Viral Vector Effects on Exoenzyme C3 Transferase-Mediated Actin Disruption and on Outflow Facility.

In the human eye, the main pathway of aqueous humor outflow from the anterior chamber involves filtering through intercellular spaces in the trabecular meshwork before reaching Schlemm's canal, which drains into the aqueous veins.1 Increased resistance to outflow through this pathway results in an increase in IOP, often leading to development of open-angle glaucoma. Cellular relaxation and other architectural modifications to the trabecular meshwork that reduce its stiffness and increase intercellular spaces have the potential to facilitate outflow and alleviate chronically elevated IOP.2 Rho signaling pathways regulate the assembly and contractility of the actomyosin network, and Rho guanosine triphosphatases (GTPases) specifically regulate actin dynamics, actomyosin contractility, and cellular adhesions.3 The Clostridium botulinum exoenzyme C3 transferase (C3) inactivates the Rho GTPases RhoA, RhoB, and RhoC by adenosine diphosphate (ADP)–ribosylation, resulting in disruption of actin stress fibers, cellular relaxation, and loss of focal adhesions.4 We previously showed the ability of adenoviral (AdV)–mediated delivery of C3 to the trabecular meshwork by intracameral injection to increase outflow facility in monkey organ cultured anterior segments (MOCAS).5 Transduction of differentiated human trabecular meshwork (HTM) cell monolayers with AdV.C3 resulted in a loss of stress fibers as observed by actin fluorescence. By phase contrast imaging, cells treated with AdV.C3 appeared to be partially retracted or rounded up compared to cells treated with media or control AdV.5 Interestingly, it has been shown previously that purified C3 protein is taken up by cells, resulting in a physiological effect.6,7 The uptake mechanism for C3 toxin is thought to be by a specific endocytic mechanism involving acidified endosomal vesicles.8 The fact that purified C3 is taken up by cells afforded us the opportunity to separate the effect of C3 expression in TM cells from the potential cytoskeletal altering activities of viral vectors. Both AdV and lentiviral infection involve temporary rearrangement of the actin cytoskeleton.9 The AdV receptor (CAR)10 or CD4611 serve as the major primary human AdV (HAdV) receptors on most cell types. Subsequent AdV internalization via clathrin-mediated endocytosis is facilitated by secondary interactions of the penton base with αv integrins.12 This event is accompanied by partial disassembly of the virus particle in which the vertex proteins (i.e., penton base, pIIIa, fiber, peripentonal hexons) are released in the acidified environment of the early or late endosome. The precise molecular events involved in HAdV disassembly are not very well defined, but it is thought that the release of the internal capsid protein pVI at this stage facilitates endosomalysis13 allowing entry of the uncoated particle into the cytoplasm. Entry of VSV-G envelope pseudotyped lentiviral vectors activates the PI3 kinase pathway, which also results in alterations in the actin cytoskeleton.14 Thus, the viral vectors themselves could be having a role in the actin disrupting activity of C3 transferase. The current studies sought to determine whether or not coadministration of viral vectors with recombinant proteins could alter the delivery and cell uptake of potential therapeutic proteins in the eye. We determined in primary differentiated HTM cells if cotransduction of recombinant C3 (C3rec) with AdV or lentivirus vectors enhanced the effect of C3rec on the actin cytoskeleton. Two different vectors were used to determine if the effects were vector-specific. In MOCAS, we determined whether coadministration of C3rec and AdV altered outflow facility.