Assuntos
Anticorpos Monoclonais Humanizados , Antivirais , Doenças do Recém-Nascido , Recém-Nascido Prematuro , Infecções por Vírus Respiratório Sincicial , Humanos , Lactente , Recém-Nascido , Anticorpos Monoclonais Humanizados/uso terapêutico , Antivirais/uso terapêutico , Hospitalização , Palivizumab , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Nascimento a Termo , Doenças do Recém-Nascido/tratamento farmacológico , Doenças do Recém-Nascido/prevenção & controleRESUMO
Endoglin (CD105) is an auxiliary receptor for members of the TFG-ß superfamily. Whereas it has been demonstrated that the deficiency of endoglin leads to minor and defective angiogenesis, little is known about the effect of its increased expression, characteristic of several types of cancer. Angiogenesis is essential for tumor growth, so high levels of proangiogenic molecules, such as endoglin, are supposed to be related to greater tumor growth leading to a poor cancer prognosis. However, we demonstrate here that endoglin overexpression do not stimulate sprouting or vascularization in several in vitro and in vivo models. Instead, steady endoglin overexpression keep endothelial cells in an active phenotype that results in an impairment of the correct stabilization of the endothelium and the recruitment of mural cells. In a context of continuous enhanced angiogenesis, such as in tumors, endoglin overexpression gives rise to altered vessels with an incomplete mural coverage that permit the extravasation of blood. Moreover, these alterations allow the intravasation of tumor cells, the subsequent development of metastases and, thus, a worse cancer prognosis.
Assuntos
Endoglina/genética , Metástase Neoplásica/genética , Neoplasias/irrigação sanguínea , Neoplasias/genética , Neovascularização Patológica/genética , Animais , Movimento Celular/genética , Células Cultivadas , Endoglina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Invasividade Neoplásica , Neoplasias/patologia , Neovascularização Patológica/patologia , Regulação para Cima/genéticaRESUMO
Preeclampsia is a pregnancy-specific disease of high prevalence characterized by the onset of hypertension, among other maternal or fetal signs. Its etiopathogenesis remains elusive, but it is widely accepted that abnormal placentation results in the release of soluble factors that cause the clinical manifestations of the disease. An increased level of soluble endoglin (sEng) in plasma has been proposed to be an early diagnostic and prognostic biomarker of this disease. A pathogenic function of sEng involving hypertension has also been reported in several animal models with high levels of plasma sEng not directly dependent on pregnancy. The aim of this work was to study the functional effect of high plasma levels of sEng in the pathophysiology of preeclampsia in a model of pregnant mice, in which the levels of sEng in the maternal blood during pregnancy replicate the conditions of human preeclampsia. Our results show that wild type pregnant mice carrying human sEng-expressing transgenic fetuses (fWT(hsEng+)) present high plasma levels of sEng with a timing profile similar to that of human preeclampsia. High plasma levels of human sEng (hsEng) are associated with hypertension, proteinuria, fetal growth restriction, and the release of soluble factors to maternal plasma. In addition, fWT(hsEng+) mice also present placental alterations comparable to those caused by the poor remodeling of the spiral arteries characteristic of preeclampsia. In vitro and ex vivo experiments, performed in a human trophoblast cell line and human placental explants, show that sEng interferes with trophoblast invasion and the associated pseudovasculogenesis, a process by which cytotrophoblasts switch from an epithelial to an endothelial phenotype, both events being related to remodeling of the spiral arteries. Our findings provide a novel and useful animal model for future research in preeclampsia and reveal a much more relevant role of sEng in preeclampsia than initially proposed.
