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The Mechanosensitive Ion Channel TRPV4 is a Regulator of Lung Development and Pulmonary Vasculature Stabilization

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Abstract

Introduction

Clinical observations and animal models suggest a critical role for the dynamic regulation of transmural pressure and peristaltic airway smooth muscle contractions for proper lung development. However, it is currently unclear how such mechanical signals are transduced into molecular and transcriptional changes at the cell level. To connect these physical findings to a mechanotransduction mechanism, we identified a known mechanosensor, TRPV4, as a component of this pathway.

Methods

Embryonic mouse lung explants were cultured on membranes and in submersion culture to modulate explant transmural pressure. Time-lapse imaging was used to capture active changes in lung biology, and whole-mount images were used to visualize the organization of the epithelial, smooth muscle, and vascular compartments. TRPV4 activity was modulated by pharmacological agonism and inhibition.

Results

TRPV4 expression is present in the murine lung with strong localization to the epithelium and major pulmonary blood vessels. TRPV4 agonism and inhibition resulted in hyper- and hypoplastic airway branching, smooth muscle differentiation, and lung growth, respectively. Smooth muscle contractions also doubled in frequency with agonism and were reduced by 60% with inhibition demonstrating a functional role consistent with levels of smooth muscle differentiation. Activation of TRPV4 increased the vascular capillary density around the distal airways, and inhibition resulted in a near complete loss of the vasculature.

Conclusions

These studies have identified TRPV4 as a potential mechanosensor involved in transducing mechanical forces on the airways to molecular and transcriptional events that regulate the morphogenesis of the three essential tissue compartments in the lung.

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Abbreviations

BPD:

Bronchopulmonary dysplasia

TRPV4:

Transient receptor potential cation channel subfamily V member 4

Pa:

Pascal

PV:

Pulmonary vasculature

PH:

Pulmonary hypertension

HIF1α :

Hypoxia-inducible factor 1 alpha

VEGF:

Vascular endothelial growth factor

ASM:

Airway smooth muscle

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Acknowledgments

The authors would like to thank Mr. Peter Sariano and Ms. Julia Pelesko for their technical assistance. This work was supported in part by grants from the National Institutes of Health (R01HL133163, R21ES027962, P20GM103446, U54GM104941, S10OD016361), the National Science Foundation (1537256), the Oak Ridge Associated Universities Ralph E. Powe Junior Faculty Enhancement Award (J.P.G.) and the March of Dimes Basil O’Connor Award (5-FY16-33 to J.P.G).

Conflict of interest

Joshua T. Morgan, Wade G. Stewart, Robert A. McKee and Jason P. Gleghorn report no conflicts of interest.

Human and Animal Studies

No human studies were carried out by the authors for this article. All institutional and national guidelines for the care and use of laboratory animals were followed and approved by the appropriate institutional committees at the University of Delaware.

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Author notes

  1. Joshua T. Morgan

    Present address: Department of Bioengineering, University of California, Riverside, CA, USA

  2. Joshua T. Morgan and Wade G. Stewart have equally contributed to this work.

Authors and Affiliations

  1. Department of Biomedical Engineering, University of Delaware, 161 Colburn Lab, Newark, DE, 19716, USA

    Joshua T. Morgan, Wade G. Stewart, Robert A. McKee & Jason P. Gleghorn

  2. Department of Biological Sciences, University of Delaware, 161 Colburn Lab, Newark, DE, 19716, USA

    Jason P. Gleghorn

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Correspondence to Jason P. Gleghorn.

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Associate Editor Michael R. King oversaw the review of this article.

Jason P. Gleghorn is an Assistant Professor at the University of Delaware in the Department of Biomedical Engineering. Gleghorn received his Ph.D. from Cornell University under the mentorship of Lawrence Bonassar. He then completed postdoctoral fellowships at Princeton University with Celeste Nelson and Cornell University with Brian Kirby. During his postdoctoral training, Gleghorn applied microfluidic and microfabrication techniques to identify new physical mechanisms that regulate organ development and he created novel microfluidic systems to isolate rare circulating tumor cells from patient blood samples respectively. His lab, started in 2014 at the University of Delaware, develops and uses microfluidic and microfabrication technologies to determine how cells behave and communicate within multicellular populations to form complex 3D tissues and organs. The long-term goals of this research are to develop techniques to engineer physiologically relevant 3D culture systems with well-defined structure, flows, and cell-cell interactions to study tissue-scale biology and disease. These techniques in combination with what they learn in studies of the native cellular behaviors and interactions in the embryo are used to define new therapeutic approaches for regenerative medicine. Gleghorn’s honors include the ORAU Powe Junior Faculty Award, the March of Dimes Basil O’Connor Award, the UD Bernard Canavan Faculty Research Award, and the BMES CMBE Rising Star Award.

This article is part of the 2018 CMBE Young Innovators special issue.

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12195_2018_538_MOESM1_ESM.tif

Figure S1: TRPV4 antibody Specificity. Mouse lung epithelial (MLE12) cells were transfected with siRNA specific to TRPV4 or to a non-targeting control (siNT) using Lipofectamine RNAiMAX according to manufacturer instructions. After 72 h, the cells were fixed and stained for TRVP4 and counterstained with DAPI to identify the nuclei. There was a substantial decrease in TRPV4 staining intensity with siTRPV4 compared to siNT treated cells. Scale bars 50 µm. Supplementary material 1 (TIFF 29097 kb)

12195_2018_538_MOESM2_ESM.mp4

Movie S1: TRPV4 regulates active contractility of the developing lung. Embryonic mouse lungs were isolated at E12.5 and cultured on floating membranes for ~ 48 h before 1 Hz imaging. (A) Cultured lungs demonstrated active airway contraction, and this was influenced by TRVP4 modulation. This video spans 100 s, and the control lung visibly contracts at ~ 50 s, whereas 100 nM GSK1016790A (Activator) treated lung visibly contracts at ~ 36 and ~ 68 s, and the lung treat with 10 µM GSK205 (Inhibitor) does not visibly contract. Supplementary material 2 (MP4 1226 kb)

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Morgan, J.T., Stewart, W.G., McKee, R.A. et al. The Mechanosensitive Ion Channel TRPV4 is a Regulator of Lung Development and Pulmonary Vasculature Stabilization. Cel. Mol. Bioeng. 11, 309–320 (2018). https://doi.org/10.1007/s12195-018-0538-7

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