Effect Of Rat Non–muscle Caldesmon Overexpression On The Cytoskeleton Of Human Trabecular Meshwork Cells.

Abstract

Abstract: : Purpose: Non–muscle caldesmon interferes with the formation of focal adhesions and actin stress fiber assembly. Because drugs that disrupt the actin cytoskeleton and/or focal adhesions of human trabecular meswork cells (HTM) are known to increase aqueous humor outflow facility, we investigated the possibility of altering the HTM cytoskeleton by using caldesmon gene transfer. Methods:A recombinant replication–deficient adenovirus carrying the linked coding cDNAs of GFP and non–muscle rat caldesmon genes was constructed by homologous recombination (AdGFPCald). Expression of the fused protein was driven by the promoter–enhancer sequence CMV5 (Qbiogene). Primary HTM cells grown on coverslips were infected with AdGFPCald at different multiplicities of infection, fixed and assayed by immunofluorescence staining of cytoskeletal proteins 24–48 h post–infection. SV40–transformed HTM cells were plated in glass bottom dishes, AdGFPCald infected, and examined by live time–lapse recording with an Axiovert 100 TV microscope. Results:Caldesmon co–localized with all actin–containing structures. High caldesmon overexpression induced severe changes in the actin cytoskeleton and formation of new type of actin structures such as curvy fiber networks. In these cells, focal adhesions were disrupted. HTM cells containing lower levels of recombinant caldesmon induced different and milder changes with shorter stress fibers and triangular structures. Real–time GFP–caldesmon dynamics showed motile curvy fibers undergoing continuous remodeling (fusion, formation of loops etc). Myosin staining in infected and control cells remained unchanged. Conclusions:Recombinant caldesmon induced changes in the HTM cytoskeleton in a dose–dependent manner. Modulation of caldesmon expression in the human trabecular meshwork could potentially be used as a therapy tool to increase aqueous humor outflow facility and reduce intraocular pressure in glaucoma. JLV current affiliation: Inspire Pharmaceuticals, Inc., Durham, NC.

Keywords: trabecular meshwork • gene transfer/gene therapy • cytoskeleton