Endothelial cell barrier protection by simvastatin: GTPase regulation and NADPH oxidase inhibition - PubMed (original) (raw)

Endothelial cell barrier protection by simvastatin: GTPase regulation and NADPH oxidase inhibition

Weiguo Chen et al. Am J Physiol Lung Cell Mol Physiol. 2008 Oct.

Abstract

The statins, hydroxy-3-methylglutaryl-CoA reductase inhibitors that lower serum cholesterol, exhibit myriad clinical benefits, including enhanced vascular integrity. One potential mechanism underlying increased endothelial cell (EC) barrier function is inhibition of geranylgeranylation, a covalent modification enabling translocation of the small GTPases Rho and Rac to the cell membrane. While RhoA inhibition attenuates actin stress fiber formation and promotes EC barrier function, Rac1 inhibition at the cell membrane potentially prevents activation of NADPH oxidase and subsequent generation of superoxides known to induce barrier disruption. We examined the relative regulatory effects of simvastatin on RhoA, Rac1, and NADPH oxidase activities in the context of human pulmonary artery EC barrier protection. Confluent EC treated with simvastatin demonstrated significantly decreased thrombin-induced FITC-dextran permeability, a reflection of vascular integrity, which was linked temporally to simvastatin-mediated actin cytoskeletal rearrangement. Compared with Rho inhibition alone (Y-27632), simvastatin afforded additional protection against thrombin-mediated barrier dysfunction and attenuated LPS-induced EC permeability and superoxide generation. Statin-mediated inhibition of both Rac translocation to the cell membrane and superoxide production were attenuated by geranylgeranyl pyrophosphate (GGPP), indicating that these effects are due to geranylgeranylation inhibition. Finally, thrombin-induced EC permeability was modestly attenuated by reduced Rac1 expression (small interfering RNA), whereas these effects were made more pronounced by simvastatin pretreatment. Together, these data suggest EC barrier protection by simvastatin is due to dual inhibitory effects on RhoA and Rac1 as well as the attenuation of superoxide generation by EC NADPH oxidase and contribute to the molecular mechanistic understanding of the modulation of EC barrier properties by simvastatin.

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Figures

Fig. 1.

Fig. 1.

Endothelial cell (EC) barrier protection and cytoskeletal rearrangement by simvastatin. A: EC grown on Transwell filters were stimulated with thrombin (1 U/ml, 1 h) before measurements of FITC-dextran permeability. Pretreatment with simvastatin (5 μM) confers time-dependent protection with a significant decrease in thrombin-induced permeability evident within 6 h (*P < 0.05) and more pronounced at 16 h (**P < 0.05, n = 3 for each condition). B: immunofluorescent imaging of confluent EC monolayers demonstrates evidence of early cytoskeletal rearrangement after treatment with simvastatin alone (5 μM) characterized by decreased transcellular actin stress fibers and enhanced peripheral actin evident as early as 2 h. These effects are associated with a dramatic decrease in the number of appreciable paracellular gap and are even more pronounced at 16 h.

Fig. 2.

Fig. 2.

Role of Rho inhibition in EC barrier protection by simvastatin (simva). Thrombin-induced (1 U/ml, 1 h) FITC-dextran translocation across EC monolayers was measured in control cells as well as subsequent to pretreatment with either simvastatin (5 μM, 16 h), a pharmacological Rho kinase inhibitor, Y-27632 (10 μM, 30 min), or a combination of both (n = 3 for each condition). Y-27632 effects a marked decrease in thrombin-induced EC permeability that is significantly augmented with the coadministration of simvastatin (*P < 0.05). In addition, the protection conferred by simvastatin alone is significantly augmented by Y-27632 (**P < 0.05). Basal permeability did not significantly differ among EC monolayers treated with simvastatin, Y-27632, or the combination of both.

Fig. 3.

Fig. 3.

