Abstract
Purpose: :
We have previously shown that Cross-linked Actin Networks (CLANs) can be induced by aqueous humor and TGF-β2 in vitro and have been identified in human trabecular meshwork (TM) tissue. Although CLANs occur more frequently in glaucomatous TM tissue ex vivo, they are also present in the TM of normal donors. In this study we wish to determine whether CLANs are an age-related feature of TM cells.
Methods: :
The TM was dissected from fixed human globes prior to staining with Alexa-phalloidin-488 and propidium iodide. Z-series’ images were captured via confocal microscopy and used to analyse CLAN incidence through the tissue.Primary cultures of confluent bovine TM (BTM) cells were treated with sub-toxic levels of hydrogen peroxide (200, 600, 800μM) for 1hour, which was then removed, prior to 7 days treatment with either media containing 1% fetal calf serum (FCS) or media containing 1% FCS plus 2ng/ml TGF-β2.In further experiments confluent cultures of primary human TM (HTM) cells, from 13 donors of various ages (0-91 years), were treated for 7 days, with either media containing 1% FCS or with 2ng/ml TGF-β2 added.Cultured cells were fixed, stained with Alexa-phalloidin-488 and propidium iodide and imaged using fluorescence microscopy. HTM cells were also assessed for senescence via β-galactosidase (β-gal) staining.
Results: :
Analysis of TM tissue revealed a variable CLAN to nuclei ratio (0.2-1.16) across the age range available (35-94yrs) with no clear age association. β-gal staining also showed no significant association with CLAN presence. 13.4% of β-gal negative cells contained a CLAN compared to 8% of β-gal positive cells.A baseline CLAN incidence in BTM cells in our 1% FCS culture media was 6-12%. Pre-treatment with hydrogen peroxide significantly increased CLAN incidence under these conditions producing an average of 32%. However, if hydrogen peroxide pre-treated cells were exposed to TGF-β2 there was a dose dependent response resulting in CLAN incidence of up to 54%.CLAN incidence in HTM cells in media containing 1% FCS ranged from 0.2-7% but had no age-relationship. Treatment with TGF-β2 showed a significant increase in CLAN incidence (35-47%) in cells from older donors (51-83yrs) which was not so evident in donors >10yrs (12-20%).
Conclusions: :
In tissue CLAN incidence does not correlate directly with age, but cultures show CLAN are influenced by stress such as that induced by hydrogen peroxide. Older cells having depleted protective mechanisms may be more susceptible to such stress, thus making them more prone to CLAN induction by factors, such as TGF-β2 in the aqueous humor. In glaucoma the up-regulation of TGF-β2 may explain the high CLAN incidence in glaucomatous tissue ex vivo.
Keywords: trabecular meshwork • cytoskeleton • aging