RESUMO
Osteogenesis imperfecta (OI) is a heterogeneous connective tissue disease mainly caused by structural mutations in type I collagen. Mutant collagen accumulates intracellularly, causing cellular stress that has recently been shown to be phenotype-related. Therefore, the aim of the study was to search for potential drugs reducing collagen accumulation and improving OI fibroblast homeostasis. We found that rosemary extract (RE), which is of great interest to researchers due to its high therapeutic potential, at concentrations of 50 and 100 µg/mL significantly reduced the level of accumulated collagen in the fibroblasts of four patients with severe and lethal OI. The decrease in collagen accumulation was associated with RE-induced autophagy as was evidenced by an increase in the LC3-II/LC3-I ratio, a decrease in p62, and co-localization of type I collagen with LC3-II and LAMP2A by confocal microscopy. The unfolded protein response, activated in three of the four tested cells, and the level of pro-apoptotic markers (Bax, CHOP and cleaved caspase 3) were attenuated by RE. In addition, the role of RE-modulated proteasome in the degradation of unfolded procollagen chains was investigated. This study provides new insight into the beneficial effects of RE that may have some implications in OI therapy targeting cellular stress.
Assuntos
Osteogênese Imperfeita , Rosmarinus , Autofagia , Caspase 3/metabolismo , Colágeno/metabolismo , Colágeno Tipo I/metabolismo , Fibroblastos/metabolismo , Mutação , Osteogênese Imperfeita/metabolismo , Pró-Colágeno/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
The Akt substrate of 160 kDa (AS160) is a key regulator of GLUT4 translocation from intracellular depots to the plasma membrane in myocytes. Likely, AS160 also controls LCFAs transport, which requires relocation of fatty acid transporters. The aim of the present study was to determine the impact of AS160 knockdown on lipid milieu in L6 myotubes incubated with palmitate (PA). Therefore, we compared two different settings, namely: 1) AS160 knockdown prior to palmitate incubation (pre-PA-silencing, AS160- /PA); 2) palmitate incubation with subsequent AS160 knockdown (post-PA-silencing, PA/AS160- ). The efficiency of AS160 silencing was checked at mRNA and protein levels. The expression and localization of FA transporters were determined using Western Blot and immunofluorescence analyses. Intracellular lipid content (FFA, DAG, TAG, and PL) and FA composition were estimated by GLC, whereas basal palmitate uptake was analyzed by means of scintigraphy. Both groups with silenced AS160 were characterized by a greater expression of FA transporters (FAT/CD36, FATP-1, 4) which had contributed to an increased FA cellular influx. Accordingly, we observed that post-PA-silencing of AS160 resulted in a marked decrement in DAG, TAG, and PL contents, but increased FFA content (PA/AS160- vs. PA). The opposite effect was observed in the group with pre-PA-silencing of AS160 in which AS160 knockdown did not affect the lipid pools (AS160- /PA vs. PA). Our results indicate that post-PA-silencing of AS160 has a capacity to decrease the lipotoxic effect(s) of PA by decreasing the content of lipids (DAG and PL) that promote insulin resistance in myotubes. J. Cell. Physiol. 232: 2373-2386, 2017. © 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals Inc.
Assuntos
Proteínas Ativadoras de GTPase/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Ácido Palmítico/farmacologia , Animais , Transporte Biológico , Antígenos CD36/metabolismo , Linhagem Celular , Proteínas de Transporte de Ácido Graxo/metabolismo , Proteínas de Ligação a Ácido Graxo/metabolismo , Proteínas Ativadoras de GTPase/genética , Resistência à Insulina , Mitocôndrias Musculares/efeitos dos fármacos , Mitocôndrias Musculares/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Ácido Palmítico/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacos , TransfecçãoRESUMO
Cancer cells have developed a number of adaptation mechanisms involving the signal activation of the transduction pathways, which promotes the progression and metastasis. Our results showed that the percentage of apoptotic MCF-7 cells incubated in the low glucose medium for 48 h was lower in comparison to those cultured in the high glucose medium, despite the high expression of the proapoptotic transcription factor-CHOP. Furthermore, the MCF-7 cells incubated in the low glucose medium for 48 h showed a higher expression of NF-κB p100/p52 subunits compared to cells incubated in the high glucose medium. Moreover, our findings demonstrated that the shortage of glucose strongly induces autophagy in MCF-7 cells. The activation of this process is not associated with the changes in the expression of mTOR kinase. We suggest, that the antiapoptotic chaperone ORP150 induction, transcription factor NF-κB2 activation, and increased autophagy constitute mechanisms protecting the MCF-7 cells against apoptosis.
