Abstract
Purpose:
It has been long known that there is decreased human trabecular meshwork (HTM) cellularity in glaucoma. The mechanism for this is unknown but senescence is suspected to play a role. We wished to characterize the expression of progerin and secreted frizzled-related protein 1 (SFRP1) in HTM cell senescence. Progerin is a transcript variant of Lamin A which is associated with the accelerated aging diseases as well as normal aging, and has recently been identified as a component of cellular senescence. SFRP1 has recently been associated with glaucoma and other recent work has identified it as necessary and sufficient for the induction and maintenance of senescence.
Methods:
HTM cells from three donors were maintained to terminal passage (13 or 14) in DMEM/F12 + 10% fetal bovine serum. Terminal passage was assessed based on the absence of proliferation in the presence of serum. The cells were split and plated at 1x106 cells per 100 mm dish and cultured for three days in the absence of serum. RNA was isolated using an RNeasy kit and cDNA was synthesized using Maxima First Strand kit according to manufacturer instructions. The relative expression of SFRP1 was quantified from the mRNA by qPCR using using SensiFAST Hi-ROX One-Step mastermix and Taqman primers (Hs00610060_m1). Expression at terminal passage was compared to earlier passages (6-9) for cells isolated from three donors. The relative expression of progerin was quantified from the cDNA using SYBR Green PCR Master Mix with the following primers 5’-ACTGCAGCAGCTCGGGG-3’ (forward) and 5’-GGCTCTGGGCTCCTGAGCC-3’ (reverse). Expression at terminal passage was compared to earlier passage (7) for cells isolated from one donor.
Results:
Terminal passage SFRP1 expression was 2.12±0.30, 4.91±0.22, and 5.52±0.40 greater for cells from the three donors when normalized to an earlier passage. Terminal passage progerin was increased 21.86±0.42 fold when normalized to an earlier passage.
Conclusions:
Typical of other primary cultures, HTM cells will senesce in vitro after extended culture. Late passage cultures have elevated mRNA expression of SFRP1 and progerin, suggesting these play a role in establishing or maintaining the senescent phenotype in HTM cells.
Keywords: 735 trabecular meshwork •
493 cytoskeleton •
413 aging