Lovastatin inhibits the thrombin-induced loss of barrier integrity in bovine corneal endothelium.
OBJECTIVE
Increased actomyosin contraction of the dense band of actin cytoskeleton at the apical junctional complex (perijunctional actomyosin ring, PAMR) breaks down the barrier integrity of corneal endothelium. This study has investigated the efficacy of statins, which inhibit activation of RhoA, in opposing the thrombin-induced loss of barrier integrity of monolayers of cultured bovine corneal endothelium.


METHODS
Myosin light chain (MLC) phosphorylation, a biochemical measure of actomyosin contraction, was assayed by urea-glycerol gel electrophoresis, followed by western blot analysis. The locus of MLC phosphorylation and changes in the organization of the PAMR were visualized by immunostaining. Phosphorylation of MYPT1, a regulatory subunit of myosin light-chain phosphatase (MLCP), was assessed by Western blot analysis to determine down-regulation of RhoA. The barrier integrity was assessed in terms of trans-endothelial electrical resistance (TER), and further confirmed by determining permeability to FITC dextran (10 kDa) and distribution of ZO-1, a marker of tight junctional assembly.


RESULTS
Lovastatin, a prototype of lipophilic statins, induced MLC dephosphorylation under basal conditions. It opposed increase in phosphorylation of MLC and MYPT1 in response to thrombin and nocodazole, agents known to activate RhoA in the endothelium. Pretreatment with the statin opposed the thrombin- and nocodazole-induced disruption of the PAMR and the thrombin-induced decline in TER. Lovastatin also opposed the thrombin- and nocodazole-induced increase in permeability to FITC dextran and redistribution of ZO-1. However, upon supplementation with GGPP (geranylgeranyl pyrophosphate), lovastatin failed to oppose the effects of thrombin and nocodazole on the PAMR, ppMLC, and ZO-1 distribution.


CONCLUSIONS
Lovastatin attenuates RhoA activation in the corneal endothelium presumably by reducing its isoprenylation. This underlies the suppression of the thrombin-induced loss in barrier integrity of the corneal endothelium.
School of Optometry, Indiana University, Bloomington, Indiana 47405, USA.
Mahesh
M
Shivanna
Supriya S
SS
Jalimarada
Sangly P
SP
Srinivas
D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000187:drug effects; D004728:Endothelium, Corneal / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors / Q000494:pharmacology*; D008148:Lovastatin / Q000494:pharmacology*; D018994:Myosin Light Chains / Q000378:metabolism; D043263:Myosin-Light-Chain Phosphatase / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D013917:Thrombin / Q000494:pharmacology*; D019108:Tight Junctions / Q000187:drug effects*; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D018994:Myosin Light Chains; D008148:Lovastatin; D043263:Myosin-Light-Chain Phosphatase; D013917:Thrombin; D020742:rhoA GTP-Binding Protein