Abstract
Purpose :
Elevated TGFβ2 in the aqueous humor is one of the most studied biomarkers of glaucoma. TGFβ2 induces excessive extracellular matrix proteins as well as the formation of cross-linked actin networks (CLANs) in trabecular meshwork (TM) cells. CLANs are complex web-like structures. Our previous studies identified four actin-associated proteins including caldesmon, calponin, tropomyosin and myosin light chain which are enriched in CLANs. In this study, we determined if these proteins promote CLAN formation in the TM.
Methods :
Lentiviral vectors were constructed to express fusion proteins of caldesmon/calponin/ myosin light chain/tropomyosin+GFP. Primary human TM (HTM) cells were cultured close to confluency and transduced with different lentiviral vectors or a control vector (GFP only) at an MOI of 1:125 in triplicates (N=3) in 24-well plates. The cells were cultured up to 2 months. Some HTM cells were treated with 5ng/ml TGFβ2 as a positive control. At the end of the study, the cells were fixed and stained with phalloidin-Alexa-568 and DAPI for actin filaments and nuclei, respectively. To quantitate the number of CLAN-positive cells, ten fluorescent images per well were captured. The total number of fluorescent positive cells per image was counted and the percentage of CLAN-positive cells over total cell numbers per image (PCPC) per well was determined. PCPCs were compared using one-way ANOVA and post-hoc tests.
Results :
The baseline level of PCPC in the GFP overexpression group was 4.3% ±0.2%. In comparison, there was a 7-fold increase in PCPC (38.0% ±2.0%; P<0.001) in TGFβ2 treated cells. There was about 2-fold increase of PCPC in the myosin light chain group (7.3% ±1.5%; P>0.05) and tropomyosin group (8.5% ±3.8%, P>>0.05), about 4-fold increase in the caldesmon group (19.3% ±2.5%; P<0.05), and about 6-fold increase in calponin (26.0% ±4.0%; P<0.01). The greatest increase was observed in the cells overexpressing all four proteins with about 11-fold increase in PCPC (51.0% ±14.2%; P<0.001), and this increase was higher than that in TGFβ2-treated cells (P<0.01).
Conclusions :
Caldesmon, calponin, tropomyosin and myosin light chain work individually or synergistically to promote CLAN formation in primary HTM cells.
This is a 2021 ARVO Annual Meeting abstract.