aorta and mesenteric arteries. Rho kinase inhibition with fasudil also prevented IH-induced NFAT activation. Mesenteric artery cross-sectional wall thickness was increased by IH in wild-type (WT) and vehicle-treated mice but not in bosentan-treated and NFATc3 KO mice. The arterial remodeling in mesenteric arteries after IH was characterized by increased expression of the hypertrophic NFATc3 target smooth muscle-α-actin in WT but not in KO mice. These results indicate that ET-1 is an upstream activator of NFATc3 during intermittent hypoxia, contributing to the resultant hypertension and increased wall thickness.