Introduction Mesenchymal stromal cell (MSC) therapy mitigates lung injury and improves survival in murine models of sepsis. Precise mechanisms of therapeutic benefit remain poorly understood.
Objectives To identify host-derived regulatory elements that may contribute to the therapeutic effects of MSCs, we profiled the microRNAome (miRNAome) and transcriptome of lungs from mice randomised to experimental polymicrobial sepsis-induced lung injury treated with either placebo or MSCs.
Methods and results A total of 11 997 genes and 357 microRNAs (miRNAs) expressed in lungs were used to generate a statistical estimate of association between miRNAs and their putative mRNA targets; 1395 miRNA:mRNA significant association pairs were found to be differentially expressed (false discovery rate ≤0.05). MSC administration resulted in the downregulation of miR-27a-5p and upregulation of its putative target gene VAV3 (adjusted p=1.272E-161) in septic lungs. In human pulmonary microvascular endothelial cells, miR-27a-5p expression levels were increased while VAV3 was decreased following lipopolysaccharide (LPS) or tumour necrosis factor (TNF) stimulation. Transfection of miR-27a-5p mimic or inhibitor resulted in increased or decreased VAV3 message, respectively. Luciferase reporter assay demonstrated specific binding of miR-27a-5p to the 3′UTR of VAV3. miR27a-5p inhibition mitigated TNF-induced (1) delayed wound closure, increased (2) adhesion and (3) transendothelial migration but did not alter permeability. In vivo, cell infiltration was attenuated by intratracheal coinstillation of the miR-27a-5p inhibitor, but this did not protect against endotoxin-induced oedema formation.
Conclusions Our data support involvement of miR-27a-5p and VAV3 in cellular adhesion and infiltration during acute lung injury and a potential role for miR-27a-based therapeutics for acute respiratory distress syndrome.
- critical care
- respiratory infection
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What is the key question?
The key question of our study is whether mesenchymal stromal/stem cells (MSCs) exert their beneficial anti-inflammatory effect in septic lungs by in part modulating endogenous host-derived microRNAs and their target genes—thus amplifying their therapeutic effect by capitalising on shared endogenous post-transcriptional regulatory mechanisms.
What is the bottom line?
MSCs regulate the expression of miR-27a and its various target genes including VAV3, ACE and Nrf2 known to play critical roles in sepsis-induced lung injury and inhibition of miR-27a prevents inflammatory cell infiltration during endotoxin-induced lung injury.
Why read on?
This is the first study that links MSC regulation with microRNA alterations of various acute lung injury-related genes containing miR-27a 3′UTR regulatory sequences, suggesting that targeting this pathway may be a suitable strategy to preserve the alveolar epithelial function and to reduce inflammatory cell infiltration in acute lung injury.
With an annual incidence of close to one million cases in North America, sepsis-related multiorgan failure is responsible for more than 200 000 deaths per year and rivals myocardial infarction as a leading cause of mortality in the developed world.1 2 The lung is the organ that fails most often dictating the need for life support, and sepsis is the highest risk factor for development and progression of acute respiratory distress syndrome (ARDS), the clinical syndrome of acute lung injury (ALI).3 In preclinical studies, mesenchymal stromal/stem cell (MSC) therapy mitigates pulmonary oedema formation, protein-rich fluid exudation and cellular infiltration.4 Network analysis of transcriptional changes following MSC administration suggests reprogramming of the immune system involving thousands of genes and multiple pathways in various organs.5 MicroRNAs (miRNAs) are centrally positioned to orchestrate this host response because of their fundamental role as modulators of biological processes and cell fate.6
miRNAs are endogenous RNA molecules that can directly bind to the 3′ untranslated region (UTR) of mRNAs to modulate gene expression at the post-transcriptional level. Binding of miRs to their seed sequences results in gene repression either by destabilising their complementary mRNAs or by preventing translation. Various studies have implicated miRNAs in the regulation of endothelial barrier integrity and demonstrated their potential as pharmacological targets for the maintenance of endothelial cell barrier function.6 Here, we hypothesise that the beneficial therapeutic effects of MSC administration in septic lungs is in part due to the modulation of host-derived endogenous miRNAs.
