Actin fence therapy with exogenous V12Rac1 protects against acute lung injury
Galina A. Gusarova, Shonit R. Das, Mohammad N. Islam, Kristin Westphalen, Guangchun Jin, Igor O. Shmarakov, Li Li, Sunita Bhattacharya, Jahar Bhattacharya
Galina A. Gusarova, Shonit R. Das, Mohammad N. Islam, Kristin Westphalen, Guangchun Jin, Igor O. Shmarakov, Li Li, Sunita Bhattacharya, Jahar Bhattacharya
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Research Article Pulmonology

Actin fence therapy with exogenous V12Rac1 protects against acute lung injury

Abstract

High mortality in acute lung injury (ALI) results from sustained proinflammatory signaling by alveolar receptors, such as TNF-α receptor type 1 (TNFR1). Factors that determine the sustained signaling are not known. Unexpectedly, optical imaging of live alveoli revealed a major TNF-α–induced surge of alveolar TNFR1 due to a Ca2+-dependent mechanism that decreased the cortical actin fence. Mouse mortality due to inhaled LPS was associated with cofilin activation, actin loss, and the TNFR1 surge. The constitutively active form of the GTPase, Rac1 (V12Rac1), given intranasally (i.n.) as a noncovalent construct with a cell-permeable peptide, enhanced alveolar filamentous actin (F-actin) and blocked the TNFR1 surge. V12Rac1 also protected against ALI-induced mortality resulting from i.n. instillation of LPS or of Pseudomonas aeruginosa. We propose a potentially new therapeutic paradigm in which actin enhancement by exogenous Rac1 strengthens the alveolar actin fence, protecting against proinflammatory receptor hyperexpression, and therefore blocking ALI.

Authors

Galina A. Gusarova, Shonit R. Das, Mohammad N. Islam, Kristin Westphalen, Guangchun Jin, Igor O. Shmarakov, Li Li, Sunita Bhattacharya, Jahar Bhattacharya

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Figure 3

Exogenous delivery of Rac1 mutants modifies the actin cytoskeleton in alveolar epithelium.

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Exogenous delivery of Rac1 mutants modifies the actin cytoskeleton in al...
(A) Confocal images and the plots show changes in alveolar fluorescence of differentially tagged TAT and V12Rac1. The tagging was done prior to conjugation. The plot shows data from ROIs (dotted circles) for 10 alveoli of a single lung (mean ± SEM). Replicated in 3 lungs. Scale bar: 50 μm. *P < 0.05 V12Rac1 versus TAT using 2-tailed t test. (B) Images show F-actin levels 15 min after alveolar microinfusions of Rac1 mutants. Scale bars, 50 μm. Bars are whole-image quantifications of F-actin (rhodamine-phalloidin) (left) at indicated time points and TNFR1 expression (right) 30 min after TNF-α microinfusion in alveoli pretreated with TAT-Rac1 mutants. Mean ± SEM, n = 5 lungs for each group. Each dot shows data for a single lung. *P < 0.05 versus corresponding baseline (dashed line) using ANOVA with Bonferroni correction. (C–E) Immunoblots and densitometry are for lungs given indicated instillations. Tissues were harvested at 4 (C and D), or 24 (E) hours after instillations. Bands in (C) are immunoblots on lysates that were fractionated as triton-insoluble (F-actin), or -soluble (G-actin), or not fractionated (Actin). Bands in (E) were obtained on immunoprecipitates of His-tagged, exogenous Rac1 mutants. PBS, control; V12, TAT-V12Rac1; N17, TAT-N17Rac1; JP, jasplakinolide. All samples in blots were run simultaneously. Each blot was replicated 4 times (C) or 6 times (D and E). Each dot shows data for a single lung. *P < 0.05 compared with PBS using ANOVA with Bonferroni correction.