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1.
Biotechnol Bioeng ; 120(10): 2953-2968, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37256741

RESUMO

Adeno-associated virus-based gene therapies have demonstrated substantial therapeutic benefit for the treatment of genetic disorders. In manufacturing processes, viral capsids are produced with and without the encapsidated gene of interest. Capsids devoid of the gene of interest, or "empty" capsids, represent a product-related impurity. As a result, a robust and scalable method to enrich full capsids is crucial to provide patients with as much potentially active product as possible. Anion exchange chromatography has emerged as a highly utilized method for full capsid enrichment across many serotypes due to its ease of use, robustness, and scalability. However, achieving sufficient resolution between the full and empty capsids is not trivial. In this work, anion exchange chromatography was used to achieve empty and full capsid resolution for adeno-associated virus serotype 5. A salt gradient screen of multiple salts with varied valency and Hofmeister series properties was performed to determine optimal peak resolution and aggregate reduction. Dual salt effects were evaluated on the same product and process attributes to identify any synergies with the use of mixed ion gradients. The modified process provided as high as ≥75% AAV5 full capsids (≥3-fold enrichment based on the percent full in the feed stream) with near baseline separation of empty capsids and achieved an overall vector genome step yield of >65%.


Assuntos
Capsídeo , Dependovirus , Humanos , Capsídeo/química , Dependovirus/genética , Sorogrupo , Vetores Genéticos , Cromatografia , Proteínas do Capsídeo/genética , Cloreto de Sódio
3.
Proc Natl Acad Sci U S A ; 116(1): 199-204, 2019 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-30559191

RESUMO

Cysteinyl leukotrienes (cys-LTs) are proinflammatory mediators that enhance vascular permeability through distinct receptors (CysLTRs). We found that CysLT2R regulates angiogenesis in isolated mouse endothelial cells (ECs) and in Matrigel implants in WT mice and enhances EC contraction and permeability via the Rho-dependent myosin light chain 2 and vascular endothelial (VE)-cadherin axis. Since solid tumors utilize aberrant angiogenesis for their growth and metastasis and their vessels exhibit vascular hyperpermeability, we hypothesized that CysLT2R, via its actions on the endothelium, might regulate tumor growth. Both tumor growth and metastases of adoptively transferred syngeneic Lewis lung carcinoma (LLC) cells are significantly reduced in CysLT2R-null mice (Cysltr2-/-) compared with WT and CysLT1R-null mice (Cysltr1-/-). In WT recipients of LLC cells, CysLT2R expression is significantly increased in the tumor vasculature, compared with CysLT1R. Further, the tumor vasculature in Cysltr2-/- recipients exhibited significantly improved integrity, as revealed by increased pericyte coverage and decreased leakage of i.v.-administered Texas Red-conjugated dextran. Administration of a selective CysLT2R antagonist significantly reduced LLC tumor volume, vessel density, dextran leakage, and metastases in WT mice, highlighting CysLT2R as a VEGF-independent regulator of the vasculature promoting risk of metastasis. Thus, both genetic and pharmacological findings establish CysLT2R as a gateway for angiogenesis and EC dysregulation in vitro and ex vivo and in an in vivo model with a mouse tumor. Our data suggest CysLT2R as a possible target for intervention.


Assuntos
Células Endoteliais/efeitos dos fármacos , Neovascularização Patológica/induzido quimicamente , Receptores de Leucotrienos/metabolismo , Animais , Permeabilidade Capilar/efeitos dos fármacos , Ácidos Cicloexanocarboxílicos/farmacologia , Técnicas de Inativação de Genes , Antagonistas de Leucotrienos/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Metástase Neoplásica/tratamento farmacológico , Transplante de Neoplasias , Neoplasias Experimentais , Neovascularização Patológica/tratamento farmacológico , Ácidos Ftálicos/farmacologia , Receptores de Leucotrienos/efeitos dos fármacos
4.
FASEB J ; 31(4): 1556-1570, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28073835