To identify biologically relevant, and novel, miRNAs involved in mediating the beneficial effects of MSC in preclinical models of ARDS, we capitalised on canonical binding predictions between miRs and their target mRNAs to develop a statistical algorithm that detects tightly regulated differentially expressed miRNA:mRNA pairs. Functional genomics (gain and loss of function) in vitro and in vivo studies demonstrates inhibition of host-derived miR-27a-5p in mediating the salutary effect of MSC administration in a model of preclinical lung injury.
Material and methods
All studies were approved by the animal care committee at St. Michael’s Hospital (Toronto, Ontario, Canada) in accordance with Canadian Council of Animal Care guidelines and ARRIVE criteria.7 Briefly, male C57Bl/6J mice of 10–14 weeks old (The Jackson Laboratory, Bar Harbor, Maine, USA) were randomised to caecum ligation and puncture (CLP) or sham surgery and at 6 hours post-CLP. Mice were further randomised to receive a single intrajugular injection of 2×105 bone marrow-derived MSCs or placebo (saline).4 Animals were humanely sacrificed at 28 hours and right lungs snap frozen for mRNA and miRNA profiling. Transcriptional microarray data are available in GEO (Gene Expression Omnibus, http://www.ncbi.nlm.nih.gov/geo GSE40180)). Detailed materials and methods are available https://thorax.bmj.com/.
MicroRNA analysis and miRNA target gene expression association
miRNA isolated from lungs (five animals per group—sham/saline, CLP/saline and CLP/MSCs) using Exiqon miRCURY LNA microRNA arrays (Exiqon, Vedbaek, Denmark). Variance-stabilising was used to normalise expression values in R/Bioconductor8 and differential expression was determined using LIMMA.9 miRNA:mRNA associated pairs were identified by matching a gene by miRNA matrix table based on in silico predictions versus empiric lung mRNA and miRNA experimental data. Predicted targets were determined using miRanda (http://mirdb.org 10 score <−0.5, conservation score >0.5) and TargetScan11 (context score <−0.3) databases with appropriate thresholds. A statistical estimate of association or correlation between miRNAs and their putative mRNA targets was determined using the equation:
where Y is the expression value of a given target gene and X is the expression of its miRNA.
We fitted a Bayes hierarchical generalised linear model12 for the equation to identify the miRNAs which significantly regulated the gene expression of the mRNA (false discovery rate, FDR set at <0.05). Adjusted p-values (Adj-p) are p-values corrected for multiple comparisons.10 Functional enrichment was performed in Enrichr.13
Cell culture, treatments and transfections
Primary human pulmonary microvascular endothelial cells (HPMECs; PromoCELL, Heidelberg, Germany), primary human distal bronchial epithelial cells (ATCC CRL-9609; BEAS2b, Toronto, Canada), RAW 264.7 (91062702; Sigma, Darmstadt, Germany) and murine MSCs (isolated from male C57Bl/6J mice, a gift from Dr Darwin Prockop, Texas A&M Health Science Center, College Station, Texas, USA) were used as previously described.4 5 All in vitro experiments were performed in triplicate. Details of in vitro experiments are presented in the online supplementary material.
In vivo inhibition of miR-27a-5p in murine lungs
Mice were randomised (random number generator) to receive miR-27a-5p inhibitor (INH), negative control (NC) or equal volume saline combined with transfection reagent (HiPerfect Transfection reagent). Sample size calculation was based on bronchial alveolar lavage fluid (BALf) cell count between mice treated with LPS+NC versus LPS+INH. A total sample size of six animals per group provided 80% power to detect statistically significant differences using an alpha 0.05 and beta 0.2 (https://clincalc.com/stats/samplesize.aspx). The oligonucleotide mixtures were coadministered intratracheally with LPS (10 mg/kg body weight) or equal volume saline up to a total volume of 60 μL. Dose and route of oligonucleotide administration was based on manufactures’ suggestion and pilot experiments. Animals were anaesthetised with inhaled isofluorane. LPS and oligonucleotide mixture were coadministered. The surgical area was sutured and animals were allowed to recover in a bedding free cage. All animals received subcutaneous fluid resuscitation with 50 mL/kg saline and 0.2 mg/kg buprenorphine (Buprenex) for pain. Endpoints were measured at 24 hours postinstillation. BALf total cell counts and differentials were obtained as previously described.4 14 Total protein in BALf was measured by Bradford (Bio-Rad) and IgM levels ELISA (E90-101, Bethyl BioSciences).