Assuntos
Apoptose , Autofagia , Neoplasias da Mama/metabolismo , Glucose/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Feminino , Glucose/genética , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Células MCF-7 , Subunidade p52 de NF-kappa B/genética , Subunidade p52 de NF-kappa B/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismoRESUMO
Background: Recent studies indicate that aquaporin (AQP) water channels have a regulatory function in human platelet secretion and in procoagulant response of murine platelets. However, the engagement of AQPs in morphological changes, procoagulant response, and thrombus formation in human blood has never been investigated. Methods: Confocal microscopy was used to study platelet spreading, filopodia formation, ballooning, and thrombus formation under flow. Flow cytometry was utilized to assess platelet phosphatidylserine (PS) exposure and microparticles shedding. Kinetics of clot formation in vitro was evaluated by thromboelastometry. Mouse model of ferric chloride (III) (FeCl3)-induced thrombosis was used to investigate thrombus formation in vivo. Results: We found that chloroauric(III) acid (HAuCl4), a classical AQP inhibitor (10-100 µM), reduced spreading of human platelets on collagen-coated surfaces and inhibited filopodia formation in a fluid phase. Under flow conditions, HAuCl4 (100 µM) attenuated thrombi growth on collagen, platelet secretion, and PS exposure. Thrombus formation was restored by the addition of exogenous adenosine diphosphate (ADP). Collagen-evoked platelet procoagulant response (evaluated as PS exposure, shedding of microparticles, platelet-dependent thrombin generation, and membrane ballooning) was distinctly reduced by HAuCl4 (25-200 µM), as well as the dynamics of clot formation. In mouse model of thrombosis, reduction of surface of PS-positive cells within thrombus was observed in the presence of HAuCl4 (1-10 mg/kg). Conclusion: These results suggest that in human platelets AQPs are crucial for agonist-evoked morphological changes, thrombus formation under flow, and in development of procoagulant response. Antithrombotic effect in vivo suggests that nontoxic inhibitors of AQPs may be considered as potential candidates for a novel class of antiplatelet drugs.
RESUMO
This study was undertaken to establish the presence and the role of aquaporins (AQPs) in human platelets. Immunodetection with polyclonal antibodies and fluorescent microscopy suggest the presence of AQP isoforms - 0-7 and 9-12 - localized (in resting platelets) in the plasma membrane and in the dense and alpha granules. In thrombin- or monensin-treated platelets, the granules' AQPs become visible in the whole cell body, indicating the granules' swelling. In our studies on the role of AQPs in platelet responses we used tetrachloroauric acid (HAuCl4), a classical water channel blocker. We found that 10-100 µM of Au(III) inhibited the hypotonicity-, monensin (simulating the action of Na+/H+ exchanger)-, and collagen-evoked platelet swelling and reduced tritiated water uptake by platelets treated by collagen or monensin, indicating its ability to block water channels in these cells. HAuCl4, at the concentrations reducing water influx, did not induce cell lysis, alter the plasma membrane shape or the -SH group content. The inhibitor also failed to affect Na+ and Cl--related osmotic gradient formation and protein kinase D2 phosphorylation. In platelets activated by threshold concentrations of collagen, the thrombin receptor activating peptide, ADP, calcium ionophore A23187, phorbol ester and arachidonic acid, HAuCl4 (100 µM) completely inhibited secretion of ATP from dense granules but failed to reduce platelet aggregation. In collagen-stimulated platelets, HAuCl4 (10-100 µM) reduced secretion from dense and alpha granules, as well as lysosomes, in a dose-dependent manner. We conclude that human platelets possess numerous AQPs subtypes localized in the plasma and granule membranes. AQP-mediated water fluxes may be crucial for platelet volume regulation as well as secretion from dense and alpha granules and lysosomes.
Assuntos
Aquaporinas/metabolismo , Plaquetas/metabolismo , Lisossomos/metabolismo , Plaquetas/efeitos dos fármacos , Cloretos/farmacologia , Colágeno/metabolismo , Grânulos Citoplasmáticos/metabolismo , Compostos de Ouro/farmacologia , Humanos , Agregação Plaquetária , Trombina/farmacologiaRESUMO
: Rise in mean platelet volume (MPV) has been demonstrated to be associated with increased platelet reactivity. In diabetes patients, augmented MPV was proposed to contribute to increased risk of thrombotic complications. Therefore, the aim of this study was to investigate whether under hyperglycemic conditions, aldose reductase (AR)-mediated sorbitol formation and associated rise in cell volume, which subsequently results in platelet hyperactivation. Platelets were obtained from 30 healthy volunteers and 13 patients with diabetes. We evaluated changes in platelet size, their reactivity (measured as aggregation and secretion), and sorbitol content evoked by glucose. Measurement of procoagulant activity and thromboelastography were performed to assess how hyperglycemia affects coagulation. We have found that incubation of platelets with glucose (>10âmmol/l) leads to increased MPV, potentiation of collagen-evoked platelet aggregation, secretion, and procoagulant response (measured as platelet-dependent thrombin generation and phosphatidylserine expression). Glucose-treated platelets had higher sorbitol content and demonstrated enhanced tubulin polymerization. All the above-mentioned phenomena were reduced following the blocking of AR or by vincristine (microtubule destabilizing agent). Thromboelastography measurements demonstrated that hyperglycemia is associated with reduction of clotting time (R) and increase in the alpha angle (reflects platelet activation). Addition of sorbinil (AR inhibitor) or vincristine normalized R variable and alpha angle. The hyperglycemic conditions may accelerate platelet-related thrombin generation through the activation of polyol pathway, enhanced tubulin polymerization and associated with it rise in platelet volume.