RESUMO

Contributions of mechanical signals to airway remodeling during asthma are poorly understood. Transient receptor potential vanilloid 4 (TRPV4), a mechanosensitive ion channel, has been implicated in cardiac and pulmonary fibrosis; however, its role in asthma remains elusive. Employing a Dermatophagoides farinae-induced asthma model, we report here that TRPV4-knockout mice were protected from D. farinae-induced airway remodeling. Furthermore, lung fibroblasts that were isolated from TRPV4-knockout mice showed diminished differentiation potential compared with wild-type mice. Fibroblasts from asthmatic lung exhibited increased TRPV4 activity and enhanced differentiation potential compared with normal human lung fibroblasts. Of interest, TGF-ß1 treatment enhanced TRPV4 activation in a PI3K-dependent manner in normal human lung fibroblasts in vitro Mechanistically, TRPV4 modulated matrix remodeling in the lung via 2 distinct but dependent pathways: one enhances matrix deposition by fibrotic gene activation, whereas the other slows down matrix degradation by increased plasminogen activator inhibitor 1. Of importance, both pathways are regulated by Rho/myocardin-related transcription factor-A and contribute to fibroblast differentiation and matrix remodeling in the lung. Thus, our results support a unique role for TRPV4 in D. farinae-induced airway remodeling and warrant further studies in humans for it to be used as a novel therapeutic target in the treatment of asthma.-Gombedza, F., Kondeti, V., Al-Azzam, N., Koppes, S., Duah, E., Patil, P., Hexter, M., Phillips, D., Thodeti, C. K., Paruchuri, S. Mechanosensitive transient receptor potential vanilloid 4 regulates Dermatophagoides farinae-induced airway remodeling via 2 distinct pathways modulating matrix synthesis and degradation.


Assuntos
Remodelação das Vias Aéreas , Asma/metabolismo , Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Canais de Cátion TRPV/metabolismo , Adulto , Animais , Asma/etiologia , Asma/genética , Asma/patologia , Células Cultivadas , Dermatophagoides farinae/imunologia , Matriz Extracelular/patologia , Fibroblastos/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosfatidilinositol 3-Quinases/metabolismo , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Canais de Cátion TRPV/genética , Fator de Crescimento Transformador beta/metabolismo
5.
J Am Soc Nephrol ; 28(5): 1421-1436, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-27895157

RESUMO

Overexpression of the proximal tubular enzyme myo-inositol oxygenase (MIOX) induces oxidant stress in vitro However, the relevance of MIOX to tubular pathobiology remains enigmatic. To investigate the role of MIOX in cisplatin-induced tubular AKI, we generated conditional MIOX-overexpressing transgenic (MIOX-TG) mice and MIOX-knockout (MIOX-/-) mice with tubule-specific MIOX overexpression or knockout, respectively. Compared with cisplatin-treated wild-type (WT) mice, cisplatin-treated MIOX-TG mice had even greater increases in urea, creatinine, and KIM-1 levels and more tubular injury and apoptosis, but these effects were attenuated in cisplatin-treated MIOX-/- mice. Similarly, MIOX-TG mice had the highest and MIOX-/- mice had the lowest renal levels of Bax, cleaved caspase-3, and NADPH oxidase-4 expression and reactive oxygen species (ROS) generation after cisplatin treatment. In vitro, cisplatin dose-dependently increased ROS generation in LLC-PK1 cells. Furthermore, MIOX overexpression in these cells accentuated cisplatin-induced ROS generation and perturbations in the ratio of GSH to oxidized GSH, whereas MIOX-siRNA or N-acetyl cysteine treatment attenuated these effects. Additionally, the cisplatin-induced enhancement of p53 activation, NF-κB binding to DNA, and NF-κB nuclear translocation in WT mice was exacerbated in MIOX-TG mice but absent in MIOX-/- mice. In vitro, MIOX-siRNA or NAC treatment reduced the dose-dependent increase in p53 expression induced by cisplatin. We also observed a remarkable influx of inflammatory cells and upregulation of cytokines in kidneys of cisplatin-treated MIOX-TG mice. Finally, analysis of genomic DNA in WT mice revealed cisplatin-induced hypomethylation of the MIOX promoter. These data suggest that MIOX overexpression exacerbates, whereas MIOX gene disruption protects against, cisplatin-induced AKI.


Assuntos
Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/enzimologia , Cisplatino/efeitos adversos , Inositol Oxigenase/deficiência , Animais , Inositol Oxigenase/fisiologia , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos
6.
Dalton Trans ; 45(11): 4729-35, 2016 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-26863280

RESUMO

We have synthesized two Re(CO)3-modified lysine complexes (1 and 2), where the metal is attached to the amino acid at the Nε position, via a one-pot Schiff base formation reaction. These compounds can be used in the solid phase synthesis of peptides, and to date we have produced four conjugate systems incorporating neurotensin, bombesin, leutenizing hormone releasing hormone, and a nuclear localization sequence. We observed uptake into human umbilical vascular endothelial cells as well as differential uptake depending on peptide sequence identity, as characterized by fluorescence and rhenium elemental analysis.