Observers assessing endpoints were blinded to group assignment. Where Kolmogorov-Smirnov (K-S) test demonstrated normal distribution (based on p-value ≥0.05 for statistic (D)), data are presented as individual values (symbols), means±SD and corrected using Tukey’s test for multiple comparisons. Non-parametric data are presented as median±IQR and Dunn’s post hoc test was used to correct for multiple comparisons.
Identification of significantly associated miRNA:mRNA pairs in MSC-treated septic lungs
To identify miRNA:mRNA pairs in MSC-treated versus non-treated septic lungs, we generated a gene by miRNA matrix based on in silico predictions and matched to our lung mRNA and miRNA experimental data (schematic of the experiment shown in figure 1A). Overlap between in silico (predicted) versus empiric (observed) was 72% (p≤0.01). A total of 1395 putative miRNA:mRNA associations were identified in both sham versus CLP, as well as CLP treated with placebo versus MSC comparisons (FDR ≤0.0515; online supplementary table 1). Table 1 shows top 10 differentially expressed miRNA:mRNA associations in septic lungs after treatment with MSCs. miR-27a-5p and its putative target the GEF 3 (VAV3, table 1) were selected for detailed functional genomics based on the following criteria: (1) robust statistical prediction (unbiased); (2) empirical evidence of regulation of both miRNA and its target in lungs following CLP and MSC administration; (3) novelty, that is, not known to play a role in ARDS; (4) potential for clinical relevance based on homology of both miR and gene target between human and murine orthologues; (5) biological plausibility, that is, both miR-27a and VAV3 are linked to cytoskeletal rearrangement, receptor tyrosine kinase and integrin signalling and (6) clinical relevance: involved in cell adhesion, apoptosis, phagocytosis and cell migration—processes known to play a role in ARDS.16 17 miRNA 27a-5p and its target VAV3 are regulated by MSCs in septic lungs.
Principal component analysis shows clustering of septic (CLP) versus sham-treated groups (figure 1B). MSC administration results in a shift in global transcription profiles favouring reconstitution of a ‘sham-like’ expression patterns (figure 1B). This transition is more evident from the hierarchical clustering of miR-27a putative target genes differentially expressed in septic and MSC treated lungs (figure 1C, table 2). Putative miR-27a-5p targets include VAV3 as well as other ARDS/ALI relevant targets: nuclear factor 2 (Nrf2), ACE 1 (ACE1), epidermal growth factor receptor (EGFR) and fibroblast growth factor 7 (FGF7, also known as keratinocyte growth factor (KGF)). Ingenuity pathway analysis was used to generate a putative gene interaction network using predicted (dashed lines) and documented (solid lines) interactions between miR-27a-5p targets (figure 1D). Independent experiments confirmed upregulation of miR-27a-5p and downregulation of VAV3 in CLP-placebo (figure 1E,F) and downregulation of miR-27a-5p and upregulation of VAV3 and ACE1 in the MSC-treated groups (figure 1E–G). miR-191 was used for normalisation; this miRNA was found to be statistically superior to other reference miRs.18 Additional separate mice were exposed to intratracheal LPS (10 mg/kg) to confirm miR-27a-3p and miR-27a-5 p (figure 1H–J) at 24 hours.
Predicted involvement of miR-27a-5p:VAV3 in inflammation and innate immunity pathways
Enrichment analysis highlighted MSC-mediated regulation of receptor tyrosine kinase signalling (Torso signalling), integrin-mediated signalling and sterol regulatory element-binding protein (table 3) in septic lungs. Canonical pathway analysis predicted alteration in: DR3 and DR4/5 death receptors, modulation by heat shock protein 70, FAS/FADD signalling and response to DNA damage (p53 pathway). Inflammation and innate immunity pathways were associated with the inhibition of cell adhesion and migration, phagocytosis, antigen presentation and cell junction function (integrin signalling, Fc gamma R-mediated phagocytosis and JAK/STAT signalling; table 4). Enrichment analysis using only miR-27a-5p predicted targets underscored involvement of this miR in regulation of cytoskeleton, chemokine and cytokine activity, focal adhesion, gap junction and leucocyte transmigration (table 5), implicating miR-27a-5p in inflammatory cell migration and barrier function.
