Abstract
Purpose: :
There is considerable evidence that the acto-myosin system in the human trabecular meshwork (HTM) plays an important role in modulating trabecular outflow resistance. The information on endogenous factors that modulate contractility and architecture of the HTM actin cytoskeleton is incomplete. Connective tissue growth factor (CTGF) is expressed at high amounts in HTM cells in situ. Recently, we showed that CTGF is a potent inducer of extracellular matrix in HTM cells. In the present study, we analyzed, if the changes in HTM biology induced by CTGF do also affect their actin cytoskeleton.
Methods: :
Primary human TM cells were treated with CTGF at different concentrations (5-100ng/ml). Changes in the expression and distribution of cytoskeletal proteins were examined by quantitative real-time RT-PCR, western blotting and immunohistochemistry. Cells of an immoratlized human TM cell line (HTM5) cells were stable transfected with a pSilencer(siCTGF)-Vector. Subsequently, the actin cytoskeleton of HTM5-siCTGF cells was compared to that of control HTM5 cells under normal conditions and after stress (heat-shock).
Results: :
CTGF treatment of HTM cells caused an increase of -smooth-muscle actin, -actinin, B-crystallin and their respective mRNAs. In addition, by immunohistochemistry, more and thicker actin stress fibres were observed that contained increased amounts of the -smooth-muscle actin isoform. In addition, the number of focal contacts was increased considerably. The knock-down of CTGF in HTM5-siCTGF cells caused a dramatic decrease in the number of focal contacts as compared to untreated HTM5 cells. Stress applied as heat shock to HTM5 cells substantially induced the expression of -actinin, of the small heat shock protein B-crystallin that is known to bind to actin, and caused a marked increase in the number of actin stress fibers. Similar effects were completely absent in HTM5-siCTGF cells.
Conclusions: :
CTGF is a potent mediator of the actin cytoskeleton in HTM cells. Together with the inducing effects of CTGF on extracellular matrix synthesis in HTM cells, CTGF might lead to an increased stiffness of HTM cells. An increase in HTM stiffness might contribute to an increase in HTM outflow resistance and POAG .
Keywords: trabecular meshwork • cytoskeleton • growth factors/growth factor receptors