Expression of dominant negative Rho-binding domain of Rho-kinase in organ cultured human eye anterior segments increases aqueous humor outflow.
OBJECTIVE
Based on pharmacological inhibition of activity, a role has been proposed for Rho-kinase in the modulation of aqueous outflow and intraocular pressure (IOP). This study employed a molecular approach to specifically address the role of Rho-kinase in the modulation of aqueous humor outflow.


METHODS
Adenoviral vectors expressing green fluorescent protein alone (Ad-GFP) or the dominant negative Rho-binding domain of Rho-kinase and GFP (Ad-DNRK-GFP) were utilized in these experiments. Human and porcine primary trabecular meshwork (TM) cells were infected with 30 MOI (multiplicity of infection) of Ad-GFP alone or with Ad-DNRK-GFP. Changes in cell shape, actomyosin cytoskeletal integrity, and the status of myosin light chain (MLC) phosphorylation were evaluated. The aqueous outflow facility was determined in organ cultured anterior segments of human cadaver eyes infected with 10(7) pfu adenoviral vectors (Ad-GFP or Ad-DNRK-GFP) using a constant flow perfusion system.


RESULTS
Expression of DNRK resulted in changes in cell shape (cell rounding, cell-cell detachment) and decreased actin stress fiber and focal adhesion staining in TM cells. These cellular changes were associated with substantially reduced myosin light chain phosphorylation. Additionally, organ cultured human eye anterior segments infected with Ad-DNRK-GFP exhibited a significant increase in the outflow facility (80%, n=9) compared to control anterior segments infected with Ad-GFP (5%).


CONCLUSIONS
This study demonstrated that specific inhibition of Rho-kinase activity in trabecular meshwork cells led to alterations in cell shape and presumed contractile properties, and we hypothesize that these morphological and contractile events underlie the observed effects of dominant negative Rho-kinase on the aqueous humor outflow facility. These data provide molecular evidence for the hypothesis of Rho-kinase being a potential cellular target involved in the regulation of aqueous humor outflow resistance, with implications for novel glaucoma therapy.
Rao
Ponugoti Vasantha
PV
Department of Ophthalmology, Duke University School of Medicine, Durham, NC 27710, USA. rao00011@mc.duke.edu
Deng
PeiFeng
P
Maddala
Rupalatha
R
Epstein
David L
DL
Li
Chuan-Yuan
CY
Shimokawa
Hiroaki
H
D000199:Actins / Q000378:metabolism; D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D001082:Aqueous Humor / Q000378:metabolism*; D001665:Binding Sites; D048430:Cell Shape; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008856:Microscopy, Fluorescence; D009218:Myosins / Q000378:metabolism; D009924:Organ Culture Techniques; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics*; D013552:Swine; D014129:Trabecular Meshwork / Q000166:cytology / Q000378:metabolism*; D020741:rho GTP-Binding Proteins / Q000235:genetics*; D054460:rho-Associated Kinases
D000199:Actins; D047908:Intracellular Signaling Peptides and Proteins; D049452:Green Fluorescent Proteins; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D009218:Myosins; D020741:rho GTP-Binding Proteins