Effect of simvastatin on LPS-induced EC permeability and cytoskeletal rearrangement. A: compared with untreated controls, EC monolayer permeability is significantly increased in response to LPS (1 μg/ml, 1.5 h) as measured by FITC-dextran translocation (*P < 0.05). Independently, simvastatin pretreatment (5 μM, 16 h) and Rho kinase inhibition (Y-27632, 10 mM, 30 min) both significantly attenuated LPS-induced (1 μg/ml, 1.5 h) EC barrier disruption (**P < 0.05 and †P < 0.05) (n = 3 for each condition). EC barrier protection by simvastatin was abrogated by concomitant treatment with geranylgeranyl pyrophosphate (GGPP; 10 μM, 16 h). B: EC monolayer protection by simvastatin corresponds to early evidence of decreased LPS-induced (1 μg/ml, 1.5 h) paracellular gaps by immunofluorescence (white arrows) in simvastatin-treated EC (5 μM, 16 h).

Fig. 4.

Fig. 4.

Rac and p47phox localization in response to simvastatin and effect of simvastatin on superoxide-induced EC barrier dysfunction. A: simvastatin pretreatment (5 μM, 16 h) induces a marked decrease in EC membrane Rac1 content concomitant with a pronounced increase in cytosolic Rac1, effects that are independent of LPS treatment (*P < 0.05). B: p47phox levels are similarly redistributed in response to simvastatin (decreased at the membrane and increased in the cytosol), again independent of LPS treatment (*P < 0.05). C: compared with control cells, superoxide generation in LPS-treated (100 ng/ml, 6 h) EC is associated with a marked increase in superoxide production as measured by dihydroethidium (DHE) fluorescence. Comparable to the effects observed with SOD (150 U/ml, 6 h), LPS-induced superoxide production is significantly attenuated by simvastatin (5 μM, 16 h) pretreatment (*P < 0.05). D: Superoxide-induced EC barrier disruption by concomitant treatment with xanthine (X; 200 μM, 1 h) and xanthine oxidase (XO; 30 mU/ml, 1 h) was significantly increased compared with controls as measured by FITC-dextran monolayer permeability (*P < 0.05). (n = 3 for each condition).

Fig. 5.

Fig. 5.

Role of geranylgeranylation inhibition in simvastatin-mediated EC effects. A: comparable to the effects of Diphenyleneiodonium (DPI; 10 μM, 16 h), simvastatin pretreatment (5 μM, 16 h) significantly attenuates LPS-induced (100 ng/ml, 6 h) superoxide generation (*P < 0.05 and **P < 0.05). However, compared with untreated EC, there is not a significant difference when cells are concomitantly treated with simvastatin (5 μM, 16 h) and GGPP (10 μM, 16 h). B: GGPP (10 μM, 16 h) attenuates the cellular redistribution of Rac1 induced by simvastatin (5 μM, 16 h) as measured by densitometry of Western blots (*P < 0.05). C: the attenuation of LPS-induced (100 ng/ml, 6 h) superoxide generation by simvastatin (5 μM, 16 h) is comparable to effects of small interfering RNA (siRNA) specific for Rac1 (*P < 0.05 and **P < 0.05). (n = 3 for each experimental condition). ns RNA, nonspecific siRNA.

Fig. 6.

Fig. 6.

Role of Rac1 in EC barrier protection by simvastatin. Compared with the protective effects of simvastatin (5 μM, 16 h) in EC transfected with nonspecific siRNA (nsRNA), thrombin-induced (1 U/ml, 1 h) EC permeability as measured by FITC-dextran permeability is only modestly decreased via siRac1 transfection (*P < 0.05 and **P < 0.05). A more pronounced effect is observed in EC that are both transfected with siRac1 and pretreated with simvastatin (5 μM, 16 h) (†P < 0.05, n = 3 for each experimental condition).

Fig. 7.

Fig. 7.

Role of Cdc42 in EC barrier protection by simvastatin. Compared with control EC transfected with nonspecific siRNA (nsRNA), thrombin-induced (1 U/ml, 1 h) EC permeability as measured by FITC-dextran permeability is not effected via transfection with small interfering RNA specific for Cdc42 (siCdc42) (*P < 0.05). Moreover, compared with control EC pretreated with simvastatin no additional effect is noted in EC that are both transfected with siCdc42 and pretreated with simvastatin (5 μM, 16 h) (**P < 0.05, n = 3 for each experimental condition).

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