Assuntos
Endoglina/metabolismo , Retardo do Crescimento Fetal/patologia , Doenças Placentárias/patologia , Pré-Eclâmpsia/patologia , Trofoblastos/patologia , Doenças Vasculares/patologia , Animais , Endoglina/genética , Feminino , Retardo do Crescimento Fetal/etiologia , Retardo do Crescimento Fetal/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Doenças Placentárias/etiologia , Doenças Placentárias/metabolismo , Pré-Eclâmpsia/etiologia , Pré-Eclâmpsia/metabolismo , Gravidez , Trofoblastos/metabolismo , Doenças Vasculares/etiologia , Doenças Vasculares/metabolismoRESUMO
Complex interactions between platelets and activated endothelium occur during the thrombo-inflammatory reaction at sites of vascular injuries and during vascular hemostasis. The endothelial receptor endoglin is involved in inflammation through integrin-mediated leukocyte adhesion and transmigration; and heterozygous mutations in the endoglin gene cause hereditary hemorrhagic telangiectasia type 1. This vascular disease is characterized by a bleeding tendency that is postulated to be a consequence of telangiectasia fragility rather than a platelet defect, since platelets display normal functions in vitro in this condition. Here, we hypothesize that endoglin may act as an adhesion molecule involved in the interaction between endothelial cells and platelets through integrin recognition. We find that the extracellular domain of human endoglin promotes specific platelet adhesion under static conditions and confers resistance of adherent platelets to detachment upon exposure to flow. Also, platelets adhere to confluent endothelial cells in an endoglin-mediated process. Remarkably, Chinese hamster ovary cells ectopically expressing the human αIIbß3 integrin acquire the capacity to adhere to myoblast transfectants expressing human endoglin, whereas platelets from Glanzmann's thrombasthenia patients lacking the αIIbß3 integrin are defective for endoglin-dependent adhesion to endothelial cells. Furthermore, the bleeding time, but not the prothrombin time, is significantly prolonged in endoglin-haplodeficient (Eng +/-) mice compared to Eng +/+ animals. These results suggest a new role for endoglin in αIIbß3 integrin-mediated adhesion of platelets to the endothelium, and may provide a better understanding on the basic cellular mechanisms involved in hemostasis and thrombo-inflammatory events.
Assuntos
Plaquetas/metabolismo , Comunicação Celular , Endoglina/metabolismo , Células Endoteliais/metabolismo , Animais , Plaquetas/citologia , Células CHO , Adesão Celular , Linhagem Celular , Células Cultivadas , Cricetinae , Cricetulus , Endoglina/genética , Células Endoteliais/citologia , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismoRESUMO
Renal fibrosis and anaemia are two of the most relevant events in chronic kidney disease. Fibrosis is characterized by the accumulation of extracellular matrix proteins in the glomeruli and tubular interstitium. Anaemia is the consequence of a decrease in erythropoietin production in fibrotic kidneys. This work analyses the possibility that the accumulation of abnormal collagens in kidney interstitium could be one of the mechanisms responsible for erythropoietin decreased synthesis. In renal interstitial fibroblast grown on collagen I, erythropoietin mRNA expression and HIF-2α protein decreased, whereas focal adhesion kinase protein (FAK) phosphorylation and proteasome activity increased, compared to cells grown on collagen IV. Proteasome inhibition or FAK inactivation in cells plated on collagen I restored erythropoietin and HIF-2α expression. FAK inhibition also decreased the collagen I-dependent proteasome activation. In a model of tubulointerstitial fibrosis induced by unilateral ureteral obstruction in mice, increased collagen I protein content and an almost complete disappearance of erythropoietin mRNA expression were observed in the ureteral ligated kidney with respect to the contralateral control. Interestingly, erythropoietin synthesis was recovered in obstructed mice treated with proteasome inhibitor. These data suggest that reduced kidney erythropoietin synthesis could be caused by the accumulation of abnormal extracellular matrix proteins.
Assuntos
Eritropoetina/biossíntese , Matriz Extracelular/metabolismo , Insuficiência Renal Crônica/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular , Colágeno Tipo I/farmacologia , Regulação para Baixo/efeitos dos fármacos , Eritropoetina/genética , Eritropoetina/metabolismo , Matriz Extracelular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibrose , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Humanos , Rim/patologia , Camundongos Endogâmicos C57BL , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Proteólise/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Insuficiência Renal Crônica/patologia , Obstrução Ureteral/patologiaRESUMO
Nephrotoxicity is the main limitation to the dosage and anticancer efficacy of cisplatin. Cisplatin produces tubular epithelial cell apoptosis and necrosis depending on the concentration of the drug. Protection from cisplatin nephrotoxicity must therefore tackle both cell death modes. For its ability to reduce cisplatin reactivity, in addition to its antioxidant effect, we tested and found that N-acetylcysteine (NAC) was most effective at inhibiting cisplatin cytotoxicity. NAC has no significant effect on cell death induced by either cycloheximide or Fas activation, indicating a rather selective action. Pt-DNA-binding experiments suggest that the differential effectiveness of NAC is due to its capacity to quench cisplatin reactivity inside the cell. NAC abolishes cisplatin-induced apoptosis, and transforms the necrosis induced by high concentrations of cisplatin into apoptosis. In fact, NAC allows the anti-apoptotic molecule Bcl-2 to reduce the cell death caused by pro-necrotic concentrations of cisplatin, to a significantly greater extent than in the absence of NAC. In rats, a dosage of NAC that significantly ameliorates cisplatin nephrotoxicity, has little effect on gentamicin nephrotoxicity. These characteristics provide NAC with a rationale as a potential nephroprotectant specifically tailored to and especially effective for therapeutic courses with platinated antineoplastics, which prompts to deepening into further preclinical knowledge, and to initiate clinical studies with NAC and mixed therapies composed of NAC and antiapoptotic drugs.
