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1.
Bioorg Med Chem ; 25(20): 5849-5858, 2017 10 15.
Artículo en Inglés | MEDLINE | ID: mdl-28958848

RESUMEN

The NAD+-dependent deacetylase SIRT6 is an emerging cancer drug target, whose inhibition sensitizes cancer cells to chemo-radiotherapy and has pro-differentiating effects. Here we report on the identification of novel SIRT6 inhibitors with a salicylate-based structure. The new SIRT6 inhibitors show improved potency and specificity compared to the hit inhibitor identified in an in silico compound screen. As predicted based on SIRT6 biological roles, the new leads increase histone 3 lysine 9 acetylation and glucose uptake in cultured cells, while blocking TNF-α production and T lymphocyte proliferation. Notably, the new SIRT6 inhibitors effectively sensitize pancreatic cancer cells to gemcitabine. Finally, studies of compound fingerprinting and pharmacokinetics defined the drug-like properties of one of the new SIRT6 inhibitors, potentially allowing for subsequent in vivo proof-of-concept studies. In conclusion, new SIRT6 inhibitors with a salicylate-like structure were identified, which are active in cells and could potentially find applications in disease conditions, including cancer and immune-mediated disorders.


Asunto(s)
Sistemas de Liberación de Medicamentos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Salicilatos/química , Sirtuinas/antagonistas & inhibidores , Acetilación/efectos de los fármacos , Animales , Línea Celular , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Humanos , Inmunosupresores/química , Inmunosupresores/farmacología , Concentración 50 Inhibidora , Ratones , Estructura Molecular , Salicilatos/farmacología , Sirtuinas/metabolismo , Linfocitos T/citología , Linfocitos T/efectos de los fármacos
2.
Exp Eye Res ; 146: 128-136, 2016 05.
Artículo en Inglés | MEDLINE | ID: mdl-26771090

RESUMEN

This study was conducted to compare the effects of two anti-VEGF-A drugs, Ranibizumab and Aflibercept, on the expression and secretion of VEGFs family members, and on their influence in proliferation and migration of endothelial cells (HECV) in vitro. HECV cells were exposed 24 h (T1), 4 days (T2) and 6 days (T3) to Ranibizumab or Aflibercept at pharmacodynamically relevant concentrations (Ranibizumab: 12.5 µg/ml and 125 µg/ml; Aflibercept: 50 µg/ml and 500 µg/ml). Cell viability and then expression and secretion of VEGF-A, VEGF-B, VEGF-C and PlGF were evaluated respectively by Real Time-PCR and ELISA. Intracellular signaling activated by VEGF-A and VEGF-C was investigated evaluating phosphorylation of VEGFR2. Influence in would healing was evaluated through scratch assay. In general no differences were observed among the tested concentrations of anti-vegf drugs. Ranibizumab and Aflibercept did not affect HECV cell viability in all experimental times. At T1, Ranibizumab decreased mRNA levels of VEGF-A, induced VEGF-C secretion, abrogated phosphorylation of VEGFR2 stimulated by VEGF-A, and impaired ability of HECV cells to repair wound healing. Aflibercept decreased mRNA levels of VEGF-A, -B and PlGF; slightly increased basal level of phVEGFR2, and completely abrogated phosphorylation stimulated by VEGF-A and VEGF-C. No effects on secretion of VEGF-B and on would healing were observed after exposure to Aflibercept. Prolonged exposure to anti-VEGFs decreased expression and secretion of VEGF-A and VEGF-B, up-regulated VEGF-C mRNA levels and its secretion, and increased basal phosphorylation of VEGFR2. Acute treatment with Ranibizumab or Aflibercept evoked different responses on endothelial cells, however these differences were lost after prolonged exposure. Scratch test results suggest that treatment with Ranibizumab may be more effective than Aflibercept in reducing angiogenic potential of endothelial cells in vitro.


