MicroRNA-1 protects the endothelium in acute lung injury
Asawari Korde, Maria Haslip, Prachi Pednekar, Alamzeb Khan, Maurizio Chioccioli, Sameet Mehta, Francesc Lopez-Giraldez, Santos Bermejo, Mauricio Rojas, Charles Dela Cruz, Michael A. Matthay, Jordan S. Pober, Richard W. Pierce, Shervin S. Takyar
Asawari Korde, Maria Haslip, Prachi Pednekar, Alamzeb Khan, Maurizio Chioccioli, Sameet Mehta, Francesc Lopez-Giraldez, Santos Bermejo, Mauricio Rojas, Charles Dela Cruz, Michael A. Matthay, Jordan S. Pober, Richard W. Pierce, Shervin S. Takyar
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Research Article Pulmonology Vascular biology

MicroRNA-1 protects the endothelium in acute lung injury

Abstract

Acute lung injury (ALI) and its most severe form, acute respiratory distress syndrome (ARDS), cause severe endothelial dysfunction in the lung, and vascular endothelial growth factor (VEGF) is elevated in ARDS. We found that the levels of a VEGF-regulated microRNA, microRNA-1 (miR-1), were reduced in the lung endothelium after acute injury. Pulmonary endothelial cell–specific (EC-specific) overexpression of miR-1 protected the lung against cell death and barrier dysfunction in both murine and human models and increased the survival of mice after pneumonia-induced ALI. miR-1 had an intrinsic protective effect in pulmonary and other types of ECs; it inhibited apoptosis and necroptosis pathways and decreased capillary leak by protecting adherens and tight junctions. Comparative gene expression analysis and RISC recruitment assays identified miR-1 targets in the context of injury, including phosphodiesterase 5A (PDE5A), angiopoietin-2 (ANGPT2), CNKSR family member 3 (CNKSR3), and TNF-α–induced protein 2 (TNFAIP2). We validated miR-1–mediated regulation of ANGPT2 in both mouse and human ECs and found that in a 119-patient pneumonia cohort, miR-1 correlated inversely with ANGPT2. These findings illustrate a previously unknown role of miR-1 as a cytoprotective orchestrator of endothelial responses to acute injury with prognostic and therapeutic potential.

Authors

Asawari Korde, Maria Haslip, Prachi Pednekar, Alamzeb Khan, Maurizio Chioccioli, Sameet Mehta, Francesc Lopez-Giraldez, Santos Bermejo, Mauricio Rojas, Charles Dela Cruz, Michael A. Matthay, Jordan S. Pober, Richard W. Pierce, Shervin S. Takyar

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Figure 9

miR-1/ANGPT2 axis.

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miR-1/ANGPT2 axis. (A and B) Endothelial cells were transduced with V-mi...
(A and B) Endothelial cells were transduced with V-miR-1 or V-ctrl. Cell lysates were collected after 48 hours and ANGPT2 protein levels were measured by Western blotting. The top panels show a representative Western blot and the bottom panels show quantification based on band density values, normalized to β-actin for (A) HUVECs (n = 3, *P = 0.044) and (B) MLECs (n = 3, *P = 0.05). (C) HUVECs were transfected with miR-1 or control RNA (Ctrl) and grown in starvation media (containing 2% FBS) for 16 hours. ANGPT2 levels in the media were measured by ELISA (n = 3 per group). *P = 0.0252. (D) Mice received V-miR-1 or V-ctrl intranasally. BAL was collected after 2 weeks and analyzed for ANGPT2 and albumin content by Western blotting assay. The top panel shows the Western blot and the bottom panel shows band density values normalized to albumin (n = 5 for V-ctrl and 4 for V-miR-1). *P = 0.037. (E) Human lung tissue samples were cultured and transfected with a double-stranded miR-1 RNA mimic (miR-1) or control RNA (ctrl). Culture media were collected after 24 hours and ANGPT2 levels were measured by ELISA (n = 4 patients, 2 replicates each). *P = 0.0011. (F) The sequence of the miR-1 binding sites in Angpt2 luciferase vectors: Green letters indicate mutations in the scrambled (Scr)–miR-1, and red letters indicate compensatory mutations in the Angpt2 3′UTR. (G and H) 293T cells were cotransfected with miR-1 or scr-miR-1 RNA and the luciferase vector containing (G) Angpt2 WT 3′UTR or (H) Angpt2 mutated 3′UTR. Relative firefly/Renilla luciferase activities were normalized to the mean of the control groups (2 experiments, n = 4). **P = 0.002, ***P = 0.0007. (I) HPMECs were transfected with miR-1 (or control RNA) and treated with TNF-α (10 ng/mL) in the presence of increasing concentrations of recombinant ANGPT2 and harvested after 24 hours. Cell death was measured and expressed as percentage TUNEL-positive cells, as described in Figure 4A (n > 4, from 2 experiments). *P = 0.01, **P < 0.0004, ***P < 0.0005). Error bars represent the SEM. Data were analyzed by unpaired, 2-tailed t test with Welch’s correction or Mann-Whitney U test based on normality.