miR-144-mediated Inhibition of ROCK1 Protects against LPS-induced Lung Endothelial Hyperpermeability

M. Rizwan Siddiqui, Suhail Akhtar, Mohd Shahid, Mohammad Tauseef, Kelli McDonough, Thomas Patrick Shanley

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

Dysfunctional endothelial cell (EC) barrier and increased lung vascular permeability is a cardinal feature of acute lung injury and sepsis that may result in a pathophysiological condition characterized by alveolar flooding, pulmonary edema, and subsequent hypoxemia. In lung ECs, activation of Rho-associated kinase-1 (ROCK1) phosphorylates myosin light chain (MLC)-associated phosphatase at its inhibitory site, which favors phosphorylation of MLC, stress fiber formation, and hyperpermeability during acute lung injury. The role of microRNA-144 (miR-144) has been well investigated in many human diseases, including cardiac ischemia/reperfusion-induced injury, lung cancer, and lung viral infection; however, its role in pulmonary EC barrier regulation remains obscure. Here, we investigated the miR-144-mediated mechanism in the protection of endothelial barrier function in an LPS-induced lung injury model. By using transendothelial electrical resistance and transwell permeability assay to examine in vitro permeability and immunofluorescence microscopy to determine barrier integrity, we showed that ectopic expression of miR-144 effectively blocked lung EC barrier disruption and hyperpermeability in response to proinflammatory agents. Furthermore, using a gain-and-loss-of-function strategy, overexpression of miR-144 significantly decreased ROCK1 expression. Concomitantly, miR-144 inhibits ROCK1-mediated phosphorylation of MLC phosphataseThr853 and thus phosphorylation of MLCThr18/Ser19 to counteract stress fiber formation in LPS-activated EC. Finally, in LPS-challenged mice, intranasal delivery of miR-144 mimic via liposomes attenuated endotoxemia-induced increases in lung wet/dry ratio, vascular permeability, and inflammation. In conclusion, these data suggest that miR-144-attenuated activation of inflammatory ROCK1/MLC pathway in vascular ECs is a promising therapeutic strategy to counter inflammatory lung injury.

Original languageEnglish (US)
Pages (from-to)257-265
Number of pages9
JournalAmerican journal of respiratory cell and molecular biology
Volume61
Issue number2
DOIs
StatePublished - Aug 1 2019

Fingerprint

rho-Associated Kinases
MicroRNAs
Myosin Light Chains
Endothelial cells
Lung
Phosphorylation
Endothelial Cells
Stress Fibers
Acute Lung Injury
Capillary Permeability
Lung Injury
Pulmonary Edema
Permeability
Lung Neoplasms
Chemical activation
Myosin-Light-Chain Phosphatase
Endotoxemia
Acoustic impedance
Fibers
Virus Diseases

Keywords

  • ROCK1
  • acute lung injury
  • endothelial permeability
  • miR-144

ASJC Scopus subject areas

  • Molecular Biology
  • Pulmonary and Respiratory Medicine
  • Clinical Biochemistry
  • Cell Biology

Cite this

Siddiqui, M. Rizwan ; Akhtar, Suhail ; Shahid, Mohd ; Tauseef, Mohammad ; McDonough, Kelli ; Shanley, Thomas Patrick. / miR-144-mediated Inhibition of ROCK1 Protects against LPS-induced Lung Endothelial Hyperpermeability. In: American journal of respiratory cell and molecular biology. 2019 ; Vol. 61, No. 2. pp. 257-265.
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miR-144-mediated Inhibition of ROCK1 Protects against LPS-induced Lung Endothelial Hyperpermeability. / Siddiqui, M. Rizwan; Akhtar, Suhail; Shahid, Mohd; Tauseef, Mohammad; McDonough, Kelli; Shanley, Thomas Patrick.

In: American journal of respiratory cell and molecular biology, Vol. 61, No. 2, 01.08.2019, p. 257-265.

Research output: Contribution to journalArticle

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T1 - miR-144-mediated Inhibition of ROCK1 Protects against LPS-induced Lung Endothelial Hyperpermeability

AU - Siddiqui, M. Rizwan

AU - Akhtar, Suhail

AU - Shahid, Mohd

AU - Tauseef, Mohammad

AU - McDonough, Kelli

AU - Shanley, Thomas Patrick

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AB - Dysfunctional endothelial cell (EC) barrier and increased lung vascular permeability is a cardinal feature of acute lung injury and sepsis that may result in a pathophysiological condition characterized by alveolar flooding, pulmonary edema, and subsequent hypoxemia. In lung ECs, activation of Rho-associated kinase-1 (ROCK1) phosphorylates myosin light chain (MLC)-associated phosphatase at its inhibitory site, which favors phosphorylation of MLC, stress fiber formation, and hyperpermeability during acute lung injury. The role of microRNA-144 (miR-144) has been well investigated in many human diseases, including cardiac ischemia/reperfusion-induced injury, lung cancer, and lung viral infection; however, its role in pulmonary EC barrier regulation remains obscure. Here, we investigated the miR-144-mediated mechanism in the protection of endothelial barrier function in an LPS-induced lung injury model. By using transendothelial electrical resistance and transwell permeability assay to examine in vitro permeability and immunofluorescence microscopy to determine barrier integrity, we showed that ectopic expression of miR-144 effectively blocked lung EC barrier disruption and hyperpermeability in response to proinflammatory agents. Furthermore, using a gain-and-loss-of-function strategy, overexpression of miR-144 significantly decreased ROCK1 expression. Concomitantly, miR-144 inhibits ROCK1-mediated phosphorylation of MLC phosphataseThr853 and thus phosphorylation of MLCThr18/Ser19 to counteract stress fiber formation in LPS-activated EC. Finally, in LPS-challenged mice, intranasal delivery of miR-144 mimic via liposomes attenuated endotoxemia-induced increases in lung wet/dry ratio, vascular permeability, and inflammation. In conclusion, these data suggest that miR-144-attenuated activation of inflammatory ROCK1/MLC pathway in vascular ECs is a promising therapeutic strategy to counter inflammatory lung injury.

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