miR-27a-5p and VAV3 are differentially regulated in primary human endothelial cells
Both miR-27a-5p and VAV3 target gene share 100% homology with their human orthologues (figure 2A). In HPMECs exposed to TNF, the expression of miR27a-5p is upregulated by increasing doses of TNF (10–40 ng/mL, figure 2B) and remains elevated up to 24 hours poststimulation (figure 2C). The expression of VAV3 gene (figure 2E) and protein (figure 2F) are downregulated 61% and 50%, respectively, in cells treated with TNF where the expression of miR-27a-5p and miR-27a-3p is increased (figure 2D). The expression of miR-27a-5p and miR-27a-3p is also increased by TNF in RAW 264.7 cells (murine macrophage cell line; online supplementary figure 1A) and human distal bronchial epithelial cells BEAS2b (online supplementary figure 1B). While the expression of both VAV3 and ACE1 decreased as per in silico predictions in HPMECs in response to TNF, FGF7 and EGFR increased (online supplementary figure 1B,C and online supplementary figure 2A). Protein expression of ACE1 decreased, while protein expression of EGFR and FGF7 (KGF) increased (online supplementary figure 1F,G), suggesting that in these cells these genes are not regulated as predicted.
The mirna27a gene is located on chromosome 8 in mice and chromosome 19 in humans; it lives in a cluster that encodes in both species three distinct pre-miRNAs: miR-23a, miR-24–2 and miR-27a.19 Paired miRNA:mRNA analysis did not identify other members of the cluster as differentially regulated. Notwithstanding, we found miR-24a and miR-23 were both upregulated by exposure to TNF in HPMECs (figure 2G); only miRs-27a and miRs-24 levels were upregulated by CLP and LPS in murine lungs (figure 2H).
Conditioned medium from cultured MSCs attenuated inflammation-mediated upregulation of miR-27a in vitro
Conditioned medium from cultured MSCs mitigated TNF-induced upregulation of miR-27a-5p in HPMECs and BEAS2b cells (figure 3A,B). Transfection of HPMECs with the miR-27a-5p inhibitor significantly reduced TNF-induced upregulation of miR-27a-5p (figure 3C) but did not significantly affect the expression of the miR-27a-3p fragment (figure 3D). Transfection of the miR-27a-5p mimic resulted in a 47% to 52% reduction in VAV3 expression at baseline and following TNF treatment, respectively (figure 3E). ACE1 mRNA levels were reduced following TNF and transfection of the miR-27a-5p mimic (online supplementary figure 2A) and increased following miR-27a-5p inhibitor transfection (online supplementary figure 2A). The expression of Nrf2 was not significantly modulated by the miR-27a-5p mimic or inhibitor (online supplementary figure 2B). Baseline VAV3 expression is protected from miR-27a-5p overexpression (figure 3E,F). Although TNF induced downregulation of ACE1 mRNA levels and transfection, this was significantly mitigated by miR-27a-5p inhibition. Transfection of miR-27a-5p mimic or inhibitor did not affect cell viability at 24 hours (online supplementary figure 3).
Binding of miR-27a-5p to the human 3′UTR of VAV3
Two mutation constructs containing either a deletion of the heptameric (mutant 1) or both the heptameric and sextameric (mutant 2) miR-27a-5p seed binding sequences on the 3′UTR of VAV3 were generated (figure 3G, online supplementary material and figure 4A) to demonstrate specific binding of miR-27a-5p to the 3′UTR of VAV3 (figure 3I). miR-27a-5p inhibition in TNF-treated cells rescued luciferase activity indicating that inhibition of miR-27a-5p prevents binding of miR-27a-5p to the wild-type 3′UTR and VAV3 mRNA degradation. No significant changes in luciferase activity were seen when cells were transfected with a luciferase constructs containing the 3′UTR of the housekeeping gene actinomycin B or a non-targeting control R01 (online supplementary figure 4B).