Assuntos
Complexos de Coordenação/síntese química , Peptídeos/química , Rênio/química , Sobrevivência Celular/efeitos dos fármacos , Complexos de Coordenação/metabolismo , Complexos de Coordenação/toxicidade , Células Endoteliais da Veia Umbilical Humana , Humanos , Microscopia de Fluorescência , Peptídeos/síntese química , Rênio/metabolismo , Bases de Schiff/química , Técnicas de Síntese em Fase Sólida
7.
J Allergy Clin Immunol ; 137(1): 289-298, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26255103

RESUMO

BACKGROUND: Although arachidonic acid metabolites, cysteinyl leukotrienes (cys-LTs; leukotriene [LT] C4, LTD4, and LTE4), and prostaglandin (PG) E2 are generated at the site of inflammation, it is not known whether crosstalk exists between these 2 classes of inflammatory mediators. OBJECTIVE: We sought to determine the role of LTD4-PGE2 crosstalk in inducing vascular inflammation in vivo, identify effector cells, and ascertain specific receptors and pathways involved in vitro. METHODS: Vascular (ear) inflammation was assessed by injecting agonists into mouse ears, followed by measuring ear thickness and histology, calcium influx with Fura-2, phosphorylation and expression of signaling molecules by means of immunoblotting, PGD2 and macrophage inflammatory protein 1ß generation by using ELISA, and expression of transcripts by using RT-PCR. Candidate receptors and signaling molecules were identified by using antagonists and inhibitors and confirmed by using small interfering RNA. RESULTS: LTD4 plus PGE2 potentiated vascular permeability and edema, gearing the system toward proinflammation in wild-type mice but not in Kit(W-sh) mice. Furthermore, LTD4 plus PGE2, through cysteinyl leukotriene receptor 1 (CysLT1R) and E-prostanoid receptor (EP) 3, enhanced extracellular signal-regulated kinase (Erk) and c-fos phosphorylation, inflammatory gene expression, macrophage inflammatory protein 1ß secretion, COX-2 upregulation, and PGD2 generation in mast cells. Additionally, we uncovered that this synergism is mediated through Gi, protein kinase G, and Erk signaling. LTD4 plus PGE2-potentiated effects are partially sensitive to CysLT1R or EP3 antagonists but completely abolished by simultaneous treatment both in vitro and in vivo. CONCLUSIONS: Our results unravel a unique LTD4-PGE2 interaction affecting mast cells through CysLT1R and EP3 involving Gi, protein kinase G, and Erk and contributing to vascular inflammation in vivo. Furthermore, current results also suggest an advantage of targeting both CysLT1R and EP3 in attenuating inflammation.


Assuntos
Dinoprostona/imunologia , Leucotrieno D4/imunologia , Mastócitos/imunologia , Receptores de Leucotrienos/imunologia , Receptores de Prostaglandina E Subtipo EP3/imunologia , Animais , Permeabilidade Capilar , Linhagem Celular , Linhagem Celular Tumoral , Edema/imunologia , Humanos , Inflamação/imunologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos
8.
Sci Rep ; 5: 14257, 2015 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-26388427

RESUMO

Endothelial cell proliferation is a critical event during angiogenesis, regulated by both soluble factors and mechanical forces. Although the proliferation of tumor cells is studied extensively, little is known about the proliferation of tumor endothelial cells (TEC) and its contribution to tumor angiogenesis. We have recently shown that reduced expression of the mechanosensitive ion channel TRPV4 in TEC causes aberrant mechanosensitivity that result in abnormal angiogenesis. Here, we show that TEC display increased proliferation compared to normal endothelial cells (NEC). Further, we found that TEC exhibit high basal ERK1/2 phosphorylation and increased expression of proliferative genes important in the G1/S phase of the cell cycle. Importantly, pharmacological activation of TRPV4, with a small molecular activator GSK1016790A (GSK), significantly inhibited TEC proliferation, but had no effect on the proliferation of NEC or the tumor cells (epithelial) themselves. This reduction in TEC proliferation by TRPV4 activation was correlated with a decrease in high basal ERK1/2 phosphorylation. Finally, using a syngeneic tumor model revealed that TRPV4 activation, with GSK, significantly reduced endothelial cell proliferation in vivo. Our findings suggest that TRPV4 channels regulate tumor angiogenesis by selectively inhibiting tumor endothelial cell proliferation.


