Abstract
Purpose :
Primary open-angle glaucoma (POAG) is associated with mechanical changes in the trabecular meshwork, leading to increased aqueous humor (AH) outflow resistance. The role of Notch signaling, an evolutionarily conserved pathway implicated in mechanotransduction, has not been investigated in glaucoma pathogenesis. Here, we determine the expression of Notch pathway molecules in TM cells subjected to cyclical strain or when grown on hydrogels of varying rigidity.
Methods :
Primary human total TM (hTM) cells were isolated and validated from donor corneal rings. hTM cells (3-7 passages) were plated on collagen coated 3 kPa or 80 kPa hydrogels for 72hrs with 10% FBS containing Ham’s F12 medium. In cyclical strain experiments, hTM cells were cultured on collagen coated PDMS stretch chambers and subjected to uniaxial 20% strain for 24 h at 1Hz. mRNA and protein levels of Notch receptors, ligands, and effectors in the hTM cells was determined by RT-qPCR or western blot.
Results :
On stiffer hydrogels, expression of Notch receptors 1(0.6-fold), 2(0.8-fold) 3(0.8-fold) or 4(0.3-fold) was significantly decreased. While expression of Notch ligand Jagged1 was similar on both substrates, expression of Jagged2 (0.4-fold), DLL1 (0.8-fold), 3 (0.8-fold) and 4 (0.3-fold) were all down-regulated. Correspondingly, downstream targets of Notch pathway, Hes3, 5 (0.3, 0.3-fold) or Hey1, 2 (0.4, 0.4-fold) were decreased. In cyclic stretch, expression of Notch receptors 1(0.8-fold), 3(0.9-fold) decreased, whereas Notch2(1.17-fold) increased. While expression of Notch ligands Jagged1 (0.9-fold), Jagged2 (0.8-fold), DLL1 (0.8-fold), 4 (0.8-fold) was down regulated. DLL3 (1.2-fold) was upregulated. Congruently, effectors of Notch pathway, Hes3 (1.2-fold) and 5 (1.2-fold) increased, and Hey1 (0.8-fold) was decreased, compared to non-stretched strains.
Conclusions :
Our data demonstrates that Notch signaling in hTM cells differs by mechanical insult. Notably, where substratum stiffness elicited maximal intrinsic differences, cyclical strain did not. These results imply: (i) The different Notch components may have distinct mechanotransduction roles to maintain tissue homeostasis. (ii) Notch signaling may be temporal in cell fate determination. Overall, these findings warrant further studies on the exact role of Notch signal in human trabecular meshwork in health and disease.
This is a 2021 ARVO Annual Meeting abstract.