Asunto(s)
Endotelio Vascular/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Degeneración Macular/tratamiento farmacológico , ARN/genética , Ranibizumab/farmacología , Proteínas Recombinantes de Fusión/farmacología , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Inhibidores de la Angiogénesis/farmacología , Supervivencia Celular , Células Cultivadas , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Ensayo de Inmunoadsorción Enzimática , Humanos , Degeneración Macular/metabolismo , Degeneración Macular/patología , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Factores de Crecimiento Endotelial Vascular , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Factor A de Crecimiento Endotelial Vascular/genética
3.
Mediators Inflamm ; 2014: 162021, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25214711

RESUMEN

In type 2 diabetes, hyperglycemia, insulin resistance, increased inflammation, and oxidative stress were shown to be associated with the progressive deterioration of beta-cell function and mass. Short-chain fatty acids (SCFAs) are organic fatty acids produced in the distal gut by bacterial fermentation of macrofibrous material that might improve type 2 diabetes features. Their main beneficial activities were identified in the decrease of serum levels of glucose, insulin resistance as well as inflammation, and increase in protective Glucagon-like peptide-1 (GLP-1) secretion. In this review, we updated evidence on the effects of SCFAs potentially improving metabolic control in type 2 diabetes.


Asunto(s)
Ácidos Grasos Volátiles/metabolismo , Tracto Gastrointestinal/microbiología , Microbiota/fisiología , Animales , Diabetes Mellitus Tipo 2/metabolismo , Glucosa/metabolismo , Humanos
4.
Mediators Inflamm ; 2014: 923120, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24648662

RESUMEN

Glucagon-like peptide-1 (GLP-1), an intestinal hormone contributing to glucose homeostasis, is synthesized by proglucagon and secreted from intestinal neuroendocrine cells in response to nutrients. GLP-1 secretion is impaired in type 2 diabetes patients. Here, we aimed at investigating whether diabetic toxic products (glycated serum (GS) or high levels of glucose (HG)) may affect viability, function, and insulin sensitivity of the GLP-1 secreting cell line GLUTag. Cells were cultured for 5 days in presence or absence of different dilutions of GS or HG. GS and HG (alone or in combination) increased reactive oxygen species (ROS) production and upregulated proglucagon mRNA expression as compared to control medium. Only HG increased total production and release of active GLP-1, while GS alone abrogated secretion of active GLP-1. HG-mediated effects were associated with the increased cell content of the prohormone convertase 1/3 (PC 1/3), while GS alone downregulated this enzyme. HG upregulated Glucokinase (GK) and downregulated SYNTHAXIN-1. GS abrogated SYNTHAXIN-1 and SNAP-25. Finally, high doses of GS alone or in combination with HG reduced insulin-mediated IRS-1 phosphorylation. In conclusion, we showed that GS and HG might regulate different pathways of GLP-1 production in diabetes, directly altering the function of neuroendocrine cells secreting this hormone.


Asunto(s)
Glucemia/metabolismo , Regulación de la Expresión Génica , Péptido 1 Similar al Glucagón/sangre , Inflamación/sangre , Línea Celular , Supervivencia Celular , Regulación hacia Abajo , Glucoquinasa/metabolismo , Humanos , Insulina/metabolismo , Proglucagón/metabolismo , ARN Mensajero/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteína 25 Asociada a Sinaptosomas/metabolismo , Sintaxina 1/metabolismo
5.
Mediators Inflamm ; 2014: 975872, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24771986

RESUMEN

Osteoporosis is a major public health burden that is expected to further increase as the global population ages. In the last twenty years, advanced glycation end products (AGEs) have been shown to be critical mediators both in the pathogenesis and development of osteoporosis and other chronic degenerative diseases related to aging. The accumulation of AGEs within the bone induces the formation of covalent cross-links with collagen and other bone proteins which affects the mechanical properties of tissue and disturbs bone remodelling and deterioration, underlying osteoporosis. On the other hand, the gradual deterioration of the immune system during aging (defined as immunosenescence) is also characterized by the generation of a high level of oxidants and AGEs. The synthesis and accumulation of AGEs (both localized within the bone or in the systemic circulation) might trigger a vicious circle (in which inflammation and aging merged in the word "Inflammaging") which can establish and sustain the development of osteoporosis. This narrative review will update the molecular mechanisms/pathways by which AGEs induce the functional and structural bone impairment typical of osteoporosis.