Assuntos
Acetilcisteína/farmacologia , Antineoplásicos/toxicidade , Apoptose/efeitos dos fármacos , Cisplatino/toxicidade , Sequestradores de Radicais Livres/farmacologia , Necrose/induzido quimicamente , Animais , Caspases/análise , Caspases/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Humanos , Células Jurkat , Nefropatias/induzido quimicamente , Nefropatias/patologia , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Ratos , Ratos WistarRESUMO
Cardiotrophin-1 (CT-1) holds potent anti-inflammatory, cytoprotective, and anti-apoptotic effects in the liver, kidneys, and heart. In the present study, the role of endogenous CT-1 and the effect of exogenous CT-1 were evaluated in experimental ulcerative colitis. Colitis was induced in CT-1 knockout and wild-type (WT) mice by administration of dextran sulphate sodium (DSS) in the drinking water during 7 days. CT-1 knockout mice showed higher colon damage and disease severity than WT mice. In addition, CT-1 (200 µg/kg/day, iv) or vehicle (as control) was administered during 3 days to WT, colitic mice, starting on day 4 after initiation of DSS. Disease activity index (DAI), inflammatory markers (tumor necrosis factor α (TNF-α), INFγ, IL-17, IL-10, inducible nitric oxide synthase (iNOS)), colon damage, apoptosis (cleaved caspase 3), nuclear factor κB (NFκB) and STAT-3 activation, and bacterial translocation were measured. Compared with mice treated with DSS, mice also treated with exogenous CT-1 showed lower colon damage, DAI, plasma levels of TNFα, colon expression of TNF-α, INFγ, IL-17, iNOS and cleaved caspase 3, higher NFκB and signal transducer and activator of transcription 3 (STAT3) pathways activation, and absence of bacterial translocation. We conclude that endogenous CT-1 plays a role in the defense and repair response of the colon against ulcerative lesions through an anti-inflammatory and anti-apoptotic effect. Supplementation with exogenous CT-1 ameliorates disease symptoms, which opens a potentially new therapeutic strategy for ulcerative colitis.
Assuntos
Colite Ulcerativa/tratamento farmacológico , Colite Ulcerativa/metabolismo , Citocinas/sangue , Citocinas/uso terapêutico , Animais , Colite Ulcerativa/induzido quimicamente , Citocinas/genética , Sulfato de Dextrana , Avaliação Pré-Clínica de Medicamentos , Masculino , Camundongos , Camundongos KnockoutRESUMO
Transforming growth factor beta 1 (TGF-ß1) is one of the most studied cytokines involved in renal tubulo-interstitial fibrosis, which is characterized by myofibroblast abundance and proliferation, and high buildup of extracellular matrix in the tubular interstitium leading to organ failure. Endoglin (Eng) is a 180-kDa homodimeric transmembrane protein that regulates a great number of TGF-ß1 actions in different biological processes, including ECM synthesis. High levels of Eng have been observed in experimental models of renal fibrosis or in biopsies from patients with chronic kidney disease. In humans and mice, two Eng isoforms are generated by alternative splicing, L-Eng and S-Eng that differ in the length and composition of their cytoplasmic domains. We have previously described that L-Eng overexpression promotes renal fibrosis after unilateral ureteral obstruction (UUO). However, the role of S-Eng in renal fibrosis is unknown and its study would let us analyze the possible function of the cytoplasmic domain of Eng in this process. For this purpose, we have generated a mice strain that overexpresses S-Eng (S-ENG(+)) and we have performed an UUO in S-ENG(+) and their wild type (WT) control mice. Our results indicate that obstructed kidney of S-ENG(+) mice shows lower levels of tubulo-interstitial fibrosis, less inflammation and less interstitial cell proliferation than WT littermates. Moreover, S-ENG(+) mice show less activation of Smad1 and Smad2/3 pathways. Thus, S-Eng overexpression reduces UUO-induced renal fibrosis and some associated mechanisms. As L-Eng overexpression provokes renal fibrosis we conclude that Eng-mediated induction of renal fibrosis in this model is dependent on its cytoplasmic domain.