Modulation of miR-27a-5p altered wound closure, adhesion, migration and transmigration, but not barrier function
TNF administration to confluent HPMEC monolayers resulted in increased cellular leakage as measured by the amount of 70 kDa FITC-Dextran that passed through the monolayer. Transfection of miR-27a-5p mimic did not significantly increase FITC-Dextran leakage (figure 4A). Loss of cell number due to death or apoptosis was not a confounding factor (online supplementary figure 3). Wound healing scratch assay made on 24-hour serum-starved, confluent HPMECs transfected with the miR-27a-5p mimic, inhibitor or control miRNA demonstrated that exposure to TNF or/and transfection of the miR-27a-5p mimic significantly delayed wound closure (figure 4B). In contrast, miR-27a-5p inhibition enhanced wound closure at baseline and following exposure to TNF (figure 4B). Adhesion of monocytes to the HPMEC monolayer was quantified by coculturing HPMECs with fluorescently labelled monocytes THP-1 cells (described in online supplementary methods). Exposure to TNF increased monocyte adhesion compared with saline (figure 4C). Transfection with the miR-27a-5p mimic increased monocyte adhesion both at baseline and following TNF stimulation, while miR-27a-5p inhibition mitigated monocyte adhesion primarily following TNF stimulation (figure 4C). In a transwell migration assay, MiR-27a-5p inhibition attenuated transendothelial migration of THP1 cells through an HPMEC monolayer following TNF stimulation but had no effect on transendothelial migration at baseline (figure 4D). Transfection with the miR27a-5p mimic resulted in enhanced migration of monocytes across the HPMEC monolayer following TNF stimulation (figure 4D). Transfection of either 40 nM or 80 nM of an small interfering RNA (siRNA) against VAV3 resulted in significant delay in wound closure (figure 5A,B) and decrease in the expression of VAV3 protein in HPMECs (figure 5C).
Inhibition of miR-27a-5p in vivo attenuated pulmonary cellular infiltration but not oedema formation following LPS-induced lung injury
Intratracheal pulmonary endotoxin instillation coadministration with miR-27a-5p inhibitor results in a significant decrease in total cell count and neutrophil infiltration in mice that received the miR-27a-5p inhibitor compared with NC (figure 6A,B). Lung injury scores were calculated on the basis of the relative degree of inflammation, oedema, haemorrhage and atelectasis seen on histology (figure 6C). Coinstillation of LPS and miR-27a-5p inhibitor showed a partial decrease in total protein and IgM levels after LPS but this did not reach statistical significance (figure 6C). Endotoxin instillation resulted in both increased miR-27a-5p and decreased VAV3 protein expression (figure 6F–I). Immunohistochemistry shows decreased VAV3 antibody binding after LPS treatment (figure 6F, online supplementary material; miR-27a-5p inhibitor coinstillation attenuates VAV3 protein expression decrease (figure 6H). Expression of VAV3 also decreased following LPS instillation in mice that received the NC but was preserved in mice that received the miR-27a-5p inhibitor (figure 6I).
Increased miR-27a-5p expression in patients who died with ARDS
Diffuse alveolar damage (DAD) is the histological correlate of severe ALI for most patients with ARDS. Absence of fresh human lung tissue from patients with ARDS is a major obstacle to further pathological, molecular and mechanistic studies in ARDS. To circumvent this limitation, we obtained extensively annotated autopsy lung tissues from patients who died with ARDS with or without biopsy proven DAD. We extracted RNA from formalin-fixed paraffin-embedded lung sections, known to contain DAD or no-DAD tissue, and determined by quantitative real-time PCR that the expression level of miR-27a-5p was significantly increased in ARDS lungs with DAD compared with no-DAD. These data support the importance of miR-27a-5p in human pathology (figure 7).
