RESUMEN
Our studies on the role of cholesterol homeostasis in the pathogenesis of scrapie revealed abnormal accumulation of cholesterol esters in ex vivo peripheral blood mononuclear cells (PBMCs) and skin fibroblasts from healthy and scrapie-affected sheep carrying a scrapie-susceptible genotype compared to sheep with a resistant genotype. Similar alterations were observed in mouse neuroblastoma N2a cell lines persistently infected with mouse-adapted 22L and RML strains of scrapie that showed up to threefold-higher cholesterol ester levels than parental N2a cells. We now report that proteinase K-resistant prion protein (PrPres)-producing cell populations of subclones from scrapie-infected cell lines were characterized by higher cholesterol ester levels than clone populations not producing PrPres. Treatments with a number of drugs known to interfere with different steps of cholesterol metabolism strongly reduced the accumulation of cholesterol esters in ex vivo PBMCs and skin fibroblasts from scrapie-affected sheep but had significantly less or no effect in their respective scrapie-resistant or uninfected counterparts. In scrapie-infected N2a cells, inhibition of cholesterol esters was associated with selective antiprion activity. Effective antiprion concentrations of cholesterol modulators (50% effective concentration [EC(50)] range, 1.4 to 40 microM) were comparable to those of antiprion reference compounds (EC(50) range, 0.6 to 10 microM). These data confirm our hypothesis that abnormal accumulation of cholesterol esters may represent a biological marker of susceptibility to prion infection/replication and a novel molecular target of potential clinical importance.
Asunto(s)
Colesterol/metabolismo , Fibroblastos/efectos de los fármacos , Linfocitos/efectos de los fármacos , Priones/efectos de los fármacos , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Ésteres del Colesterol/metabolismo , Relación Dosis-Respuesta a Droga , Esterificación/efectos de los fármacos , Everolimus , Fibroblastos/citología , Fibroblastos/metabolismo , Genotipo , Linfocitos/citología , Linfocitos/metabolismo , Ratones , Neuroblastoma/metabolismo , Neuroblastoma/patología , Pioglitazona , Scrapie/tratamiento farmacológico , Scrapie/genética , Scrapie/metabolismo , Ovinos , Enfermedades de las Ovejas/tratamiento farmacológico , Enfermedades de las Ovejas/genética , Enfermedades de las Ovejas/metabolismo , Sirolimus/análogos & derivados , Sirolimus/farmacología , Tiazolidinedionas/farmacologíaRESUMEN
PURPOSE: The authors have previously shown that the growth of cultured fibroblasts obtained from primary pterygia was associated with an increase in cholesterol esterification, suggesting that alterations of cholesterol homeostasis may be involved in the development and progression of this disorder. This investigation was conducted to determine whether antiproliferative agents such as pioglitazone (PIO) and everolimus (EVE) may inhibit proteins involved in the cholesterol ester cycle and the proliferation of pterygium fibroblasts (PF). METHODS: Quiescent normal conjunctival fibroblasts and PFs were treated with or without inhibitors of cell proliferation (PIO and EVE) or with inhibitors of cholesterol esterification-progesterone (Pg) and Sandoz compound (SaH)-and then were stimulated to growth by 10% fetal calf serum (FCS). Cell proliferation was assessed by counting cells. Trypan blue uptake was used to determine cell viability. mRNA and protein levels were determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. RESULTS: PIO and EVE significantly abolished the increase in cholesterol esters, acyl-coenzyme A cholesterol acyltransferase (ACAT1), and multidrug resistance protein (MDR1) mRNA observed in growing cells. Each inhibitor upregulated ATP-binding cassette-A1 (ABCA1), neutral cholesterol ester hydrolase (NCEH) mRNA, and caveolin-1 expression in a manner similar to that of specific inhibitors of cholesterol esterification such as Pg and SaH. CONCLUSIONS: Intracellular modifications of cholesterol homeostasis may be relevant to pterygium development. Moreover, antiproliferative agents such as PIO and EVE may represent a potential topical medication in the prevention and inhibition of pterygium growth at an early stage, probably by modulation of cholesterol ester metabolism.