Assuntos
Endoglina/genética , Endoglina/metabolismo , Rim/metabolismo , Rim/patologia , Nefrite/prevenção & controle , Obstrução Ureteral/metabolismo , Animais , Proliferação de Células , Colágeno Tipo I/metabolismo , Modelos Animais de Doenças , Fibronectinas/metabolismo , Fibrose , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Modelos Biológicos , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Nefrite/metabolismo , Nefrite/patologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Regulação para Cima , Obstrução Ureteral/complicações , Obstrução Ureteral/patologiaRESUMO
Following arterial occlusion, blood vessels respond by forming a new network of functional capillaries (angiogenesis), by reorganizing preexisting capillaries through the recruitment of smooth muscle cells to generate new arteries (arteriogenesis) and by growing and remodeling preexisting collateral arterioles into physiologically relevant arteries (collateral development). All these processes result in the recovery of organ perfusion. The importance of endoglin in post-occlusion reperfusion is sustained by several observations: (1) endoglin expression is increased in vessels showing active angiogenesis/remodeling; (2) genetic endoglin haploinsufficiency in humans causes deficient angiogenesis; and (3) the reduction of endoglin expression by gene disruption or the administration of endoglin-neutralizing antibodies reduces angiogenesis and revascularization. However, the precise role of endoglin in the several processes associated with revascularization has not been completely elucidated and, in some cases, the function ascribed to endoglin by different authors is controversial. The purpose of this review is to organize in a critical way the information available for the role of endoglin in several phenomena (angiogenesis, arteriogenesis and collateral development) associated with post-ischemic revascularization.
Assuntos
Endoglina/metabolismo , Isquemia/metabolismo , Isquemia/fisiopatologia , Neovascularização Fisiológica , Animais , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Humanos , Transdução de Sinais , Remodelação VascularRESUMO
Catecholamines are essential for the maintenance of physiological homeostasis under basal and stress conditions. We aim to determine the impact of deletion of a single allele of the tyrosine hydroxylase (Th) gene might have on aging arterial pressure and life-span. We found that Th haploinsufficiency prevents age-associated increase of arterial pressure (AP) in mature adult mice, and it results in the extension of the half-life of Th-heterozygous (TH-HET) mice respect to their wild-type (WT) littermates. Heart performance was similar in both genotypes. To further investigate the lack of increase in AP with age in TH-HET mice, we measured the AP response to intra-peritoneal administration of substances involved in AP regulation. The response to acetylcholine and the basal sympathetic tone were similar in both genotypes, while norepinephrine had a greater pressor effect in TH-HET mice, which correlated with altered adrenoreceptor expression in blood vessels and the heart. Furthermore, sympatho-adrenomedular response to stress was attenuated in TH-HET mice. Plasma catecholamine levels and urine glucose increased markedly in WT but not in TH-HET mice after stress. Our results showed that TH-HET mice are resistant to age-associated hypertension, present a reduction in the sympathetic response to stress and display an extended half-life.
Assuntos
Pressão Arterial , Haploinsuficiência , Hipertensão/genética , Tirosina 3-Mono-Oxigenase/genética , Fatores Etários , Envelhecimento , Animais , Hipertensão/etiologia , Hipertensão/fisiopatologia , Longevidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estresse FisiológicoRESUMO
The circulatory system is walled off by different cell types, including vascular mural cells and podocytes. The interaction and interplay between endothelial cells (ECs) and mural cells, such as vascular smooth muscle cells or pericytes, play a pivotal role in vascular biology. Endoglin is an RGD-containing counter-receptor for ß1 integrins and is highly expressed by ECs during angiogenesis. We find that the adhesion between vascular ECs and mural cells is enhanced by integrin activators and inhibited upon suppression of membrane endoglin or ß1-integrin, as well as by addition of soluble endoglin (SolEng), anti-integrin α5ß1 antibody or an RGD peptide. Analysis of different endoglin mutants, allowed the mapping of the endoglin RGD motif as involved in the adhesion process. In Eng (+/-) mice, a model for hereditary hemorrhagic telangectasia type 1, endoglin haploinsufficiency induces a pericyte-dependent increase in vascular permeability. Also, transgenic mice overexpressing SolEng, an animal model for preeclampsia, show podocyturia, suggesting that SolEng is responsible for podocytes detachment from glomerular capillaries. These results suggest a critical role for endoglin in integrin-mediated adhesion of mural cells and provide a better understanding on the mechanisms of vessel maturation in normal physiology as well as in pathologies such as preeclampsia or hereditary hemorrhagic telangiectasia.