Assuntos
Neoplasias/metabolismo , Canais de Cátion TRPV/metabolismo , Animais , Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Leucina/análogos & derivados , Leucina/farmacologia , Sistema de Sinalização das MAP Quinases , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Neovascularização Patológica/tratamento farmacológico , Sulfonamidas/farmacologia , Canais de Cátion TRPV/efeitos dos fármacos , Canais de Cátion TRPV/genética , Regulação para Cima
9.
J Cell Physiol ; 230(3): 595-602, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25161061

RESUMO

Mast cells (MCs) are important effector cells in asthma and pulmonary inflammation, and their proliferation and maturation is maintained by stem cell factor (SCF) via its receptor, c-Kit. Cysteinyl leukotrienes (cys-LTs) are potent inflammatory mediators that signal through CysLT1 R and CysLT2 R located on the MC surface, and they enhance MC inflammatory responses. However, it is not known if SCF and cys-LTs cross-talk and influence MC hyperplasia and activation in inflammation. Here, we report the concerted effort of the growth factor SCF and the inflammatory mediator LTD4 in MC activation. Stimulation of MCs by LTD4 in the presence of SCF enhances c-Kit-mediated proliferative responses. Similarly, SCF synergistically enhances LTD4 -induced calcium, c-fos expression and phosphorylation, as well as MIP1ß generation in MCs. These findings suggest that integration of SCF and LTD4 signals may contribute to MC hyperplasia and hyper-reactivity during airway hyper-response and inflammation.


Assuntos
Proliferação de Células/genética , Inflamação/genética , Mastócitos/metabolismo , Mastocitose/genética , Fator de Células-Tronco/metabolismo , Cálcio/metabolismo , Proliferação de Células/efeitos dos fármacos , Humanos , Inflamação/tratamento farmacológico , Inflamação/patologia , Leucotrieno D4/administração & dosagem , Mastócitos/efeitos dos fármacos , Mastocitose/patologia , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-kit/metabolismo , Receptores de Leucotrienos/metabolismo , Transdução de Sinais/efeitos dos fármacos
10.
Dalton Trans ; 43(30): 11452-5, 2014 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-24875597

RESUMO

We have synthesized a Re(CO)3-modified lysine via a one-pot Schiff base formation reaction that can be used in the solid phase peptide synthesis. To demonstrate its potential use, we have attached it to a neurotensin fragment and observed uptake into human umbilical vascular endothelial cells.


Assuntos
Lisina/química , Neurotensina/química , Técnicas de Síntese em Fase Sólida/métodos , Sequência de Aminoácidos , Células Endoteliais/metabolismo , Humanos , Neurotensina/metabolismo
11.
Sci Rep ; 3: 3274, 2013 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-24253666

RESUMO

Cysteinyl leukotrienes (cys-LTs), LTC4, LTD4, LTE4 are potent inflammatory lipid mediators that act through two distinct G-protein-coupled receptors, CysLT1R and CysLT2R. Although cys-LTs are shown to induce vascular leakage and atherosclerosis, the molecular mechanism by which cys-LTs modulate endothelial function is not known. Here, we show that cys-LTs (LTC4 and LTD4) induce robust calcium influx in human umbilical vein endothelial cells (HUVECs) through CysLT2R, but not CysLT1R. Further, cys-LT treatment induced endothelial cell (EC) contraction leading to monolayer disruption via CysLT2R/Rho kinase dependent pathway. Furthermore, stimulation with cys-LTs potentiated TNFα-induced VCAM-1 expression and leukocyte recruitment to ECs through CysLT2R. In contrast, we found that both LTC4 and LTD4 stimulated EC proliferation through CysLT1R. Taken together, these results suggest that cys-LTs induce endothelial inflammation and proliferation via CysLT2R/Rho kinase and CysLT1R/Erk dependent pathways, respectively, which play critical role in the etiology of cardiovascular diseases such as atherosclerosis and myocardial infarction.


Assuntos
Cisteína/farmacologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Leucotrienos/farmacologia , Receptores de Leucotrienos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Cálcio/metabolismo , Sinalização do Cálcio , Proliferação de Células/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Fator de Necrose Tumoral alfa/farmacologia , Quinases Associadas a rho/metabolismo
12.
PLoS One ; 8(8): e71536, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23977066

