RESUMO
Silver nanoparticles (Ag NPs) are increasingly being used in wound dressings, medical settings, and various household products due to their unique properties and antimicrobial activity. Despite the widespread use of Ag NP products, the molecular mechanisms underlying the biological effects of Ag NPs remain unclear. Gap junctional intercellular communication (GJIC), formed by the connexin protein family, plays a critical role in the maintenance of tissue and organ homeostasis. This study was undertaken to investigate the effects of well characterized, PVP-coated Ag NPs (69 +/- 3 nm) and silver nitrate on GJIC and connexin43 (Cx43) expression in human lung adenocarcinoma cell line A549. Our results showed that Ag NPs increased GJIC in A549 cells as assayed by dye transfer method. Western blot analysis showed that incubation of cells with Ag NPs significantly increased the expression of Cx43 protein. In addition, Ag NPs up-regulated expression of Cx43 mRNA in a dose-dependent manner. Silver nitrate failed to increase GJIC and the expression of Cx43 protein. It, however, increased Cx43 mRNA expression in A549 cells. Taken together, our results provide the first evidence that Ag NPs induced the increase of GJIC activity in A549 cells through up-regulation of Cx43 protein, suggesting that Cx43 and GJIC may be one of the targets for Ag NPs biological effects.
Assuntos
Comunicação Celular/efeitos dos fármacos , Conexina 43/biossíntese , Junções Comunicantes/efeitos dos fármacos , Nanopartículas Metálicas , Prata/farmacologia , Western Blotting , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Junções Comunicantes/metabolismo , Humanos , Nanopartículas Metálicas/química , Microscopia Eletrônica de Transmissão , Polivinil/química , Pirrolidinas/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Nitrato de Prata/farmacologia , Propriedades de Superfície , Regulação para CimaRESUMO
The objective of the present study was to investigate the toxicity of silver nanoparticles (Ag NPs) in vitro. Silver ions (Ag+) have been used in medical treatments for decades whereas Ag NPs have been used in a variety of consumer products within recent years. This study was undertaken to compare the effect of well characterized, PVP-coated Ag NPs (69 nm +/- 3 nm) and Ag+ in a human monocytic cell line (THP-1). Characterization of the Ag NPs was conducted in both stock suspension and cell media with or without serum and antibiotics. By using the flowcytometric annexin V/propidium iodide (PI) assay, both Ag NPs and Ag+ were shown to induce apoptosis and necrosis in THP-1 cells depending on dose and exposure time. Furthermore, the presence of apoptosis could be confirmed by the TUNEL method. A number of studies have implicated the production of reactive oxygen species (ROS) in cytotoxicity mediated by NPs. We used the fluorogenic probe, 2',7'-dichlorofluorescein to assess the levels of intracellular ROS during exposure to Ag NPs and Ag+. A drastic increase in ROS levels could be detected after 6-24h suggesting that oxidative stress is an important mediator of cytotoxicity caused by Ag NPs and Ag+.
Assuntos
Apoptose/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Monócitos/patologia , Nanopartículas , Cloreto de Polivinila/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Prata/farmacologia , Anexina A5/metabolismo , Linhagem Celular Tumoral , Citometria de Fluxo , Corantes Fluorescentes , Humanos , Marcação In Situ das Extremidades Cortadas , Luz , Monócitos/metabolismo , Necrose/patologia , Tamanho da Partícula , Espalhamento de RadiaçãoRESUMO
The bone-related protein osteoprotegerin (OPG) may be involved in the development of vascular calcifications, especially in diabetes, where it has been found in increased amounts in the arterial wall. Experimental studies suggest that members of the TGF-superfamily are involved in the transformation of human vascular smooth muscle cells (HVSMC) to osteoblast-like cells. In this study, we evaluated the effect of BMP-2, BMP-7 and transforming growth factor beta (TGF-beta1) on the secretion and mRNA expression of OPG and its ligands receptor activator of nuclear factor-kappabeta ligand (RANKL) and TNF-related apoptosis-inducing ligand (TRAIL) in HVSMC. All three growth factors decreased OPG protein production significantly; these results were paralleled by reduced OPG mRNA expression. TRAIL mRNA levels were also decreased. RANKL mRNA expression declined when treated with TGF-beta1 but were increased by both BMPs. Members of the TGF-superfamily, i.e. TGF-beta1, BMP-2 and BMP-7 exert effects on OPG and its ligands, indicating that these peptides may be involved in the development of vascular calcifications. The downregulation of OPG by these peptides does, however, not suggest that these factors are directly involved in OPG accumulation in diabetes.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Músculo Liso Vascular/metabolismo , Osteoprotegerina/biossíntese , Ligante RANK/biossíntese , Ligante Indutor de Apoptose Relacionado a TNF/biossíntese , Fator de Crescimento Transformador beta/metabolismo , Actinas/metabolismo , Calcinose/metabolismo , Células Cultivadas , Diabetes Mellitus/metabolismo , Humanos , RNA Mensageiro/metabolismoRESUMO