Asunto(s)
Envejecimiento , Productos Finales de Glicación Avanzada/metabolismo , Inflamación/metabolismo , Osteoporosis/metabolismo , Biomarcadores/metabolismo , Remodelación Ósea , Huesos/metabolismo , Difosfonatos/metabolismo , Humanos , Oxidantes/metabolismo , Estrés Oxidativo
6.
Mediators Inflamm ; 2013: 975032, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-24307763

RESUMEN

Glucagon-like peptide-1 (GLP-1) is a gut-derived incretin hormone that has been shown to improve glucose homeostasis in type 2 diabetes. The biological effects of GLP-1 are mediated by its specific receptor GLP-1R that is expressed in a wide range of tissues, where it is responsible of the extra-pancreatic effects of GLP-1. Since the retinal pigment epithelium (RPE), that forms the outer retinal barrier, has a key role in protecting from diabetic retinopathy (DR), we investigated the potential expression and function of GLP-1R in a RPE cell line. ARPE-19 cells were cultured in DMEM/F12 supplemented with 10% FBS. The expression of GLP-1R was evaluated at both mRNA and protein levels. Then, the activation postreceptor intracellular signal transduction pathways (extracellular signal-regulated kinases 1 and 2 [ERK1/2] and protein kinase B [PKB]) were assessed by western blot in normal cells or silenced for GLP-1R in the presence or absence of 10 nmol/L GLP-1. The potential connections between intracellular signalling pathways triggered by GLP-1 stimulation were performed before incubating cells with kinase pharmacological inhibitors of mitogen-activated protein kinase (MEK)1/2, phosphatydilinositol-3kinase (PI3K), or epidermal growth factor receptor (EGFR). The results showed that GLP1R is expressed at both mRNA and protein level in ARPE-19 cells. Stimulation with GLP-1 strongly activated PKB and ERK1/2 phosphorylation till 40 min of exposure. GLP-1-mediated activation of both kinases was dependent on the upstream activation of PI3K and EGFR. Finally, treatment with GLP-1 did not affect the spontaneous release of VEGF-A from ARPE-19 cells. In conclusion, this paper showed that the presence of functional GLP-1R is expressed in RPE cells. These data might represent the rationale to further investigate the potential direct beneficial effects of GLP-1 treatment against DR.


Asunto(s)
Péptido 1 Similar al Glucagón/farmacología , Receptores de Glucagón/metabolismo , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/patología , Línea Celular , Supervivencia Celular , Inhibidores Enzimáticos/farmacología , Receptores ErbB/metabolismo , Silenciador del Gen , Receptor del Péptido 1 Similar al Glucagón , Humanos , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Factores de Tiempo , Factor A de Crecimiento Endotelial Vascular/metabolismo
7.
Mediators Inflamm ; 2013: 317120, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23737644

RESUMEN

Advanced glycation end products (AGEs) might play a pathophysiological role in the development of diabetes and its complications. AGEs negatively affect pancreatic beta-cell function and the expression of transcriptional factors regulating insulin gene. Glucagon-like peptide-1 (GLP-1), an incretin hormone that regulates glucose homeostasis, might counteract the harmful effects of AGEs on the beta cells in culture. The aim of this study was to identify the intracellular mechanisms underlying GLP-1-mediated protection from AGE-induced detrimental activities in pancreatic beta cells. HIT-T15 cells were cultured for 5 days with glycated serum (GS, consisting in a pool of AGEs), in the presence or absence of 10 nmol/L GLP-1. After evaluation of oxidative stress, we determined the expression and subcellular localization of proteins involved in maintaining redox balance and insulin gene expression, such as nuclear factor erythroid-derived 2 (Nrf2), glutathione reductase, PDX-1, and MafA. Then, we investigated proinsulin production. The results showed that GS increased oxidative stress, reduced protein expression of all investigated factors through proteasome activation, and decreased proinsulin content. Furthermore, GS reduced ability of PDX-1 and MafA to bind DNA. Coincubation with GLP-1 reversed these GS-mediated detrimental effects. In conclusion, GLP-1, protecting cells against oxidants, triggers protective intercellular pathways in HIT-T15 cells exposed to GS.


Asunto(s)
Péptido 1 Similar al Glucagón/farmacología , Productos Finales de Glicación Avanzada/farmacología , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Animales , Línea Celular , Cricetinae , Proteínas de Homeodominio/metabolismo , Immunoblotting , Unión Proteica/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transactivadores/metabolismo
8.
Mediators Inflamm ; 2013: 750540, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23737653

RESUMEN

The primary function of pancreatic beta-cells is to produce and release insulin in response to increment in extracellular glucose concentrations, thus maintaining glucose homeostasis. Deficient beta-cell function can have profound metabolic consequences, leading to the development of hyperglycemia and, ultimately, diabetes mellitus. Therefore, strategies targeting the maintenance of the normal function and protecting pancreatic beta-cells from injury or death might be crucial in the treatment of diabetes. This narrative review will update evidence from the recently identified molecular regulators preserving beta-cell mass and function recovery in order to suggest potential therapeutic targets against diabetes. This review will also highlight the relevance for novel molecular pathways potentially improving beta-cell dysfunction.