RESUMO

Cysteinyl leukotrienes (cys-LTs) are a group of lipid mediators that are potent bronchoconstrictors, powerful inducers of vascular leakage and potentiators of airway hyperresponsiveness. Cys-LTs play an essential role in asthma and are synthesized as well as activated in mast cells (MCs). Cys-LTs relay their effects mainly through two known GPCRs, CysLT1R and CysLT2R. Although protein kinase C (PKC) isoforms are implicated in the regulation of CysLT1R function, neither the role of PKCs in cys-LT-dependent MC inflammatory signaling nor the involvement of specific isoforms in MC function are known. Here, we show that PKC inhibition augmented LTD4 and LTE4-induced calcium influx through CysLT1R in MCs. In contrast, inhibition of PKCs suppressed c-fos expression as well MIP1ß generation by cys-LTs. Interestingly, cys-LTs activated both PKCα and PKCε isoforms in MC. However, knockdown of PKCα augmented cys-LT mediated calcium flux, while knockdown of PKCε attenuated cys-LT induced c-fos expression and MIP1ß generation. Taken together, these results demonstrate for the first time that cys-LT signaling downstream of CysLT1R in MCs is differentially regulated by two distinct PKCs which modulate inflammatory signals that have significant pathobiologic implications in allergic reactions and asthma pathology.


Assuntos
Mastócitos/metabolismo , Proteína Quinase C/metabolismo , Receptores de Leucotrienos/metabolismo , Transdução de Sinais , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Quimiocina CCL4/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Cisteína/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Isoenzimas/metabolismo , Leucotrieno E4/farmacologia , Leucotrienos/farmacologia , Mastócitos/efeitos dos fármacos , Mastócitos/enzimologia , Modelos Biológicos , Fosforilação/efeitos dos fármacos , Proteína Quinase C/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-fos/metabolismo , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacos
13.
J Pathol ; 225(3): 364-77, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21984124

RESUMO

Transforming growth factor (TGF)-ß has been shown to play a central role in the development of tubulointerstitial fibrosis, which can be corrected via treatment with paclitaxel. The biology of microRNA (miR) can be modulated by paclitaxel. We hypothesized that paclitaxel may attenuate renal fibrosis in a rat model of remnant kidney disease by inhibiting TGF-ß induced-miRs. Rats in groups of 12 were subjected to 5/6 nephrectomy and received low-dose intraperitoneal injection of paclitaxel. Renal functions were assessed at 8 weeks. The TGF-ß signalling cascade and ECM proteins were evaluated by real-time polymerase chain reaction (TRT-PCR) and immunofluorescence microscopy. Animals with remnant kidneys developed hypertension, which was not relieved with paclitaxel treatment. However, paclitaxel treatment resulted in dampening the proteinuric response, reduction in serum BUN, creatinine levels and urine protein : creatinine ratio and normalization of creatinine clearance. These effects were accompanied by the inhibition of Smad2/3 activation, attenuation of renal fibrosis and normalization of integrin-linked kinase (ILK), COL(I)A1, COL(IV)A2 and α-SMA expression. Also, paclitaxel down-regulated the expression of miR-192, miR-217 and miR -377, while miR-15 was up-regulated in the remnant kidney. In vitro, in tubular epithelial cells (NRK-52E), paclitaxel also inhibited TGF-ß1-induced Smad2/3 activation and normalized ILK, COL(I)A1, COL(IV)A2 and α-SMA expression. Furthermore, ChIP analyses indicated that Taxol suppressed Smad3-mediated miR-192 transcriptional activity. Over-expression of miR-192 in NRK-52E mimicked the changes seen in the remnant kidney, while inclusion of miR-192 inhibitor in the culture medium blocked TGF-ß1-induced COL(I)A1 and COL(IV)A2 expression, while ILK and α-SMA were unaffected. These data suggest that low-dose paclitaxel ameliorates renal fibrosis via modulating miR-192 pathobiology and TGF-ß/Smad signalling.


Assuntos
Regulação para Baixo/efeitos dos fármacos , Rim/patologia , MicroRNAs/biossíntese , Paclitaxel/farmacologia , Animais , Células Cultivadas , Creatinina/farmacocinética , Modelos Animais de Doenças , Esquema de Medicação , Avaliação Pré-Clínica de Medicamentos , Matriz Extracelular/metabolismo , Fibrose , Regulação da Expressão Gênica/efeitos dos fármacos , Hipertensão Renal/metabolismo , Hipertensão Renal/prevenção & controle , Rim/efeitos dos fármacos , Rim/metabolismo , Masculino , MicroRNAs/genética , Nefrectomia/métodos , Paclitaxel/administração & dosagem , Paclitaxel/uso terapêutico , Proteinúria/prevenção & controle , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Proteínas Smad/antagonistas & inibidores , Proteínas Smad/genética , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta1/antagonistas & inibidores , Fator de Crescimento Transformador beta1/farmacologia , Moduladores de Tubulina/administração & dosagem , Moduladores de Tubulina/farmacologia , Moduladores de Tubulina/uso terapêutico
14.
Am J Pathol ; 179(4): 1706-18, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21854750

