Cyclosporine A--protection against microvascular hyperpermeability is calcineurin independent

Abstract

Background: Mitochondria-mediated apoptotic signaling contributes to microvascular hyperpermeability. We hypothesized that cyclosporine A (CsA), which protects mitochondrial transition pores, would attenuate hyperpermeability independent of its calcineurin inhibitory property.

Methods: Hyperpermeability was induced in microvascular endothelial cell monolayers using proapoptotic BAK or active caspase-3 after CsA or a specific calcineurin inhibitor, calcineurin autoinhibitory peptide (CIP), treatment. Permeability was measured based on fluorescein isothiocyanate-albumin flux across the monolayers. Mitochondrial transmembrane potential (MTP) was determined using 5,5',6,6'-tetrachoro-1,1',3,3'-tetraethylbenzimidazolyl carbocyanine iodide. Mitochondrial release of cytochrome c was measured using an enzyme-linked immunosorbent assay and caspase-3 activity fluorometrically.

Results: CsA-attenuated (10 nmol/L) but not CIP-attenuated (100 mumol/L) BAK induced hyperpermeability (P < .05), CsA- but not CIP-attenuated BAK induced a decrease in MTP and an increase in cytochrome c levels and caspase-3 activity (P < .05). CsA and CIP were ineffective against caspase-3-induced hyperpermeability.

Conclusions: CsA attenuated hyperpermeability by protecting MTP, thus preventing mitochondria-mediated apoptotic signaling. The protective effect of CsA is independent of calcineurin inhibition.

Figure 1

Cyclosporine A (CsA) attenuates BAK-induced hyperpermeability in rat lung microvascular endothelial cell monolayers whereas calcineurine specific inhibitor CIP shows no significant effect. Change in permeability is expressed as percentage of the basal fluorescence. (A). BAK transfection induced hyperpermeability in the monolayer compared to control (*p < 0.05; n = 5). CsA (10 nM) pre-treatment in BAK-transfected cells showed decrease in FITC-albumin fluorescence compared with untreated cells (†p < 0.05; n = 5). (B). BAK transfection induced hyperpermeability in the monolayer compared to control (*p < 0.05; n = 5). CIP (100μM) treatment showed no significant effect on BAK-induced hyperpermeability.

Figure 2

CsA or CIP showed no significant effect on active caspase-3 induced-hyperpermeability in rat lung microvascular endothelial cell monolayers. Active caspase-3 transfection induced hyperpermeability in the monolayer compared to control (*p < 0.05; n = 5; A, B). CsA (10 nM) or CIP (100μM) pre-treatment in active acspase-3 transfected cells showed no significant change in FITC-albumin fluorescence compared with untreated cells (A, B).

Figure 3

CsA protects mitochondrial membrane integrity in rat lung microvascular endothelial cells (RLMEC). Fluorescence microscopy images of the mitochondrial membrane potential indicator JC-1 in its monomeric (green) and dimeric (red) forms are shown. BAK (BH3) transfection leads to the collapse of mitochondrial membrane potential showing predominantly monomeric forms. CsA (10nM) treatment prevents the collapse of mitochondrial membrane potential evidenced by the restoration of dimeric form-red fluorescence.

Figure 4

CIP shows no protective effect on mitochondrial membrane integrity in rat lung microvascular endothelial cells (RLMEC). Fluorescence microscopy images of the mitochondrial membrane potential indicator JC-1 in its monomeric (green) and dimeric (red) forms are shown. BAK (BH3) transfection leads to the collapse of mitochondrial membrane potential showing predominantly monomeric forms. CIP (100μM) treatment did not prevent the collapse of mitochondrial membrane potential evidenced by the absence of dimeric form-red fluorescence.

Figure 5

CsA treatment prevents cytochrome c release to the cytoplasm in rat lung microvascular endothelial cells (RLMEC) whereas CIP treatment shows no effect. Cytosolic cytochrome c levels are increased following BAK (BH3) transfection compared with control (*p < 0.05; n = 5). CsA pre-treatment in BAK (BH3) transfected cells shows significant decrease in cytochrome c levels compared with BAK (BH3) transfected cells without CsA pre-treatment or with CIP treated-BAK transfected cells (†p < 0.05; n = 5).

Figure 6

CsA inhibits pro-apoptotic BAK (BH3) peptide induced caspase-3 activity, in rat lung microvascular endothelial cells (RLMEC) whereas CIP treatment shows no significant effect. BAK (BH3) transfection significantly increased caspase-3 activity compared with control cells (*p < 0.05; n = 5). CsA pre-treatment in BAK (BH3) transfected cells shows significant decrease in caspase-3 activity compared with BAK (BH3) transfected cells without CsA pre-treatment or with CIP treated-BAK transfected cells (†p < 0.05; n = 5).