Asunto(s)
Diabetes Mellitus Tipo 2/metabolismo , Células Secretoras de Insulina/metabolismo , Calcio/metabolismo , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/fisiopatología , Péptido 1 Similar al Glucagón/uso terapéutico , Humanos , Hipoglucemiantes/uso terapéutico , Células Secretoras de Insulina/efectos de los fármacos , Metformina/uso terapéutico , MicroARNs/metabolismo
9.
Int J Mol Sci ; 14(6): 10694-709, 2013 May 23.
Artículo en Inglés | MEDLINE | ID: mdl-23702842

RESUMEN

The aim of this work was to evaluate the ability of oxidative and glycative stressors to modify properties of human serum albumin (HSA) by analyzing markers of glycation (pentosidine) and oxidation (advanced oxidative protein products (AOPPs)) and assessing fluorescence and circular dichroism. HSA was incubated for up to 21 days with ribose, ascorbic acid (AA) and diethylenetriamine pentacetate (DTPA) in various combinations in order to evaluate influences of these substances on the structure of HSA. Ribose was included as a strong glycative molecule, AA as a modulator of oxidative stress, and DTPA as an inhibitor of metal-catalyzed oxidation. Ribose induced a significant increase in pentosidine levels. AA and DTPA prevented the accumulation of pentosidine, especially at later time points. Ribose induced a mild increase in AOPP formation, while AA was a strong inducer of AOPP formation. Ribose, in combination with AA, further increased the formation of AOPP. DTPA prevented the AA-induced generation of AOPP. Ribose was also a potent inducer of fluorescence at 335nm ex/385nm em, which is typical of pentosidine. AA and DTPA prevented this fluorescence. Circular dichroism showed complex results, in which AA and DTPA were strong modifiers of the percentages of the alpha-helical structure of HSA, while ribose affected the structure of HSA only at later time points.


Asunto(s)
Estrés Oxidativo , Albúmina Sérica/química , Albúmina Sérica/metabolismo , Acetatos/farmacología , Arginina/análogos & derivados , Arginina/farmacología , Ácido Ascórbico/farmacología , Dicroismo Circular , Fluorescencia , Glicosilación/efectos de los fármacos , Humanos , Lisina/análogos & derivados , Lisina/farmacología , Oxidación-Reducción/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Estructura Secundaria de Proteína
10.
Mol Vis ; 18: 2509-17, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-23112566

RESUMEN

PURPOSE: Neovascularization is a common complication of many degenerative and vascular diseases of the retina. Advanced glycation end-products (AGEs) have a pathologic role in the development of retinal neovascularization, mainly for their ability in upregulating vascular endothelial growth factor-A (VEGF-A) secretion. The aim of this study was to investigate whether AGEs are able to modulate the secretion of VEGF-C, another angiogenic factor that increases the effect of VEGF-A. METHODS: A human retinal pigment epithelial cell line (ARPE-19) and human endothelial vascular cell line (HECV) cells were cultured for 24 h in presence of AGEs, and then mRNA expression of VEGF-C was analyzed with reverse transcription-polymerase chain reaction (RT-PCR). To verify whether AGEs-induced VEGF secretion is mediated by RAGE (Receptor for AGEs), RAGE expression was depleted using the small interfering RNA method. To investigate whether VEGF-A is involved in upregulating VEGF-C secretion, the cells were cultured for 24 h in the presence of bevacizumab, a monoclonal antibody against VEGF-A, alone or in combination with AGEs. VEGF-A and VEGF-C levels in the supernatants of the treated cells were evaluated with enzyme-linked immunosorbent assay. RESULTS: Exposure to AGEs significantly increased VEGF-C gene expression in ARPE-19 cells. AGEs-induced VEGF-C secretion was upregulated in retinal pigment epithelium (RPE) and endothelial cells. Downregulation of RAGE expression decreased VEGF-A secretion in cell models, and increased VEGF-C secretion in ARPE-19 cells. Adding bevacizumab to the culture medium upregulated constitutive VEGF-C secretion but did not affect AGEs-induced VEGF-C secretion. CONCLUSIONS: These findings suggest that AGEs take part in the onset of retinal neovascularization, not only by modulating VEGF-A but also by increasing VEGF-C secretion. In addition, our results suggest that VEGF-C may compensate for treatments that reduce VEGF-A.