RESUMO

The mechanisms involved in tubular hypertrophy in diabetic nephropathy are unclear. We investigated the role of exchange protein activated by cAMP 1(Epac1), which activates Rap-family G proteins in cellular hypertrophy. Epac1 is expressed in heart, renal tubules, and in the HK-2 cell line. In diabetic mice, increased Epac1 expression was observed, and under high glucose ambience (HGA), HK-2 cells also exhibited increased Epac1 expression. We isolated a 1614-bp DNA fragment upstream of the initiation codon of Epac1 gene, inclusive of glucose response elements (GREs). HK-2 or COS7 cells transfected with the Epac1 promoter revealed a dose-dependent increase in its activity under HGA. Mutations in GRE motifs resulted in decreased promoter activity. HK-2 cells exhibited a hypertrophic response and increased protein synthesis under HGA, which was reduced by Epac1-siRNA or -mutants, whereas the use of a protein kinase A inhibitor had minimal effect. Epac1 transfection led to cellular hypertrophy and increased protein synthesis, which was accentuated by HGA. HGA increased the proportion of cells in the G0/G1 cell-cycle phase, and the expression of pAkt and the cyclin-dependent kinase inhibitors p21 and p27 was increased while the activity of cyclin-dependent kinase 4 decreased. These effects were reversed following transfection of cells with Epac1-siRNA or -mutants. These data suggest that HGA increases GRE-dependent Epac1 transcription, leading to cell cycle arrest and instigation of cellular hypertrophy.


Assuntos
Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Glucose/farmacologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Túbulos Renais Proximais/enzimologia , Túbulos Renais Proximais/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patologia , Perfilação da Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Hipertrofia , Túbulos Renais Proximais/efeitos dos fármacos , Camundongos , Especificidade de Órgãos/genética , Regiões Promotoras Genéticas/genética , Estreptozocina
15.
J Biol Chem ; 286(39): 34131-46, 2011 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-21795690

RESUMO

Tubulo-interstitial nephritis antigen (TINag) is an extracellular matrix protein expressed in tubular basement membranes. Combined mutations in TINag and nephrocystin-1 genes lead to nephronophthisis with reduced cell survival. Because certain extracellular matrix proteins are known to modulate cell survival, studies were initiated in Lewis rats lacking TINag to assess if they are more susceptible to cisplatin-induced injury. Cisplatin induced a higher degree of tubular cell damage and apoptosis in regions where TINag is expressed in a parental Wistar strain. This was accompanied by an accentuated increase in serum creatinine and Kim-1 RNA and renal expression of Bax, p53, and its nuclear accumulation, mtDNA fragmentation, and a decrease of Bcl-2. Cisplatin induced fulminant apoptosis of HK-2 cells with increased caspase3/7 activity, mtDNA fragmentation, and a reduced cell survival. These effects were partially reversed in cells maintained on TINag substratum. Far Western/solid phase assays established TINag binding with integrin αvß3 comparable with vitronectin. Transfection of cells with αv-siRNA accentuated cisplatin-induced apoptosis, aberrant translocation of cytochrome c and Bax, and reduced cell survival. The αv-siRNA decreased expression of integrin-recruited focal adhesion kinase (FAK) and p-FAK, while increasing the expression of p53 and p-p53. Similarly, p-AKT was reduced although ILK was unaffected. Inhibition of PI3K had similar adverse cellular effects. These effects were ameliorated in cells on TINag substratum. In vivo, a higher degree of decrease in the expression of p-FAK and pAKT was observed in Lewis rats following cisplatin treatment. These in vivo and in vitro studies demonstrate an essential role of TINag in cellular survival to maintain proper tubular homeostasis utilizing integrin αvß3 and downstream effectors.


Assuntos
Proteínas da Matriz Extracelular/metabolismo , Matriz Extracelular/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Membrana Basal Glomerular/metabolismo , Integrina alfaVbeta3/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/fisiologia , Animais , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Caspase 3/genética , Caspase 3/metabolismo , Caspase 7/genética , Caspase 7/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Cisplatino/efeitos adversos , Cisplatino/farmacologia , Citocromos c/genética , Citocromos c/metabolismo , Matriz Extracelular/genética , Proteínas da Matriz Extracelular/genética , Quinase 1 de Adesão Focal/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Integrina alfaVbeta3/genética , Nefropatias/induzido quimicamente , Nefropatias/genética , Nefropatias/metabolismo , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Ratos , Ratos Endogâmicos Lew , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Especificidade da Espécie , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
16.
J Biol Chem ; 286(31): 27594-611, 2011 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-21652700