Assuntos
Aldeído Redutase/fisiologia , Coagulação Sanguínea , Plaquetas/citologia , Hiperglicemia/complicações , Adolescente , Adulto , Plaquetas/fisiologia , Tamanho Celular , Diabetes Mellitus/sangue , Feminino , Humanos , Masculino , Ativação Plaquetária , Agregação Plaquetária , Sorbitol/análise , Trombina/metabolismo , Adulto JovemRESUMO
We tested the hypothesis that in vitro peroxynitrite (ONOO(-), a product of activated inflammatory cells) may affect fibrinolysis in human blood through the reduction of platelet-related fibrinolysis resistance. It was found that ONOO(-) (25-300 µM) accelerated lysis of platelet-fibrin clots (in PRP) dose-dependently, whereas fibrinolysis of platelet-free clots was slightly inhibited by ≥ 1000 µM stressor. Concentrations of ONOO(-) affecting the lysis of platelet-rich clots, inhibited clot retraction (CR) in a dose-dependent manner. Thromboelastometry (ROTEM) measurements performed in PRP showed that treatment with ONOO(-) (threshold conc. 100 µM) prolongs clotting time, and reduces alpha angle, and clot formation velocity parameters indicating for reduced thrombin formation rate. In PRP, ONOO(-) (threshold conc. 100 µM) reduced the collagen-evoked exposure of phosphatidylserine (PS) on platelets' plasma membrane, the shedding of platelet-derived microparticles (PMP), and inhibited platelet-dependent thrombin generation (measured in artificial system), dose-dependently. As judged by confocal microscopy, similar ONOO(-) concentrations altered the architecture of clots formed in collagen-treated PRP. Clots formed in the presence of ONOO(-) were less dense and were composed of thicker fibers, which make them more susceptible to lysis. In platelet-depleted plasma, ONOO(-) (up to milimolar concentration) did not alter clot structure. Blockage of PS exposed on platelets resulted in an alteration of clot architecture toward more prone to lysis. ONOO(-), at lysis-affecting concentrations, inhibited the collagen-evoked secretion of fibrinolytic inhibitors from platelets. We conclude that physiologically relevant ONOO(-) concentrations may accelerate the lysis of platelet-fibrin clots predominantly via downregulation of platelet-related mechanisms including: platelet secretion, clot retraction, platelet procoagulant response, and the alteration in clot architecture associated with it.
Assuntos
Coagulação Sanguínea/fisiologia , Plaquetas/metabolismo , Fibrinólise/fisiologia , Ácido Peroxinitroso/metabolismo , Adolescente , Adulto , Feminino , Humanos , Masculino , Microscopia Confocal , Adulto JovemRESUMO
Using patients with polycythemia vera (PV) as an experimental model, we evaluated the impact of clot retraction (CR) and architecture of the clot on fibrinolysis. We studied the kinetics of clot retraction and the fibrinolysis rate in whole blood from 48 PV patients and 48 healthy controls. Measurements were performed before and after isovolemic eryhrocytapheresis (ECP). CR was measured by optical method. Clot lysis time (CLT) and maximum clot firmness (MCF) were measured by thromboelastometry in recalcified blood supplemented with t-PA and tissue factor. Compared with healthy controls, CR rate in PV patients was higher (0.0219 vs. 0.0138; p<0.001), the clot volume smaller and MCF elevated (64 vs. 58 mm). CR rate correlated with platelet count (r=0.546; p=0.001) but not with erythrocyte concentration (r=0.192; p>0.3). Compared with healthy controls, CLT in PV patients was significantly prolonged (158 min vs. 71 min). Fibrinolysis rate inversely correlated with CR rate (r=-0.566; p<0.001); MCF (r=-0.704; p<0.001) and platelet count (r=-0.461; p<0.001). As judged by confocal microscope, in comparison to healthy controls, clots formed in blood from PV patients demonstrated booth a distinctly higher degree of crosslinking and possessed thinner fibers. Altered CR, MCF and fibrinolysis speeds were not normalized following the ECP procedure. Tirofiban (a blocker of platelet GPIIb/IIIa receptors), unlike aspirin, normalized abnormal CR and fibrinolysis in blood from PV patients. Collectively, our data indicate that in PV patients, abnormal CR may result in formation of thrombi that are more resistant to fibrinolysis. ECP and aspirin failed to normalize platelet-related fibrinolysis resistance.