Asunto(s)
Células Epiteliales/efectos de los fármacos , Productos Finales de Glicación Avanzada/farmacología , Neovascularización Patológica , Receptores Inmunológicos/antagonistas & inhibidores , Epitelio Pigmentado de la Retina/efectos de los fármacos , Factor C de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Anticuerpos Monoclonales Humanizados/farmacología , Bevacizumab , Células Epiteliales/citología , Células Epiteliales/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , ARN Mensajero/biosíntesis , ARN Interferente Pequeño/genética , Receptor para Productos Finales de Glicación Avanzada , Receptores Inmunológicos/genética , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/metabolismo , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/genética , Factor C de Crecimiento Endotelial Vascular/genética , Factor C de Crecimiento Endotelial Vascular/metabolismo
11.
Life (Basel) ; 12(1)2021 Dec 28.
Artículo en Inglés | MEDLINE | ID: mdl-35054437

RESUMEN

The insulin-like growth factor 1 (IGF-1) stimulates expression and secretion of vascular endothelial growth factor-A (VEGF-A), the main actor in ocular neovascularization, by RPE cells. Activity of IGF-1 is regulated by interaction between its receptor and Caveolin-1 (Cav-1), the main component of caveolae. The aim of this study was to investigate whether modulation of Cav-1 expression affects synthesis and secretion of VEGF-A. ARPE-19 cells were transfected with small interfering RNA for Cav-1 (si-Cav-1) and with control siRNA (si-CTR) and stimulated with IGF-1. We found that down-regulation of Cav-1 did not affect activation of IGF-1R but regulated in an opposite manner the phosphorylation of Akt and Erk1/2. Moreover, we found that IGF-1 increased mRNA levels of VEGF-A in both si-CTR and in si-Cav-1 ARPE-19 cells and that Cav-1 silencing significantly reduced basal and IGF-1-stimulated VEGF-A release. Then we investigated the response of the microvascular endothelial cell line HMEC-1 to secretory products of ARPE-19 cells by evaluating wound healing closure, finding that conditioned media from si-Cav-1-ARPE-19 cells reduced endothelial cell migration rate. These data demonstrate that Cav-1 regulates secretion of VEGF-A, and that the depletion of Cav-1 reduces IGF-1 induced VEGF-A secretion in ARPE-19 cells and the migratory potential of their secretory products.

12.
J Diabetes Res ; 2021: 6649222, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33709000

RESUMEN

Vascular endothelial growth factor-A (VEGF-A) has a pathologic role in microvascular diabetic complication, such as diabetic retinopathy (DR). miR-126 plays an important role in vascular development and angiogenesis by regulating the expression of VEGF-A. Since levels of miR-126 have been found downregulated in diabetes, this study is aimed at investigating whether hyperglycemia affects expression of miR-126 in a retinal pigment epithelium cell line. ARPE-19 cells were transfected with miR-126 inhibitor or with miR-126 mimic and the respective scramble negative control. After 24 hours, medium was replaced and cells were cultured for 24 hours in normal (CTR) or diabetic condition (HG). Then, we analyzed mRNA levels of miR-126, VEGF-A, PI3KR2, and SPRED1. We also evaluated protein amount of HIF-1α, PI3KR2, and SPRED1 and VEGF-A secretion. The results showed that exposure of ARPE-19 cells to HG significantly decreased miR-126 levels; mRNA levels of VEGF-A and PI3KR2 were inversely correlated with those of miR-126. Overexpression of miR-126 under HG restored HIF-1α expression and VEGF-A secretion to the level of CTR cells. These results indicate that reduced levels of miR-126 may contribute to DR progression by increasing expression of VEGF-A in RPE cells. In addition, we provide evidence that upregulation of miR-126 in RPE cells counteracts the rise of VEGF-A secretion induced by hyperglycemia. In conclusion, our data support a role of miR-126 mimic-approach in counteracting proangiogenic effects of hyperglycemia.