Assuntos
Antígenos CD/metabolismo , Adesão Celular/fisiologia , Endotélio Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Podócitos/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Antígenos CD/genética , Linhagem Celular Tumoral , Modelos Animais de Doenças , Endoglina , Feminino , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Integrina beta1/genética , Células Jurkat , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Camundongos Transgênicos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/citologia , Neovascularização Patológica/metabolismo , Pericitos/metabolismo , Pré-Eclâmpsia/genética , Pré-Eclâmpsia/patologia , Gravidez , Ligação Proteica , Interferência de RNA , RNA Interferente Pequeno , Receptores de Superfície Celular/genética , Retina/metabolismo , Telangiectasia Hemorrágica Hereditária/genética , Telangiectasia Hemorrágica Hereditária/patologiaRESUMO
The involvement of Ras-GTPases in the development of renal fibrosis has been addressed in the last decade. We have previously shown that H- and N-Ras isoforms participate in the regulation of fibrosis. Herein, we assessed the role of K-Ras in cellular processes involved in the development of fibrosis: proliferation, migration, and extracellular matrix (ECM) proteins synthesis. K-Ras knockout (KO) mouse embryonic fibroblasts (K-ras(-/-) ) stimulated with transforming growth factor-ß1 (TGF-ß1) exhibited reduced proliferation and impaired mobility than wild-type fibroblasts. Moreover, an increase on ECM production was observed in K-Ras KO fibroblasts in basal conditions. The absence of K-Ras was accompanied by reduced Ras activation and ERK phosphorylation, and increased AKT phosphorylation, but no differences were observed in TGF-ß1-induced Smad signaling. The MEK inhibitor U0126 decreased cell proliferation independently of the presence of K-ras but reduced migration and ECM proteins expression only in wild-type fibroblasts, while the PI3K-AKT inhibitor LY294002 decreased cell proliferation, migration, and ECM synthesis in both types of fibroblasts. Thus, our data unveil that K-Ras and its downstream effector pathways distinctively regulate key biological processes in the development of fibrosis. Moreover, we show that K-Ras may be a crucial mediator in TGF-ß1-mediated effects in this cell type. J. Cell. Physiol. 231: 2224-2235, 2016. © 2016 Wiley Periodicals, Inc.
Assuntos
Movimento Celular , Proliferação de Células , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais , Animais , Butadienos/farmacologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Proteínas da Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Camundongos , Camundongos Knockout , Nitrilas/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas p21(ras)/deficiência , Fator de Crescimento Transformador beta1/metabolismoRESUMO
AIMS: A soluble form of endoglin (sEng) was proposed to participate in the induction of endothelial dysfunction in small blood vessels. Here, we tested the hypothesis that high levels of sEng combined with a high-fat diet induce endothelial dysfunction in an atherosclerosis-prone aorta. METHODS AND RESULTS: Six-month-old female and male transgenic mice overexpressing human sEng (Sol-Eng+) with low (Sol-Eng+low) or high (Sol-Eng+high) levels of plasma sEng were fed a high-fat rodent diet containing 1.25% cholesterol and 40% fat for 3 months. The plasma cholesterol and mouse sEng levels did not differ in the Sol-Eng+high and Sol-Eng+low mice. The expression of proinflammatory (P-selectin, ICAM-1, pNFκB and COX-2) and oxidative-stress-related markers (HO-1, NOX-1 and NOX-2) in the aortas of Sol-Eng+high female mice was significantly higher than in Sol-Eng+low female mice. Endothelium-dependent vasodilatation induced by acetylcholine was preserved better in the Sol-Eng+ high female mice than in the Sol-Eng+low female mice. CONCLUSION: These results suggest that high concentrations of sEng in plasma in combination with a high-fat diet induce the simultaneous activation of proinflammatory, pro-oxidative and vasoprotective mechanisms in mice aorta and the balance of these biological processes determines whether the final endothelial phenotype is adaptive or maladaptive.
