Microfluidic shear stress-regulated surfactant secretion in alveolar epithelial type II cells in vitro

Abstract

We investigated the role of flow-induced shear stress on the mechanisms regulating surfactant secretion in type II alveolar epithelial cells (ATII) using microfluidic models. Following flow stimulation spanning a range of wall shear stress (WSS) magnitudes, monolayers of ATII (MLE-12 and A549) cells were examined for surfactant secretion by evaluating essential steps of the process, including relative changes in the number of fusion events of lamellar bodies (LBs) with the plasma membrane (PM) and intracellular redistribution of LBs. F-actin cytoskeleton and calcium levels were analyzed in A549 cells subjected to WSS spanning 4-20 dyn/cm(2). Results reveal an enhancement in LB fusion events with the PM in MLE-12 cells upon flow stimulation, whereas A549 cells exhibit no foreseeable changes in the monitored number of fusion events for WSS levels ranging up to a threshold of ∼8 dyn/cm(2); above this threshold, we witness instead a decrease in LB fusion events in A549 cells. However, patterns of LB redistribution suggest that WSS can potentially serve as a stimulus for A549 cells to trigger the intracellular transport of LBs toward the cell periphery. This observation is accompanied by a fragmentation of F-actin, indicating that disorganization of the F-actin cytoskeleton might act as a limiting factor for LB fusion events. Moreover, we note a rise in cytosolic calcium ([Ca(2+)]c) levels following stimulation of A549 cells with WSS magnitudes ranging near or above the experimental threshold. Overall, WSS stimulation can influence key components of molecular machinery for regulated surfactant secretion in ATII cells in vitro.

A Pneumocyte–Macrophage Paracrine Lipid Axis Drives the Lung toward Fibrosis

“…At later time points, when fibrotic responses were decreasing (e.g., TGF-b1 and collagen levels were falling), lactic acid and tissue ATP levels began to normalize ( Figures 3C and 3D), suggesting that resolution of fibrotic responses is associated with the recovery of metabolic homeostasis. We next assessed the ability of bleomycin to directly induce a starvation response in alveolar epithelial cells using the ATII model cell line MLE-12 (28,29). After exposure to bleomycin for 24 hours, metabolic changes in MLE-12 cells recapitulated those observed in whole lungs, including down-regulation of factors necessary for lipid synthesis (Chrebp, Srebp1c, and Fasn) ( Figure 3E), phosphorylation of AMPK and ACC ( Figure 3F), and increased production of lactic acid ( Figure E7).…”

Biomimetics of fetal alveolar flow phenomena using microfluidics

“…Standard soft-lithography techniques 27,40 were used to fabricate a poly(dimethylsiloxane) (PDMS) microfluidic-device modeling pulmonary structures and respiratory features at realistic, true one-to-one-anatomical scales. 2,28 Our model combines straight channels (i.e., w ¼ 400 lm in width and h ¼ 100 lm in height) with cylindrical cavities (R ¼ 40 lm in radius), modeling fetal airway ducts in combination with alveolar airspaces (Figs.…”

“…To examine the production of pulmonary surfactant, A549 cells were fluorescently marked for lamellar bodies 27,38 (LBs). Cells were loaded with 1 lM quinacrine (Sigma), a fluorescent marker of LBs, for 40 min at 37 .…”

“…Since WSS acting upon AEC II is acknowledged to influence regulated pathways of surfactant secretion, as well as distribution of actin filaments within A549 cells, 27 we have sought to quantify WSS exerted on the epithelium to account for finite shear stresses emerging in utero, as a result of FBMs. Here, we have used numerical simulations (see Sec.…”

“…To date, investigations of WSS on AECII have been mostly driven by considerations of cell injury and damage caused by liquid plug propagations along airways; [24][25][26] only recently has the role of WSS begun attracting attention in the context of surfactant secretion. 27 Hence, despite ongoing progress in uncovering physiological responses of AECII to mechanical and flow-induced stimuli, few if any studies have addressed AECII function in the context of the significant morphological changes that arise in utero during fetal lung development.…”