Our group and others have shown that MSC administration reduces morbidity and mortality in experimental models of sepsis.4 20–23 Although these studies demonstrate the feasibility of cell-based technologies for the treatment of sepsis, mechanism(s) remain incompletely understood and are central to the development of novel therapeutics. Here, we exploited the canonical relationship between miRs and their putative targets to identify miR-27a:VAV3 pair as both modulated by MSCs and putatively involved in sepsis-induced lung injury. In vitro using a relevant cellular model of primary human pulmonary endothelial cells exposed to TNF (TNF can reproduce many of the key physiological and molecular derangements seen in sepsis), the human miR-27a-5p binds specifically to its target on the 3′UTR of VAV3 and is involved in cellular migration, adhesion and transmigration, but not convincingly in endothelial permeability. In vivo, using an independent model of ALI induced by endotoxin instillation, coadministration with the miR-27a-5p inhibitor prevents LPS-induced cellular infiltration, but not loss of barrier function. The clinical relevance of our findings is evidenced by increased miR-27a-5p expression in patients with ARDS who died with autopsy-proven DAD versus no DAD.
Using an miRNA:mRNA paired analysis approach, rather than matching miRs to putative mRNA lists post hoc, improved power and allowed us to derive important biological knowledge from the involvement of miR-27a in the regulation of the host response to injury and MSC therapy. Enrichment analysis predicted miR-27a would contribute to actin cytoskeleton, leucocyte transendothelial migration, transmigration, adhesion (focal adhesion) and gap junction regulation which we confirmed using in vitro and in vivo functional assays. In silico, VAV3 was predicted to be a key target—which we confirmed using a luciferase reporter system and siRNA against VAV3. This does not imply that VAV3 is the ‘only’ target nor that miR-27a-5p regulates the phenotype exclusively through VAV3 since other miR-27a-5p targets including ACE1, FGF7 and EGFR were also found to be regulated, both in vitro and in vivo, but the regulatory relationship to miR-27a was not exclusive, suggesting other (as of yet unknown) factors may further contribute. Importantly, inhibition of miR-27a-5p significantly reduced in vitro and in vivo evidence of endothelial cell dysfunction and ALI. Further studies will be required to elucidate the specific role(s) of VAV3 as well as other miR-27a-5p targets. In the interim, the main conclusion of our study is that miR-27a-5p plays a biologically and possibly clinically relevant role in ARDS.
How MSCs regulate miR expression levels in recipients remains highly speculative. Recent studies have highlighted the function of miRNAs in the paracrine effects of MSCs. These miRNAs may either modulate expression of proteins secreted by MSCs or be contained in microvesicles and exosomes, to exert their regulatory function in target cells. We tried to glean possible mechanisms by querying coregulation of the miR-27 cluster. miR-27a is part of the miR-23a~27a~24–2 cluster documented to have altered expression in many disease states24 involving various functions such as angiogenesis, cell differentiation, proliferation, inflammation25 and infection.26 27 All three miRNAs of this cluster are derived from a single primary transcript; transforming growth factor beta receptor 1(TGF-βR1) and SMAD transcription factors are primary regulators of the cluster, but expression pattern of different members of the cluster may vary.24 Ours is the first report underscoring the role of miR-27a-5p in acute inflammatory pulmonary injury. Previous reports implicate miR-27a in the regulation of VE-cadherin, vascular leak and recovery from ischaemic injury.28 Little is known about the role of miR-27a-5p in cell migration, transmigration and adhesion, and much of what is known has been primarily defined in malignancy. In these models, miR-27a acts as a tumour suppressor by inhibiting TGF-βRI signalling pathway.29
The Vav family of proteins (Vav1, Vav2, Vav3) are cytoplasmic guanine nucleotide exchange factors (GEFs) for Rho-family GTPases. Specific receptor signalling results in the tyrosine phosphorylation of Vav proteins and hence their activation.30 Results from mice deficient in one or more Vav proteins show critical roles in cell development, activation as well as sensing of bacterial products: LPS binding protein, CD14, Toll-like receptor 4 (TLR4) and MyD88, conveying threat signals from the cell membrane to intracellular intermediate mediators Rac1, Cdc42, ras homology gene (RhoG) and rho associated coiled-coil containing protein kinase 1 (ROCK). These processes are associated with multiple cell shape changes that rely on the reorganisation of the cytoskeleton.31 In this context, small GTPases of the RhoA family play essential regulatory roles. Vav proteins are activated by EGFR and fibroblast growth factor 2 receptor signalling.32 VAV3 also plays a key role as part of the integrin adhesome, a complex interactome involved in the transduction of integrin-related signalling.33 34 VAV3-deficient mice have hypertension and cardiac hypertrophy.35 In the immune system, deletion of Vav genes results in compromised immune responses.36
Of specific interest to ARDS, miR-27a was shown to be upregulated in microparticles derived from vascular smooth muscle cells exposed to mechanical cyclic stretch. Multipoint injection of antagomiR-27a around carotid artery decreased miR-27a expression in vivo and reversed abnormal endothelial cell proliferation suggesting a role for miR-27a in vascular remodelling.37 In studies using Townes humanised sickle cell (SS) murine models of haemolysis triggered endothelial dysfunction. In this model, haemin released during haemolysis (also happens in severe ARDS) leads to pulmonary hypertension and right ventricular hypertrophy associated with reduced lung levels of peroxisome proliferator-activated receptor γ and increased levels of miR-27a; inhibition of miR-27a reconstituted endothelin-1 levels and mitigated endothelial cell dysfunction in the SS mouse lung.38 Our study focused on the role of miR-27a in ALI, possibly longer time points would provide important clues to a role in repair and remodelling postacute injury.