Obstruction-induced fibrosis is a leading cause of end-stage renal failure in children. The pathophysiological mechanisms may involve apoptosis and the renin-angiotensin system. We studied apoptosis and fibrosis in a well-established neonatal pig model with unilateral partial ureteral obstruction (PUUO) induced during ongoing nephrogenesis in 2-day-old piglets. The role of angiotensin II (ANG II) was studied using the AT(1) receptor blocker CV-11974 (0.12 mg/h candesartan from age 23 to 30 days). At day 30 the kidneys were perfusion fixed and fibrosis, apoptosis, and tubular lengths were quantitated using stereological methods, picro Sirius red staining, and immunohistochemical techniques identifying activated caspase 3, aquaporin-2 (AQP2), and von Willebrand factor. The collagen content was assessed by hydroxyproline density. Neonatal induced PUUO increased interstitial and glomerular cell apoptosis and fibrosis. At this stage, PUUO did not increase tubular cell apoptosis or decrease tubular length and cell number. AT(1) receptor blockade prevented the PUUO-induced interstitial and glomerular cell apoptosis but did not attenuate fibrosis. In conclusion, AT(1) receptor blockade after the end of nephrogenesis may prevent interstitial and glomerular cell apoptosis but not fibrosis, suggesting that pathways not involving AT(1) receptor stimulation contribute to neonatal obstruction-induced fibrosis or that prevention of interstitial cell apoptosis counteracts a potential antifibrotic effect of AT(1) receptor blockade in this pig model of congenital obstructive nephropathy. Our results demonstrate that ANG II plays a role in PUUO-induced glomerular cell apoptosis.
Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Apoptose/efeitos dos fármacos , Glomérulos Renais/patologia , Nefrite Intersticial/patologia , Obstrução Ureteral/patologia , Angiotensina II/fisiologia , Animais , Animais Recém-Nascidos , Benzimidazóis/farmacologia , Compostos de Bifenilo , Capilares/patologia , Caspase 3/metabolismo , Contagem de Células , Colágeno/biossíntese , Feminino , Fibrose , Hidroxiprolina/análise , Hidroxiprolina/metabolismo , Imuno-Histoquímica , Glomérulos Renais/efeitos dos fármacos , Túbulos Renais/citologia , Suínos , Tetrazóis/farmacologia , Fixação de TecidosRESUMO
Arterial medial calcifications occur often in diabetic individuals as part of the diabetic macroangiopathy. The pathogenesis is unknown, but the presence of calcifications predicts risk of cardiovascular events. We examined the effects of insulin on calcifying smooth muscle cells in vitro and measured the expression of the bone-related molecule osteoprotegerin (OPG). Human vascular smooth muscle cells (VSMCs) were grown from aorta from kidney donors. Induction of calcification was performed with beta-glycerophosphate. The influence of insulin (200 microU/ml or 1,000 microU/ml) on calcification was judged by measuring calcium content in the cell layer and by von Kossa staining. OPG was measured in the medium by ELISA. Histochemistry was used for determination of alkaline phosphatase (ALP). Bone sialoprotein (BSP) and OPG mRNA expressions were done by RT-PCR. beta-Glycerophosphate was able to induce calcification in human smooth muscle cells from a series of donors after variable time in culture. Decreased OPG amounts were observed from the cells during the accelerated calcification phase. High dose of insulin (1,000 microU/ml) accelerated the calcification, whereas lower concentrations (200 microU/ml) did not. Calcified cells expressed ALP and BSP activity in high levels. In conclusion, high concentration of insulin enhances in vitro-induced calcification in VSMCs. Altered OPG levels during the calcification raise the possibility that OPG may have a potent function in regulating the calcification process or it may represent a consequence of mineralization. Effects of insulin and modulations by OPG on the calcification process in arterial cells may play a role in the development of calcifications as part of the diabetic macroangiopathy.