Asunto(s)
Retinopatía Diabética/metabolismo , Glucosa/toxicidad , MicroARNs/metabolismo , Neovascularización Retiniana/metabolismo , Epitelio Pigmentado de la Retina/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Línea Celular , Fosfatidilinositol 3-Quinasa Clase Ia/genética , Fosfatidilinositol 3-Quinasa Clase Ia/metabolismo , Retinopatía Diabética/genética , Retinopatía Diabética/patología , Retinopatía Diabética/prevención & control , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , MicroARNs/genética , Oligonucleótidos/farmacología , Neovascularización Retiniana/genética , Neovascularización Retiniana/patología , Neovascularización Retiniana/prevención & control , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patología , Transducción de Señal , Regulación hacia Arriba , Factor A de Crecimiento Endotelial Vascular/genética
13.
J Diabetes Res ; 2019: 6198495, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31828164

RESUMEN

The angiopoietin-Tie-2 system plays a crucial role in the maintenance of endothelial integrity. Hyperglycemia and advanced glycation end-products (AGEs) are involved in endothelial cell dysfunction responsible of the pathogenesis of microvascular complications of diabetes. Here, we investigated whether glycated serum (GS) or hyperglycemia (HG) affect the angiopoietin-Tie-2 system in the microvascular endothelial cells HMEC-1. We found that culture for 5 days in the presence of AGEs and HG (alone or in combination) decreased cell proliferation, increased reactive oxygen species (ROS) production, and reduced ratio between the oxidized and the reduced form of glutathione. Since angiopoietin-1 (Ang-1) signaling regulates angiopoietin-2 (Ang-2) expression through inactivation of the forkhead transcription factor FoxO1, we investigated intracellular signaling of Ang-1 and expression of Ang-2. HG and AGEs reduced phosphorylation of Akt and abrogated phosphorylation of FoxO1 induced by Ang-1 without affecting neither Tie-2 expression nor its activation. Furthermore, AGEs and/or HG induced nuclear translocation of FoxO1 and increased Ang-2 production. In conclusion, we demonstrated that both hyperglycemia and AGEs affect the angiopoietin-Tie-2 system by impairing Ang-1/Tie-2 signaling and by increasing Ang-2 expression. These results suggest that therapeutic strategies useful in preventing or delaying the onset of diabetic vascular complications should be aimed to preserve Ang-1 signaling.


Asunto(s)
Angiopoyetina 1/metabolismo , Angiopoyetina 2/biosíntesis , Células Endoteliales/efectos de los fármacos , Glucosa/farmacología , Productos Finales de Glicación Avanzada/farmacología , Hiperglucemia/metabolismo , Receptor TIE-2/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Endoteliales/metabolismo , Proteína Forkhead Box O1/efectos de los fármacos , Proteína Forkhead Box O1/metabolismo , Glutatión/efectos de los fármacos , Glutatión/metabolismo , Humanos , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Receptor TIE-2/metabolismo , Transducción de Señal
14.
Ann N Y Acad Sci ; 1126: 166-72, 2008 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-18448811

RESUMEN

Osteoporosis, a multifactorial and progressive skeletal metabolic disease, is characterized by low-mass density and structural deterioration of bone micro-architecture that leads to enhanced bone fragility and increased susceptibility to fractures. Recently, it has been proposed that age-related bone loss could be correlated with the glycoxidative process. The aim of the present study was to investigate the in vitro effects of pentosidine, a glycoxidative end product, on human osteoblasts (HOb). The mineralization rate, the specific bone markers (alkaline phosphatase [ALP], collagen Ialpha1 [COL Ialpha1], osteocalcin [BGP]), and the human receptor for advanced glycation end products (RAGE) gene expression have been evaluated. Pentosidine incubation of HOb caused a significant decrease in ALP, Col Ialpha1, and RAGE mRNA levels, but only the RAGE gene expression decreased with no dose dependency. Moreover, pentosidine incubation of osteoblasts hampered the formation of bone nodules. No effect was observed on BGP gene expression under all experimental conditions. Our data gives further support to a detrimental effect of AGEs on bone that leads to functional alterations of osteoblasts. This study addresses a crucial role of protein glycoxidation in the bone mineralization process. AGEs formation and accumulation in bone may be one of the first pathogenetic steps of bone remodeling in aging and in age-related diseases, leading to enhanced bone mass loss.