Assuntos
Aorta/metabolismo , Doenças da Aorta/metabolismo , Aterosclerose/metabolismo , Dieta Hiperlipídica , Endoglina/metabolismo , Inflamação/metabolismo , Óxido Nítrico/metabolismo , Estresse Oxidativo , Vasodilatação , Adaptação Fisiológica , Animais , Aorta/efeitos dos fármacos , Aorta/fisiopatologia , Doenças da Aorta/sangue , Doenças da Aorta/genética , Doenças da Aorta/fisiopatologia , Aterosclerose/sangue , Aterosclerose/genética , Aterosclerose/fisiopatologia , Biomarcadores/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Endoglina/sangue , Endoglina/genética , Feminino , Predisposição Genética para Doença , Humanos , Inflamação/sangue , Inflamação/genética , Inflamação/fisiopatologia , Mediadores da Inflamação/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Fenótipo , Regulação para Cima , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologiaRESUMO
Aminoglycosides are very effective antibiotics for the treatment of severe infections, but they rank among the most frequent causes of drug-induced nephrotoxicity. Thus, prevention of aminoglycoside nephrotoxicity is an unmet therapeutic objective. Cardiotrophin-1 (CT-1), a member of the IL-6 family of cytokines, has been reported to protect the kidney against toxic and ischemic acute kidney injury (AKI). We have assessed the effect of rat CT-1 in the severity of gentamicin (G)-induced AKI. Groups of male Wistar rats received the following for 6 consecutive days: i) isotonic saline solution (group CONT), ii) G, 150mg/kg/day, i.p. (group G), iii) CT-1, 100µg/kg/day i.v. (group CT-1), or iv) G and CT-1 at the doses described above. The G group showed a manifest AKI characterized by low creatinine clearance, high plasma creatinine and urea levels, increased urinary excretion of proteins, glucose and AKI markers such as N-acetyl-glucosaminidase, neutrophil gelatinase-associated lipocalin, kidney-injury molecule-1 and T-gelsolin, increased kidney levels of CD-68, iNOS, IL-1ß and TNF-α, and markedly higher histological renal damage and leukocyte infiltration than the CONT and CT-1 groups. Administration of CT-1 together with G reduced almost all of the above-described manifestations of G-induced AKI. The results of this study have potential clinical application, as CT-1 is near to being used as a drug for organ protection.
Assuntos
Injúria Renal Aguda/prevenção & controle , Antibacterianos , Citocinas/uso terapêutico , Gentamicinas , Acetilglucosaminidase/urina , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/patologia , Proteínas de Fase Aguda/urina , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Biomarcadores/sangue , Moléculas de Adesão Celular/urina , Creatinina/sangue , Citocinas/genética , Citocinas/metabolismo , Citocinas/farmacologia , Gelsolina/urina , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Lipocalina-2 , Lipocalinas/urina , Masculino , Óxido Nítrico Sintase Tipo II/metabolismo , Proteínas Proto-Oncogênicas/urina , Ratos , Ratos Wistar , Ureia/sangueRESUMO
Fibrosis is a pathological situation in which excessive amounts of extracellular matrix (ECM) are deposited in the tissue. Myofibroblasts play a crucial role in the development and progress of fibrosis as they actively synthesize ECM components such as collagen I, fibronectin and connective tissue growth factor (CTGF) and cause organ fibrosis. Transforming growth factor beta 1 (TGF-ß1) plays a major role in tissue fibrosis. Activin receptor-like kinase 1 (ALK1) is a type I receptor of TGF-ß1 with an important role in angiogenesis whose function in cellular biology and TGF-ß signaling is well known in endothelial cells, but its role in fibroblast biology and its contribution to fibrosis is poorly studied. We have recently demonstrated that ALK1 regulates ECM protein expression in a mouse model of obstructive nephropathy. Our aim was to evaluate the role of ALK1 in several processes involved in fibrosis such as ECM protein expression, proliferation and migration in ALK1(+/+) and ALK1(+/-) mouse embryonic fibroblasts (MEFs) after TGF-ß1 stimulations and inhibitors. ALK1 heterozygous MEFs show increased expression of ECM proteins (collagen I, fibronectin and CTGF/CCN2), cell proliferation and migration due to an alteration of TGF-ß/Smad signaling. ALK1 heterozygous disruption shows an increase of Smad2 and Smad3 phosphorylation that explains the increases in CTGF/CCN2, fibronectin and collagen I, proliferation and cell motility observed in these cells. Therefore, we suggest that ALK1 plays an important role in the regulation of ECM protein expression, proliferation and migration.
