The intrinsic stiffness of human trabecular meshwork cells increases with senescence.

Dysfunction of the human trabecular meshwork (HTM) plays a central role in the age-associated disease glaucoma, a leading cause of irreversible blindness. The etiology remains poorly understood but cellular senescence, increased stiffness of the tissue, and the expression of Wnt antagonists such as secreted frizzled related protein-1 (SFRP1) have been implicated. However, it is not known if senescence is causally linked to either stiffness or SFRP1 expression. In this study, we utilized in vitro HTM senescence to determine the effect on cellular stiffening and SFRP1 expression. Stiffness of cultured cells was measured using atomic force microscopy and the morphology of the cytoskeleton was determined using immunofluorescent analysis. SFRP1 expression was measured using qPCR and immunofluorescent analysis. Senescent cell stiffness increased 1.88±0.14 or 2.57±0.14 fold in the presence or absence of serum, respectively. This was accompanied by increased vimentin expression, stress fiber formation, and SFRP1 expression. In aggregate, these data demonstrate that senescence may be a causal factor in HTM stiffening and elevated SFRP1 expression, and contribute towards disease progression. These findings provide insight into the etiology of glaucoma and, more broadly, suggest a causal link between senescence and altered tissue biomechanics in aging-associated diseases

Response to chronic exposure to hexavalent chromium in human monocytes

Elevated circulating levels of metal ions, particularly chromium, have been measured in the blood of patients with metal hip implants, and this has lead to concerns about the long term safety of the prostheses. For example, depletion of lymphocytes has been reported in vivo in patients with metallic prostheses, and correlated with elevated chromium and cobalt concentrations in blood. However, the implications for immune function are unclear. We have assessed the in vitro responses of U937 human monocytes to chronic exposure (4 weeks) to Cr (VI) ions at concentrations which have been measured in patients with metal artificial hip implants (0.05-0.5 μM). Chronic exposure to these low clinically relevant concentrations of Cr (VI) induced a potent adaptive response with elevated glutathione-S-transferase (pi) expression and increased activities and expression of reactive oxygen scavengers, superoxide dismutases, catalase and glutathione peroxidase. Such direct toxicity of Cr ions may contribute to the effects of metal implants on lymphocyte populations in vivo

Chronic toxicity of hexavalent chromium to U937 human monocyte cell line

Cobalt chromium (CoCr) alloy and stainless steel are widely used in orthopaedic implants. Cr(VI) is known to be released when metals corrode in vivo (Milošev et al., 2005) and may be toxic to local cells like osteoblasts and macrophages. Ning and Grant, 2000 J. Ning and M.H. Grant, Toxicol. In Vitro 14 (2000), pp. 329-335. Article | PDF (185 K)(25)Ning and Grant (2000) have demonstrated the acute toxicity of Cr(VI) to immortalised rat osteoblasts and shown the involvement of reduced glutathione (GSH) and glutathione reductase (GRd) in the interaction. This paper details the chronic toxicity of hexavalent chromium to U937 human monocyte cell lines

YAP and TAZ are distinct effectors of corneal myofibroblast transformation.

PurposeTransforming growth factor β1 (TGFβ1) is elevated in wounds after injury and promotes the transdifferentiation of quiescent cells in the stroma (keratocytes, to activated fibroblasts and subsequently myofibroblasts-KFM transformation). Coactivators of transcription, YAP (Yes-associated protein) and TAZ (Transcriptional coactivator with PDZ-binding motif), are mechanotransducers that intersect with the TGFβ pathway via interactions with Smad proteins. Here, we examined the distinct role of YAP and TAZ on TGFβ1 induced myofibroblast transformation of primary human corneal fibroblasts (HCFs).MethodsA knockdown approach was used to silence YAP and TAZ individually in HCFs. Forty-eight hours post siRNA transfection, cells were cultured in the presence or absence of 2 ng/ml TGFβ1 for 24h. The cells were subjected to nuclear and cytoplasmic fractionation. The expression of α-smooth muscle actin (αSMA), Smad 2, 3 and 4, CTGF and phospho-Smad2, 3, and 4 were assessed by qPCR and Western blotting.ResultsTGFβ1 stimulation resulted in the decreased phosphorylation of YAP in the cytosol, and increased levels of phosphorylated TAZ and Smad2/3/4 in the nucleus. Knockdown of TAZ resulted in elevated YAP expression but not vice versa. Additionally, knockdown of TAZ but not YAP resulted in upregulation of αSMA expression in the presence and absence of TGFβ1. In the presence of TGFβ1 YAP knockdown increased Smad2/3/4 expression and Smad4 phosphorylation, while TAZ knockdown had no effect on Smad2/3/4 expression and phosphorylation. YAP knockdown inhibited CTGF expression while TAZ knockdown resulted in its increased expression. Finally, simultaneous knockdown of YAP and TAZ resulted in cell death.ConclusionOur findings suggest that YAP and TAZ function as distinct modulators of TGFβ1 induced myofibroblast transformation and have different roles in signalling. Specifically, TAZ limits YAP's ability to mediate KFM transformation via Smad proteins. The data also suggest that while having distinct effects, YAP and TAZ have redundant or combinatorial functions critical to cell survival. These results suggest that a loss of TAZ may help drive corneal haze and fibrosis and that the balance between YAP/TAZ is essential in controlling myofibroblast differentiation