Asunto(s)
Colesterol/metabolismo , Fibroblastos/efectos de los fármacos , Hipoglucemiantes/farmacología , Pterigion/prevención & control , Tiazolidinedionas/farmacología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Anciano , Amidas/farmacología , Caveolina 1/metabolismo , División Celular/efectos de los fármacos , División Celular/fisiología , Células Cultivadas , Ésteres del Colesterol/metabolismo , Inhibidores Enzimáticos/farmacología , Everolimus , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Homeostasis/efectos de los fármacos , Homeostasis/fisiología , Humanos , Inmunosupresores/farmacología , Técnicas In Vitro , Masculino , Persona de Mediana Edad , Compuestos de Organosilicio/farmacología , Pioglitazona , Progesterona/farmacología , Pterigion/metabolismo , Pterigion/patología , ARN Mensajero/metabolismo , Sirolimus/análogos & derivados , Sirolimus/farmacología , Esterol O-Aciltransferasa/genética , Esterol O-Aciltransferasa/metabolismoRESUMEN
PURPOSE: There is now increasing evidence that pterygium and pinguecula are tumorlike tissues and that cell growth and DNA replication are closely linked to cholesterol metabolism. In this study, the expression of two main genes correlated to cholesterol metabolism--namely, the low-density lipoprotein receptor (LDL-R) gene and the hydroxy-methylglutaryl-coenzyme A-reductase (HMG-CoA-R) gene--were investigated in primary pterygium, pinguecula, and normal conjunctiva. METHODS: Pterygium, pinguecula, and normal conjunctiva samples were obtained from 30 eyes (50% men) at the time of surgery. Total RNA extracted from the specimens was subjected to semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). Equal amounts of total RNA were reverse transcribed into cDNA. The cDNA was subsequently amplified by the PCR in the presence of specific primers for low-density lipoprotein receptor (LDL-R) and for hydroxy-methylglutaryl coenzyme A reductase (HMG-CoA-R). RESULTS: Semiquantitative RT-PCR analysis revealed that the mRNA levels of LDL-R and HMG-CoA-R were increased in pterygia, compared with levels in both pingueculae and normal conjunctivae. Differences were statistically significant (P <0.05), including pingueculae versus normal conjunctivae. CONCLUSIONS: This study indicates that pterygium and pinguecula have an altered metabolism of cholesterol-namely increased LDL-R and HMG-CoA-R mRNAs-as is characteristic of tumorlike tissues, and that the high expression of LDL receptors renders them amenable to be treated by photodynamic therapy with intravenously injected verteporfin.
Asunto(s)
Enfermedades de la Conjuntiva/metabolismo , Enfermedades del Tejido Conjuntivo/metabolismo , Expresión Génica , Hidroximetilglutaril-CoA Reductasas/genética , Pterigion/metabolismo , ARN Mensajero/metabolismo , Receptores de LDL/genética , Colesterol/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , ARN/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Cholesterol esterification by acyl-CoA:cholesterol acyltransferase (ACAT) and proliferation of vascular smooth muscle cells (VSMC) are key events in vascular proliferative diseases. Here we performed experiments to ascertain the role of cholesterol ester pathway in the control of human aortic VSMC cycle progression. Results showed that serum-induced VSMC proliferation was preceded by an increased ability of the cells to esterify cholesterol as well as by an increased expression of ACAT and multidrug resistance (MDR1) mRNAs and extracellular related kinases 1/2 (ERK1/2), whereas caveolin-1 levels were markedly decreased. Cell cycle analyses performed in the presence of two inhibitors of cholesterol esterification, directly inhibiting ACAT (Sandoz 58-035) or the transport of cholesterol substrate from plasma membrane to endoplasmic reticulum (progesterone), indicate that each inhibitor suppressed the serum-induced DNA synthesis by accumulation of VSMCs in the G1 phase. The effect was associated with a rapid inhibition of ERK1/2 mitogenic signaling pathway; a down-regulation of cyclin D1, ACAT, and MDR1 mRNA; and an up-regulation of caveolin-1. These data provide a plausible link between cholesterol esterification and control of cell cycle G1/S transition, supporting the hypothesis that cholesterol esterification may accelerate the progression of human vascular proliferative diseases by modulating the rate of the VSMC proliferation.