Assuntos
Receptores de Ativinas Tipo I/fisiologia , Movimento Celular , Proliferação de Células , Embrião de Mamíferos/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Heterozigoto , Receptores de Activinas Tipo II , Animais , Western Blotting , Células Cultivadas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Fator de Crescimento do Tecido Conjuntivo/genética , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Embrião de Mamíferos/citologia , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/farmacologia , Feminino , Fibroblastos/citologia , Fibronectinas/genética , Fibronectinas/metabolismo , Imunofluorescência , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Smad2/genética , Proteína Smad2/metabolismo , Proteína Smad3/genética , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/farmacologia , CicatrizaçãoRESUMO
Human endoglin is an RGD-containing transmembrane glycoprotein identified in vascular endothelial cells. Although endoglin is essential for angiogenesis and its expression is up-regulated in inflammation and at sites of leukocyte extravasation, its role in leukocyte trafficking is unknown. This function was tested in endoglin heterozygous mice (Eng(+/-)) and their wild-type siblings Eng(+/+) treated with carrageenan or LPS as inflammatory agents. Both stimuli showed that inflammation-induced leukocyte transendothelial migration to peritoneum or lungs was significantly lower in Eng(+/-) than in Eng(+/+) mice. Leukocyte transmigration through cell monolayers of endoglin transfectants was clearly enhanced in the presence of endoglin. Coating transwells with the RGD-containing extracellular domain of endoglin, enhanced leukocyte transmigration, and this increased motility was inhibited by soluble endoglin. Leukocytes stimulated with CXCL12, a chemokine involved in inflammation, strongly adhered to endoglin-coated plates and to endoglin-expressing endothelial cells. This endoglin-dependent adhesion was abolished by soluble endoglin, RGD peptides, the anti-integrin α5ß1 inhibitory antibody LIA1/2 and the chemokine receptor inhibitor AMD3100. These results demonstrate for the first time that endothelial endoglin interacts with leukocyte integrin α5ß1 via its RGD motif, and this adhesion process is stimulated by the inflammatory chemokine CXCL12, suggesting a regulatory role for endoglin in transendothelial leukocyte trafficking.
Assuntos
Antígenos CD/metabolismo , Quimiotaxia de Leucócito/fisiologia , Células Endoteliais/metabolismo , Inflamação/metabolismo , Receptores de Superfície Celular/metabolismo , Migração Transendotelial e Transepitelial/fisiologia , Animais , Adesão Celular/fisiologia , Ensaios de Migração de Leucócitos , Quimiocina CXCL12/metabolismo , Endoglina , Citometria de Fluxo , Humanos , Integrina alfa5beta1/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Microscopia de Fluorescência , Migração Transcelular de Célula/fisiologiaRESUMO
Activin receptor-like kinase-1 or ALK-1 is a type I cell surface receptor for the transforming growth factor-ß (TGF-ß) family of proteins. The role of ALK-1 in endothelial cells biology and in angiogenesis has been thoroughly studied by many authors. However, it has been recently suggested a possible role of ALK-1 in cardiovascular homeostasis. ALK-1 is not only expressed in endothelial cells but also in smooth muscle cells, myofibroblast, hepatic stellate cells, chondrocytes, monocytes, myoblasts, macrophages or fibroblasts, but its role in these cells have not been deeply analyzed. Due to the function of ALK-1 in these cells, this receptor plays a role in several cardiovascular diseases. Animals with ALK-1 haploinsufficiency and patients with mutations in Acvrl1 (the gene that codifies for ALK-1) develop type-2 Hereditary Hemorrhagic Telangiectasia. Moreover, ALK-1 heterozygous mice develop pulmonary hypertension. Higher levels of ALK-1 have been observed in atherosclerotic plaques, suggesting a possible protector role of this receptor. ALK-1 deficiency is also related to the development of arteriovenous malformations (AVMs). Besides, due to the ability of ALK-1 to regulate cell proliferation and migration, and to modulate extracellular matrix (ECM) protein expression in several cell types, ALK-1 has been now demonstrated to play an important role in cardiovascular remodeling. In this review, we would like to offer a complete vision of the role of ALK-1 in many process related to cardiovascular homeostasis, and the involvement of this protein in the development of cardiovascular diseases, suggesting the possibility of using the ALK-1/smad-1 pathway as a powerful therapeutic target.