Asunto(s)
Arginina/análogos & derivados , Lisina/análogos & derivados , Osteoblastos/fisiología , Arginina/farmacología , Huesos/efectos de los fármacos , Huesos/fisiología , División Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Colágeno/genética , Productos Finales de Glicación Avanzada/farmacología , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Humanos , Lisina/farmacología , Procedimientos Ortopédicos , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Osteocalcina/genética , Receptor para Productos Finales de Glicación Avanzada , Receptores Inmunológicos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
15.
Eur J Ophthalmol ; 26(5): 425-30, 2016 Aug 04.
Artículo en Inglés | MEDLINE | ID: mdl-27079208

RESUMEN

PURPOSE: The neovascular or wet form of age-related macular degeneration is characterized by the growth of abnormal blood vessels in the retina stimulated by vascular endothelial growth factors (VEGF). In the last decade, several anti-VEGF drugs have been developed for treating neovascular diseases of the eyes. This study was conducted to compare the effects of 2 anti-VEGF-A drugs, ranibizumab and aflibercept, on the expression and secretion of VEGF family members in retinal pigment epithelial cells (RPE) in vitro. METHODS: ARPE-19 cells were exposed for 24 hours to ranibizumab or aflibercept at clinical dose concentration. Cell viability and expression and secretion of VEGF-A, VEGF-B, VEGF-C, and placental growth factor (PlGF) were evaluated respectively by real-time polymerase chain reaction and enzyme-linked immunosorbent assay. RESULTS: Ranibizumab and aflibercept did not affect ARPE-19 cell viability after 24 hours of treatment. Ranibizumab increased expression of VEGF-A and PlGF. On the contrary, expression and secretion of VEGF-C was decreased by ranibizumab. PlGF secretion was not affected by ranibizumab. Aflibercept strongly increased VEGF-A and PlGF expression but reduced their detection on the culture media, and decreased expression and secretion of VEGF-C. No effect on expression and secretion of VEGF-B was observed after exposure to these drugs. CONCLUSIONS: Ranibizumab and aflibercept exert similar effects on VEGF expression and secretion, leading to establishing an antiangiogenic environment. Increased VEGF-A expression observed in RPE cells treated with these drugs suggests a compensatory response of the cells to the lack of VEGF-A.


Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Ranibizumab/farmacología , Proteínas Recombinantes de Fusión/farmacología , Epitelio Pigmentado de la Retina/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Humanos , Degeneración Macular/tratamiento farmacológico , Factor de Crecimiento Placentario/genética , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Factores de Crecimiento Endotelial Vascular , Epitelio Pigmentado de la Retina/metabolismo , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/genética
16.
J Diabetes Res ; 2015: 326359, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26221611

RESUMEN

Glucose-dependent insulinotropic peptide (GIP) is an incretin hormone produced in the gastrointestinal tract that stimulates glucose dependent insulin secretion. Impaired incretin response has been documented in diabetic patients and was mainly related to the inability of the pancreatic beta cells to secrete insulin in response to GIP. Advanced Glycation End Products (AGEs) have been shown to play an important role in pancreatic beta cell dysfunction. The aim of this study is to investigate whether the exposure to AGEs can induce GIP resistance in the pancreatic beta cell line HIT-T15. Cells were cultured for 5 days in low (CTR) or high glucose (HG) concentration in the presence of AGEs (GS) to evaluate the expression of GIP receptor (GIPR), the intracellular signaling activated by GIP, and secretion of insulin in response to GIP. The results showed that incubation with GS alone altered intracellular GIP signaling and decreased insulin secretion as compared to CTR. GS in combination with HG reduced the expression of GIPR and PI3K and abrogated GIP-induced AKT phosphorylation and GIP-stimulated insulin secretion. In conclusion, we showed that treatment with GS is associated with the loss of the insulinotropic effect of GIP in hyperglycemic conditions.