While in our model the 5p mature strand of miR-27a was the primary target of regulation, our initial experiments have shown that miR-27a-3p is also upregulated in response to an inflammatory stimulus in HPMECs. Future studies involving detailed analysis of both targets will elucidate their role(s) in ARDS. Here we focused primarily on the role of miR-27a-5p in endothelial cells but given the critical role of VAV3 in immune cells, future studies using gene-specific deletion mice are warranted. Exciting results from time-series miRNA-mRNA integrated analysis identified various miRNAs whose expression differs significantly between M1 and M2 polarised macrophages and miR-27a-5p was one of the top miRs regulated during M2 polarisation.19 Furthermore, upregulation of miR-27a enhances the expression of proinflammatory cytokines in TLR4-activated macrophages via downregulation of interleukin 10 (IL-10).25 Since MSC treatment is known to induce polarisation of macrophages, possibly in an IL-10 dependent manner,20 this suggests regulation of miR-27a in myeloid derived cells might play an important role in defining myeloid endophenotypes following ALI.
In summary, although various genes were predicted to be miR-27a-5p targets (ACE1, EGFR, FGF7 and NRF2), the regulation of VAV3 by miR-27a-5p in primary human microvascular endothelial cells was the most robust in vitro. This does not preclude the coexistence of other tight regulatory relationships between miR-27a-5p and its other targets in other cells, by alternative stimuli and/or at different time points. Our in vivo experiments show proof of principle that miR-27a-5p plays an important role in early ARDS. Future studies are currently underway to demonstrate whether RNA-based therapeutics at later time points represents a viable option for treatment. From a clinical perspective, the potential pleotropic beneficial effects of miR-27a-5p inhibition suggest RNA-based therapeutics targeting this miRNA early in the course of illness may modulate the progression of ALI and ARDS.
We thank Dr Ana Paula Teixeira for her review of the manuscript and Dr Yuexin Shan for experimental technical support.
NY and LZ contributed equally.
Contributors Acquisition: NY, LZ, HA, SHJM and RH; analysis: LZ, HA, NY, PH, CCdS; interpretation: CCdS, PH, WCWL, PM, DJS; approval and revising of the work: PH, SHJM, DJS, JAL, WCWL, PM, CCdS. Final approval of the version: all authors.
Funding This work was supported by the Canadian Institutes of Health Research (Grant No: MOP-106545 to CCdS; MOP-74752 to DJS) and the Ontario Thoracic Society (OTS2010/2011/2012 to CCdS). NSERC Doctoral Canada Graduate Scholarship and Ontario Graduate Scholarship (SHJM), the Weston Foundation (JJH), the McLaughlin Centre for Molecular Medicine and a Canada Research Chair in Infectious Diseases and Inflammation (WCWL).
Competing interests None declared.
Patient consent for publication Not required.
Provenance and peer review Not commissioned; externally peer reviewed.
Data availability statement Data are available in a public, open access repository. Dataset deposited in GEO—dataset ID: GSE40180. https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE40180.