Assuntos
Receptores de Activinas Tipo II/metabolismo , Doenças Cardiovasculares/metabolismo , Proteína Smad1/metabolismo , Receptores de Activinas Tipo II/química , Homeostase , Humanos , Transdução de Sinais , Proteína Smad1/química , Fator de Crescimento Transformador beta/metabolismoRESUMO
In addition to their role as oncogenes, Ras GTPases are key regulators of cell function. There is a proven relationship between the signaling pathways of transforming growth factor-ß1 (TGF- ß1) and Ras GTPases. Each of the Ras isoforms (H, N and K) exhibits specific modulatory activity on different cellular pathways. Our purpose has been to study some of the mechanisms involved in the development of renal fibrosis, assessing the individual role of N-Ras in basal and TGF-ß1-mediated extracellular matrix (ECM) synthesis, proliferation, and migration in immortalized N-Ras deficient fibroblasts (N-ras(-/-)). Compared to normal counterparts, fibroblasts deficient for N-Ras exhibited higher basal activity levels of phosphatidylinositol-3-kinase (PI3K)/Akt and MEK/Erk, accompanied by upregulated collagen synthesis and diminished proliferation and migration rates. We found that the absence of N-Ras did not affect TGF-ß1-induced proliferation and migration, which required PI3K/Akt but not Erk1/2 activation. Similar effector pathway dependence was found for fibronectin and collagen type I expression. Our results indicate that N-Ras might contribute to renal fibrosis through the down-regulation of ECM synthesis and up-regulation proliferation and migration modulating Akt activation. N-Ras also regulates TGF-ß1-induced collagen I and fibronectin expression through Erk-independent pathways.
Assuntos
Movimento Celular , Matriz Extracelular/metabolismo , Fibroblastos/citologia , Fibroblastos/enzimologia , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Animais , Proliferação de Células , Células Clonais , Ativação Enzimática , Proteínas da Matriz Extracelular/metabolismo , Técnicas de Silenciamento de Genes , Antígeno Ki-67/metabolismo , Sistema de Sinalização das MAP Quinases , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) regulates apoptosis, proliferation and inflammation in renal epithelial cells and plays a role in acute kidney injury. However, there is little information on the chronic effects of TWEAK. We hypothesized that TWEAK may influence renal fibrosis and regulate kidney fibroblast biology, in part, through Ras pathway. We studied a chronic model of experimental unilateral ureteral obstruction in wild type and TWEAK deficient mice, and a murine model of systemic TWEAK overexpression. TWEAK actions were also explored in cultured renal and embryonic fibroblasts. TWEAK and TWEAK receptor expression was increased in the obstructed kidneys. The absence of TWEAK decreased early kidney tubular damage, inflammatory infiltrates and myofibroblast number. TWEAK deficient mice had decreased renal fibrosis 21days after obstruction, as assessed by extracellular matrix staining. In mice without prior underlying kidney disease, systemic overexpression of TWEAK induced kidney inflammation and fibrosis. In cultured fibroblasts, TWEAK induced proliferation through activation of the Ras/ERK pathway. TWEAK also activated nuclear factor κB (NFκB)-dependent inflammatory chemokine production in murine renal fibroblasts. In conclusion, lack of TWEAK reduces renal fibrosis in a model of persistent kidney insult and overexpression of TWEAK led to renal fibrosis. TWEAK actions on renal fibroblasts may contribute to the in vivo observations, as TWEAK promotes inflammatory activity and proliferation in fibroblast cultures.
Assuntos
Proliferação de Células , Fibrose/fisiopatologia , Nefropatias/fisiopatologia , Rim/patologia , Fatores de Necrose Tumoral/fisiologia , Proteínas ras/fisiologia , Animais , Linhagem Celular , Citocina TWEAK , Fibroblastos/patologia , CamundongosRESUMO
Tubulointerstitial fibrosis is characterized by an accumulation of extracellular matrix in the renal interstitium, myofibroblast activation, cell infiltration, and tubular cell apoptosis, leading to chronic renal failure. Activin receptor-like kinase 1 (ALK1) is a transforming growth factor-ß1 type I receptor with a pivotal role in endothelial proliferation and migration, but its role in the development of renal fibrosis is unknown. To assess this we used the unilateral ureteral obstruction model of tubulointerstitial fibrosis in ALK1 haploinsufficient (ALK1(+/-)) and wild-type mice. After 15 days, there was an increase in extracellular matrix protein expression in the obstructed kidneys from both ALK1(+/+) and ALK1(+/-) mice, but obstructed kidneys from ALK1(+/-) mice showed significantly higher expression of type I collagen than those from wild-type mice. Ureteral obstruction increased kidney myofibroblasts markers (α-smooth muscle actin and S100A4), without differences between mouse genotypes. ALK1 expression was increased after ureteral obstruction, and this increased expression was located in myofibroblasts. Moreover, cultured renal fibroblasts from ALK1(+/-) mice expressed more collagen type I and fibronectin than fibroblasts derived from wild-type mice. Thus, ALK1 modulates obstruction-induced renal fibrosis by increased extracellular matrix synthesis in myofibroblasts, but without differences in myofibroblast number.