RESUMO
Reactivation of telomerase in endothelial cells (ECs) may be an effective approach to the treatment of vascular disorders associated with telomere attrition and EC senescence. However, overexpression of human telomerase reverse transcriptase (hTERT) does not prevent net telomere loss in ECs grown in standard culture medium with exposure to atmospheric oxygen (21% O(2)). Since these culture conditions are hyperoxic relative to normal tissue in vivo, where oxygen tension is estimated to be 1%-6%, we examined the effects of reduced exposure to oxidative stress (OS) on telomere length maintenance in hTERT-transduced bone marrow endothelial (BMhTERT) cells. Propagation of BMhTERT cells in the free radical scavenger, tert-butylhydroxylamine (tBN), and/or in 5% O(2) increased telomerase enzyme activity and facilitated telomere length maintenance. The enhancement of telomerase activity correlated with higher levels of the telomerase RNA component (hTR). We also investigated the role of the telomere binding protein, TRF1, in telomere length regulation under alternate OS conditions. Inhibition of TRF1 function had no effect on telomere length in BMhTERT cells grown under standard culture conditions. However, alleviation of OS by growth in tBN plus 5% O(2), elevated hTR levels, enhanced telomerase enzyme activity, and enabled progressive telomere lengthening. The direct impact of hTR levels on telomerase-mediated telomere lengthening was demonstrated by overexpression of hTR. BMhTERT cells transduced with hTR exhibited very high telomerase enzyme activity and underwent dramatic telomere lengthening under standard culture conditions. Overall, these results demonstrate that hTR levels are reduced by mild hyperoxia and limit telomerase-mediated telomere lengthening in hTERT-transduced ECs.
Assuntos
Células Endoteliais/metabolismo , RNA/metabolismo , Telomerase/metabolismo , Telômero/metabolismo , Western Blotting , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Hipóxia Celular , Proliferação de Células , Células Cultivadas , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Vetores Genéticos/genética , Humanos , Hidroxilaminas/farmacologia , Estresse Oxidativo , RNA/genética , Retroviridae/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Telomerase/genética , Telômero/genética , Proteína 1 de Ligação a Repetições Teloméricas/metabolismo , Transdução Genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
The factors associated with the migration of marrow-derived mesenchymal stem cells (MSCs) when transplanted into the diseased central nervous system (CNS) are unclear. Chemokines are key mediators of selective cell migration in neurodegenerative diseases and related inflammatory processes. We hypothesized that chemokines are likely to be the chief determinants of MSC migration. We, therefore, systematically assessed the expression and modulating factors for chemokines and chemokine receptors in human MSCs (HuMSCs). The present study demonstrates that unstimulated HuMSCs express a broad range of mRNAs encoding cytokines, chemokines, and their receptors. Using chemotaxis assays, we also assessed the functionality of the receptor expression in HuMSC and we show that CXCL12/stromal cell-derived factor-lalpha (SDF-lalpha), CX3CL1/fractalkine, and CXCL10/interferon-gamma (IFN-gamma)-inducible protein (IP-10) lead to significant HuMSC migration. Moreover, we provide evidence that tumor necrosis factor-alpha (TNF-alpha) and IFN-gamma act as major regulators of the expression of chemokines and their receptors in HuMSCs. Correspondingly, we demonstrate for the first time that current multiple sclerosis (MS) therapies, namely, IFN-beta and Copaxone, influence the expression of chemokines and their receptors in HuMSCs at both mRNA and protein levels. Administration of cytokines, including IFN-beta and Copaxone, may be important in stem cell transplantation therapies and perhaps important in the efficacy of existing MS therapies.
Assuntos
Quimiocinas/biossíntese , Citocinas/fisiologia , Interferon beta/fisiologia , Células-Tronco Mesenquimais/metabolismo , Peptídeos/fisiologia , Receptores de Quimiocinas/biossíntese , Regulação para Cima/fisiologia , Células Cultivadas , Quimiocinas/genética , Acetato de Glatiramer , Humanos , RNA Mensageiro/biossíntese , Receptores de Quimiocinas/genéticaRESUMO
We hypothesized that the tryptophan catabolites produced through the kynurenine pathway (KP), and more particularly the excitotoxin quinolinic acid (QUIN), may play an important role in the pathogenesis of Alzheimer's disease (AD). In this study, we demonstrated that aggregated amyloid peptide A beta 1-42 induced indoleamine 2,3-dioxygenase (IDO) expression and resulted in a significant increase in production of QUIN by human primary macrophages and microglia. In contrast, A beta 1-40 and prion peptide (PrP) 106-126 did not induce any significant increase in QUIN production. These data imply that local QUIN production may be one of the factors involved in the pathogenesis of neuronal damage in AD.