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Lovastatin inhibits the thrombin-induced loss of barrier integrity in bovine corneal endothelium.

ABSTRACT: 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.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.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.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.

SUBMITTER: Shivanna M

PROVIDER: S-EPMC3096541 | biostudies-other | 2010 Feb

REPOSITORIES: biostudies-other

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Project description:PurposeThe purpose of the study was to clarify the interplay between metabolites and microRNAs (miRs) in the aqueous humor (AqH) of bullous keratopathy (BK) patients to retain human corneal endothelium (HCE) integrity.DesignProspective, comparative, observational study.ParticipantsA total of 55 patients with BK and 31 patients with cataract (Cat) as control.MethodsA biostatic analysis of miRs and metabolites in the AqH, hierarchical clustering, and a least absolute shrinkage and selection operator (Lasso) analysis were employed. The miR levels in AqH of BK (n = 18) and Cat (n = 8) patients were determined using 3D-Gene human miR chips. Hierarchical clusters of metabolites detected by liquid chromatography-mass spectrometry or gas chromatography-mass spectrometry in AqH specimens from 2 disease groups, BK (total n = 55) and Cat (total n = 31), were analyzed twice to confirm the reproducibility. The analytical procedure applied for investigating the association between metabolites and miRs in AqH was the exploratory data analysis of biostatistics to avoid any kind of prejudice. This research procedure includes a heat-map, cluster analysis, feature extraction techniques by principal component analysis, and a regression analysis method by Lasso. The cellular and released miR levels were validated using reverse transcription polymerase chain reaction and mitochondria membrane potential was assessed to determine the functional features of the released miRs.Main outcome measuresIdentification of interacting metabolites and miRs in AqH attenuating HCE degeneration.ResultsThe metabolites that decreased in the AqH of BK patients revealed that 3-hydroxyisobutyric acid (HIB), 2-aminobutyric acid (AB) and branched-chain amino acids, and serine were categorized into the same cluster by hierarchical clustering of metabolites. The positive association of HIB with miR-34a-5p was confirmed (P = 0.018), and the Lasso analysis identified the interplay between miR-34a-5p and HIB, between miR-24-3p and AB, and between miR-34c-5p and serine (P = 0.041, 0.027, and 0.009, respectively). 3-hydroxyisobutyric acid upregulated the cellular miR-34a expression, mitochondrial membrane potential, and release of miR-184 in dedifferentiated cultured HCE cells.ConclusionsMetabolites and miRs in AqH may synchronize in ensuring the integrity of the HCE to maintain efficient dehydration from the stroma.Financial disclosuresProprietary or commercial disclosure may be found after the references.

Project description:Vascular endothelial cells are crucial mediators of inflammation during infectious diseases, due to their ability to produce lipid-based inflammatory mediators and facilitate leukocyte migration and translocation to infected tissues. Mastitis is the costliest infectious disease in North America, with over two billion dollars in annual costs due to loss of milk production, medical treatment, and potential loss of the animal. Infections caused by coliform bacteria are particularly deleterious, causing a negative impact on cow well-being and a high mortality rate. Dysfunction and breakdown of the endothelial barrier is a key part of the pathology of coliform mastitis. The endocannabinoid system (ECS), shown to modulate inflammatory responses of vascular endothelial cells in humans and rodents, may be a novel target for inflammatory modulation in dairy cows. The endocannabinoid (EC) arachidonoylethanolamide (AEA) is a potent anti- or pro-inflammatory mediator in endothelial cells, depending on location, timing, and concentration. We hypothesized that elevated AEA during LPS challenge will impair endothelial barrier integrity via increased production of reactive oxygen species (ROS) and activation of apoptotic pathways. Challenge of bovine aortic endothelial cells (BAEC) with 25 ng/mL lipopolysaccharide (LPS) for 8 h induced AEA synthesis, increased expression of cannabinoid receptor 1 and 2 (CB1/2) and the AEA synthesizing enzyme N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD), while decreasing gene expression of the AEA degradation enzyme fatty acid amide hydrolase (FAAH). Trans endothelial resistance (TER), measured through electrical resistance across the monolayer, increased 2 h after 0.5 µM AEA treatment and decreased with 5 µM AEA, compared to LPS alone. Addition of AEA to BAEC challenged with LPS induced mitochondrial dysfunction via increased ROS production, cytochrome-C release, and activation of caspase 3/7. Antagonism of CB1 by 1 µM AM251 ameliorated AEA induced ROS production and cytochrome-C release. Addition of AM251 also eliminated 2 h TER increase and improved TER following 5 µM AEA. Doses of 0.5, 1, and 5 µM AEA delayed endothelial barrier recovery, which was eliminated by the addition of AM251. Mitochondrial dysfunction and activation of apoptotic pathways in response to AEA treatment during LPS challenge of BAEC may act to delay inflammatory resolution and contribute to endothelial dysfunction.

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Lovastatin inhibits the thrombin-induced loss of barrier integrity in bovine corneal endothelium.

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