Abstract
Purpose :
To determine the effects of mechanical strain on the actin cytoskeleton and components of the extracellular matrix (ECM) in primary optic nerve head astrocytes (ONHAs).
Methods :
Primary rat ONHA cultures were exposed to 10% static equibiaxial strain in a FlexCell® (FlexCell International) chamber for 16 h, under serum-deprived (0.1% FBS) and serum-containing (20% FBS) conditions. Actin cytoskeleton was labeled with AlexaFluor® 488 phallodin and AlexaFluor® 594 DNAse I for F-actin and G-actin, respectively. Lengths of stress fibers was quantified using Matlab software
(MathWorks, Natick, MA). Quantitative PCR, immunoblotting and immunocytochemistry for components of the elastin biosynthesis pathway were performed using previously validated primers and antibodies. Expression levels of the ECM proteins elastin, fibulin-2, fibulin-5, fibrillin-1, collagen VI-a1 and lysyl oxidase-like 1 (Loxl1) were quantified.
Results :
Mechanical strain resulted in a decrease in stress fiber length with a concomitant increase in G-actin immunoreactivity, suggestive of reactive astrocytosis. Furthermore, mechanical strain resulted in a significant decrease in elastin (65% reduction, P < 0.05) and Loxl1 (35% reduction, P < 0.001) expression under serum-deprived conditions. Similar results were obtained from serum-containing cultures. Expression levels of fibulin 2 (P = 0.29) and fibulin 5 (P = 0.63) were unaltered after exposure to mechanical strain and not affected by the presence of serum (P = 0.50 and P = 0.23, respectively).
Conclusions :
Exposure to mechanical strain and Loxl1 knockdown elicited significant remodeling of the actin cytoskeleton and decreases in elastin and Loxl1. Intriguingly, similar molecular signatures of reactive astrocytosis were observed following partial Loxl1 knock-down in ONHAs to mimic exfoliation glaucoma. Our data tentatively suggest a conserved mechanism of cellular elastinopathy underlying multiple subtypes of glaucoma.
This is a 2020 ARVO Annual Meeting abstract.