BackgroundExtensive alveolar epithelial injury and remodelling is a common feature of acute lung injury and acute respiratory distress syndrome (ARDS) and it has been established that epithelial regeneration, and secondary lung oedema resorption, is crucial for ARDS resolution. Much evidence indicates that K+ channels are regulating epithelial repair processes; however, involvement of the KCa3.1 channels in alveolar repair has never been investigated before.ResultsWound-healing assays demonstrated that the repair rates were increased in primary rat alveolar cell monolayers grown on a fibronectin matrix compared to non-coated supports, whereas an anti-β1-integrin antibody reduced it. KCa3.1 inhibition/silencing impaired the fibronectin-stimulated wound-healing rates, as well as cell migration and proliferation, but had no effect in the absence of coating. We then evaluated a putative relationship between KCa3.1 channel and the migratory machinery protein β1-integrin, which is activated by fibronectin. Co-immunoprecipitation and immunofluorescence experiments indicated a link between the two proteins and revealed their cellular co-distribution. In addition, we demonstrated that KCa3.1 channel and β1-integrin membrane expressions were increased on a fibronectin matrix. We also showed increased intracellular calcium concentrations as well as enhanced expression of TRPC4, a voltage-independent calcium channel belonging to the large TRP channel family, on a fibronectin matrix. Finally, wound-healing assays showed additive effects of KCa3.1 and TRPC4 inhibitors on alveolar epithelial repair.ConclusionTaken together, our data demonstrate for the first time complementary roles of KCa3.1 and TRPC4 channels with extracellular matrix and β1-integrin in the regulation of alveolar repair processes.

Understanding the biological function of amyloid beta (Aβ) precursor protein (App) beyond its role in Alzheimer's disease is emerging. Yet, its function during embryonic development is poorly understood. The zebrafish APP orthologue, Appb, is strongly expressed during early development but thus far has only been studied via morpholino-mediated knockdown. Zebrafish enables analysis of cellular processes in an ontogenic context, which is limited in many other vertebrates. We characterized zebrafish carrying a homozygous mutation that introduces a premature stop in exon 2 of the appb gene. We report that appb mutants are significantly smaller until 2 dpf and display perturbed enveloping layer (EVL) integrity and cell protrusions at the blastula stage. Moreover, appb mutants surviving beyond 48 hpf exhibited no behavioral defects at 6 dpf and developed into healthy and fertile adults. The expression of the app family member, appa, was also found to be altered in appb mutants. Taken together, we show that appb is involved in the initial development of zebrafish by supporting the integrity of the EVL, likely by mediating cell adhesion properties. The loss of Appb might then be compensated for by other app family members to maintain normal development. The amyloid beta (Aβ) precursor protein (APP) is an integral membrane protein recognized for its role in Alzheimer's disease (AD) pathogenesis. It is a single-pass glycosylated type I transmembrane protein that is processed by proteases into numerous extracellular and intracellular biologically active fragments 1. Cleavage of APP by βand γ-secretases generates Aβ peptide fragments that aggregate into extracellular plaques in the brain with AD 2-4. Moreover, recent studies have uncovered that APP might also contribute to AD-related neurodegeneration in Aβ-independent ways 5,6. APP is ubiquitously expressed with especially strong expression in the central nervous system 7-9. However, its basic biological role has been difficult to elucidate, likely due to redundancy between APP family members 10. Nevertheless, studies suggest fundamental functions of APP in axonal transport, cell differentiation and proliferation, synaptic transmission, learning and memory, neuronal development, neurite outgrowth, as well as in cell adhesion 11. Mice with a deletion of App are viable and fertile but show reduced body weight, grip strength and locomotor activity 12,13. Similarly, single Aplp1 and Aplp2 knockouts also display mild phenotypes. However, App −/− /Aplp2 −/− or Aplp2 −/− /Aplp1 −/− double knockouts and triple knockouts (App −/− /Aplp1 −/− /Aplp2 −/−) show severe abnormalities and lethality 14. Here, we have performed a detailed characterization of zebrafish with a homozygous mutation in appb, generated with CRISPR/Cas9 technology. As an animal model, zebrafish has several advantages over, e.g., rodents;

Endothelial and epithelial cell transition to a mesenchymal phenotype was identified as cellular paradigms implicated in the appearance of fibroblasts and development of reactive fibrosis in interstitial lung disease. The intermediate filament protein nestin was highly expressed in fibrotic tissue, detected in fibroblasts and participated in proliferation and migration. The present study tested the hypothesis that the transition of endothelial and epithelial cells to a mesenchymal phenotype was delineated by nestin expression. Three weeks following hypobaric hypoxia, adult male Sprague-Dawley rats characterized by alveolar and perivascular lung fibrosis were associated with increased nestin protein and mRNA levels and marked appearance of nestin/collagen type I((+))-fibroblasts. In the perivascular region of hypobaric hypoxic rats, displaced CD31((+))-endothelial cells were detected, exhibited a mesenchymal phenotype and co-expressed nestin. Likewise, epithelial cells in the lungs of hypobaric hypoxic rats transitioned to a mesenchymal phenotype distinguished by the co-expression of E-cadherin and collagen. Following the removal of FBS from primary passage rat alveolar epithelial cells, TGF-β1 was detected in the media and a subpopulation acquired a mesenchymal phenotype characterized by E-cadherin downregulation and concomitant induction of collagen and nestin. Bone morphogenic protein-7 treatment of alveolar epithelial cells prevented E-cadherin downregulation, suppressed collagen induction but partially inhibited nestin expression. These data support the premise that the transition of endothelial and epithelial cells to a mesenchymal cell may have contributed in part to the appearance nestin/collagen type I((+))-fibroblasts and the reactive fibrotic response in the lungs of hypobaric hypoxic rats.

Alveolar ion and fluid absorption is essential for lung homeostasis in healthy conditions as well as for the resorption of lung edema, a key feature of acute respiratory distress syndrome. Liquid absorption is driven by active transepithelial sodium transport, through apical ENaC Na+ channels and basolateral Na+/K+-ATPase. Our previous work unveiled that KvLQT1 K+ channels also participate in the control of Na+/liquid absorption in alveolar epithelial cells. Our aim was to further investigate the function of KvLQT1 channels and their interplay with other channels/transporters involved in ion/liquid transport in vivo using adult wild-type (WT) and KvLQT1 knock-out (KO) mice under physiological conditions and after thiourea-induced lung edema. A slight but significant increase in water lung content (WLC) was observed in naïve KvLQT1-KO mice, relative to WT littermates, whereas lung function was generally preserved and histological structure unaltered. Following thiourea-induced lung edema, KvLQT1-KO did not worsen WLC or lung function. Similarly, lung edema was not aggravated by the administration of a KvLQT1 inhibitor (chromanol). However, KvLQT1 activation (R-L3) significantly reduced WLC in thiourea-challenged WT mice. The benefits of R-L3 were prevented in KO or chromanol-treated WT mice. Furthermore, R-L3 treatment had no effect on thiourea-induced endothelial barrier alteration but restored or enhanced the levels of epithelial alveolar AQP5, Na+/K+-ATPase, and ENaC expressions. Altogether, the results indicate the benefits of KvLQT1 activation in the resolution of lung edema, probably through the observed up-regulation of epithelial alveolar channels/transporters involved in ion/water transport.