Asunto(s)
Regulación hacia Abajo , Polipéptido Inhibidor Gástrico/antagonistas & inhibidores , Productos Finales de Glicación Avanzada/metabolismo , Hiperglucemia/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Receptores de la Hormona Gastrointestinal/antagonistas & inhibidores , Animales , Glucemia/análisis , Western Blotting , Línea Celular , Polipéptido Inhibidor Gástrico/sangre , Polipéptido Inhibidor Gástrico/genética , Polipéptido Inhibidor Gástrico/metabolismo , Péptido 1 Similar al Glucagón/sangre , Péptido 1 Similar al Glucagón/metabolismo , Productos Finales de Glicación Avanzada/sangre , Humanos , Hiperglucemia/sangre , Secreción de Insulina , Mesocricetus , Fosfatidilinositol 3-Quinasa/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación , Procesamiento Proteico-Postraduccional , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de la Hormona Gastrointestinal/agonistas , Receptores de la Hormona Gastrointestinal/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transducción de Señal
17.
Toxicol Lett ; 192(2): 101-7, 2010 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-19878710

RESUMEN

According to European laws animal testing in cosmetic industry will be prohibited in a few years and it will be replaced by alternative methods based on cell and tissue culture. Many ingredients of cosmetic formulations are potentially causes of skin inflammation and sensibilization. Since cytotoxicity is known, among other factors, to trigger irritation, in an alternative model for evaluation of skin irritation, it can be considered also the precocious release of inflammatory mediators, i.e. cytokines, originating mainly from keratinocytes. In this in vitro study we have analysed some parameters directly or indirectly related to irritation/inflammation, in NCTC 2544 human keratinocytes during short-time exposure to some potential irritants cosmetic fragrances, included in the European Laws 2003/15/EEC. IIC50 was extrapolated by MTT and NRU viability indexes after exposure of cell ultures to Geraniol Limonene and Benzylic Alcohol for 1, 3 and 6h. NCTC cells were then exposed to sub-toxic doses of selected compounds and interleukin-1alpha (IL-1alpha) and leukaemia inhibitory factor (LIF) expressions were analysed as early proinflammatory cytokines. To our knowledge our findings demonstrated for the first time that NCTC cells synthesize and modulate LIF after exposure to selected irritating stimuli. Moreover, our results give evidence on LIF role as in vitro precocious endpoint for the assessment of the risk in cosmetic field, because its response under irritation stimuli is very quick and comparable to IL-1alpha.


Asunto(s)
Alcohol Bencilo/toxicidad , Ciclohexenos/toxicidad , Interleucina-1alfa/metabolismo , Factor Inhibidor de Leucemia/metabolismo , Perfumes/toxicidad , Terpenos/toxicidad , Monoterpenos Acíclicos , Biomarcadores/metabolismo , Línea Celular , Relación Dosis-Respuesta a Droga , Humanos , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Limoneno , Pruebas de Irritación de la Piel/métodos , Dodecil Sulfato de Sodio/toxicidad
18.
Arch Neurol ; 67(7): 867-72, 2010 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-20625095

RESUMEN

OBJECTIVE: To develop a blood-based test for screening populations at risk for Alzheimer disease. DESIGN: Case-control study. Subjects A total of 180 patients with mild cognitive impairment (MCI) and 105 age-matched, cognitively normal controls. INTERVENTIONS: The titer of beta-amyloid 1-42 autoantibodies in the plasma was obtained at the time of diagnosis and evaluated by enzyme-linked immunosorbent assay before and after dissociation of the antigen-antibody complexes. A total of 107 patients with MCI were followed up for 36 months; 70 of the 107 cases progressed to Alzheimer disease. RESULTS: The average level of beta-amyloid 1-42 plasma autoantibodies in patients with MCI that progressed to Alzheimer disease, but not that of the stable cases, was significantly higher than in cognitively normal controls (P < .001). CONCLUSIONS: The results suggest that the plasma beta-amyloid 1-42 autoantibodies parallel beta-amyloid 42 deposition in the brain, which is known to precede by several years the clinical onset of Alzheimer disease. The evaluation of beta-amyloid 1-42 autoantibodies after dissociation of the complexes is a simple and inexpensive method that can be used to predict the occurrence of Alzheimer disease.


Asunto(s)
Péptidos beta-Amiloides/inmunología , Trastornos del Conocimiento/sangre , Trastornos del Conocimiento/inmunología , Inmunoglobulina G/sangre , Fragmentos de Péptidos/inmunología , Amnesia/sangre , Amnesia/complicaciones , Apolipoproteína E4/genética , Estudios de Casos y Controles , Trastornos del Conocimiento/etiología , Trastornos del Conocimiento/genética , Estudios de Seguimiento , Humanos , Probabilidad
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