RESUMO

Renal-specific oxidoreductase/myo-inositol oxygenase (RSOR/MIOX) catabolizes myo-inositol and is implicated in the pathogenesis of diabetic nephropathy. How high glucose (HG) ambience up-regulates its expression and enzyme activity was investigated. MIOX up-regulation was associated with an increase in enzyme activity, which was reduced to basal levels with phosphatase treatment. Using phosphothreonine, protein kinase A (PKA), and PKC substrate antibodies, analyses of kidney lysates of diabetic animals and LLC-PK1/HK-2 cells subjected to HG ambience indicated MIOX to be a phosphoprotein. Kinase phosphorylated recombinant RSOR/MIOX proteins had increased activity confined to exons 2-5. Mutants with substituted phosphorylation sites had a minimal increase in activity. Treatment of cells with PKC, PKA, and PDK1 kinase activators increased activity, whereas inhibitors reduced it. Inhibitors also reduced the phosphorylation and activity of MIOX induced by HG. Besides HG, exposure of cells to oxidants H(2)O(2) and methylglyoxal up-regulated MIOX expression and its phosphorylation and activity, whereas antioxidants N-acetylcysteine, ß-naphthoflavone, and tertiary butyl hydroquinone reduced MIOX expression. Treatment with HG or oxidants or overexpression of MIOX induced nuclear translocation of redox-sensitive transcription factor Nrf2, which binds to antioxidant response elements of various promoters. Promoter analyses revealed an increase in luciferase activity with HG and oxidants. Analyses of antioxidant response elements and carbohydrate response elements revealed an accentuation of DNA-protein interactions with oxidants and under HG ambience. ChIP-PCR and immunofluorescence studies revealed nuclear translocation of carbohydrate response element-binding protein. These findings suggest that phosphorylation of RSOR/MIOX enhances its activity, which is augmented by HG via transcriptional/translational events that are also modulated by diabetes-related pathobiological stresses.


Assuntos
Glucose/metabolismo , Inositol Oxigenase/metabolismo , Processamento de Proteína Pós-Traducional , Estresse Fisiológico , Transcrição Gênica , Animais , Sequência de Bases , Western Blotting , Linhagem Celular , Imunoprecipitação da Cromatina , Primers do DNA , Diabetes Mellitus Experimental/enzimologia , Diabetes Mellitus Experimental/metabolismo , Eletroforese em Gel de Poliacrilamida , Ensaio de Desvio de Mobilidade Eletroforética , Inositol Oxigenase/genética , Túbulos Renais Proximais/enzimologia , Túbulos Renais Proximais/metabolismo , Camundongos , Mutagênese Sítio-Dirigida , Fosforilação
17.
Food Chem Toxicol ; 48(5): 1281-7, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20178824

RESUMO

AIM OF THE STUDY: The present study was designed to investigate the effect of bark of Pterocarpus santalinus, an ethnomedicinal plant, on blood glucose, plasma insulin, serum lipids and the activities of hepatic glucose metabolizing enzymes in streptozotocin-induced diabetic rats. MATERIALS AND METHODS: Streptozotocin-induced diabetic rats were treated (acute/short-term and long-term) with ethyl acetate:methanol fractions of ethanolic extract of the bark of P. santalinus. Fasting blood glucose, HbA(1C), plasma insulin and protein were estimated before and after the treatment, along with hepatic glycogen, and activities of hexokinase, glucose-6-phosphatase, fructose-1,6-bisphosphatase and glucose-6-phosphate dehydrogenase. Further anti-hyperlipidemic activity was studied by measuring the levels of serum lipids and lipoproteins. RESULTS: Phytochemical analysis of active fraction showed the presence of flavonoids, glycosides and phenols. Biological testing of the active fraction demonstrated a significant antidiabetic activity by reducing the elevated blood glucose levels and glycosylated hemoglobin, improving hyperlipidemia and restoring the insulin levels in treated experimental induced diabetic rats. Further elucidation of mechanism of action showed improvement in the hepatic carbohydrate metabolizing enzymes after the treatment. Our present investigation suggests that active fraction of ethanolic extract of bark of P. santalinus decreases streptozotocin induced hyperglycemia by increasing glycolysis and decreasing gluconeogenesis.


Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Hiperlipidemias/tratamento farmacológico , Hipoglicemiantes/farmacologia , Fígado/efeitos dos fármacos , Extratos Vegetais/farmacologia , Pterocarpus/química , Animais , Glicemia/análise , Fracionamento Químico , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/complicações , Flavonoides/análise , Frutose-Bifosfatase/metabolismo , Glucose-6-Fosfatase/metabolismo , Glicosídeos/análise , Hiperlipidemias/sangue , Hiperlipidemias/etiologia , Hipoglicemiantes/química , Insulina/sangue , Lipídeos/sangue , Fígado/enzimologia , Glicogênio Hepático/metabolismo , Masculino , Fenóis/análise , Extratos Vegetais/química , Ratos , Ratos Wistar
18.
Food Chem Toxicol ; 48(4): 1078-84, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20122979

RESUMO

The present study was taken up to identify potent antihyperglycemic fraction from the aqueous extract of Syzygium alternifolium (SA) seeds, using bioassay guided fractionation. The isolated fraction C at a dose of 50 mg/kg.b.w produced the maximum fall of 83% in the blood glucose level in the diabetic rats after 6 h of the treatment. The administration of fraction C (50 mg/kg.b.w) once daily for 30 days in STZ diabetic rats resulted in a significant decrease in blood glucose, glycosylated haemoglobin with a significant rise in plasma insulin level. Further fraction C showed antihyperlipidemic activity as evidenced by significant decrease in serum TC, TG, LDL-C, VLDL-C levels coupled together with elevation of HDL-C level in diabetic rats. A significant decrease in the activities of SGOT, SGPT, ALP and decreased levels of serum urea and creatinine in diabetic treated rats when compared to diabetic untreated rats, indicate the protective role against liver and kidney damage and non-toxic property of the fraction C. A comparison was made between the action of fraction C and antidiabetic drug glibenclamide (20 mg/kg.b.w). The effect of fraction C was more prominent when compared to that of glibenclamide.


Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Hipoglicemiantes/farmacologia , Hipolipemiantes/farmacologia , Syzygium/química , Alanina Transaminase/sangue , Fosfatase Alcalina/sangue , Animais , Aspartato Aminotransferases/sangue , Glicemia/efeitos dos fármacos , Fracionamento Químico , Diabetes Mellitus Experimental/sangue , Glibureto/farmacologia , Hemoglobinas Glicadas/análise , Hemoglobinas Glicadas/efeitos dos fármacos , Insulina/sangue , Lipídeos/sangue , Testes de Função Hepática , Masculino , Metanol/química , Extratos Vegetais/farmacologia , Ratos , Ratos Wistar , Sementes/química , Estreptozocina , Água/química
19.
Food Chem Toxicol ; 48(2): 495-501, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19896519

RESUMO

Ethanolic extract prepared from the seeds of Vernonia anthelmintica was evaluated for its antihyperglycemic activity in STZ (Streptozotocin) induced diabetic rats. Administration of ethanolic extract at a dosage of 0.50 g/kg bw produced the maximum fall (82%) in the blood glucose levels in diabetic rats after 6 h of treatment. Bioassay-directed fractionation using silica gel column chromatography was performed. Among the five fractions (A1, B1, C1, A2 and B2) obtained, of an initial chromatographic separation of the ethanolic extract, fraction A2 (100 mg/kg bw) showed the maximum antihyperglycemic activity which is significantly higher than that of the reference drug glibenclamide (20 mg/kg bw). Administration of the active fraction (100 mg/kg bw) for 45 days resulted in significant reduction in plasma glucose, HbA1(C), cholesterol, triglycerides, LDL, VLDL, free fatty acids, phospholipids and HMG-CoA reductase in STZ diabetic rats. In addition to that, significant decrease in plasma insulin, protein, HDL and hepatic glycogen observed in STZ diabetic rats, was normalized after 45 days of treatment with the active fraction of V. anthelmintica seeds. From the present study, it is evident that, the seeds of V. anthelmintica possess significant antidiabetic and antihyperlipidemic property without evident toxic effects.


Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Hipoglicemiantes/farmacologia , Hipolipemiantes/farmacologia , Fitoterapia/métodos , Extratos Vegetais/farmacologia , Vernonia/química , 2-Propanol/química , Acetatos/química , Animais , Glicemia/análise , Glicemia/efeitos dos fármacos , Fracionamento Químico , Cromatografia em Gel , Diabetes Mellitus Experimental/sangue , Etanol/química , Glibureto/farmacologia , Hidroximetilglutaril-CoA Redutases/metabolismo , Insulina/sangue , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipídeos/sangue , Ratos , Sementes/química , Estreptozocina
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