Ca2+ Signaling and Barrier Function of Lung Microvascular Endothelial Cells are Modulated by Mesenchymal Stromal Cell Microparticles
Authors: Maishan Mazharul , McVey Mark J. , Curley Gerard F. , Kuebler Wolfgang M. 
Published: June 2021

Introduction

Acute respiratory distress syndrome (ARDS) is a fatal condition characterized by hyperinflammation and pulmonary microvascular leak. With no pharmacological cure, treatment remains only supportive. Mesenchymal stromal cells (MSCs) have recently generated excitement as a potential therapy, presumably functioning through paracrine mechanisms which may rely on the release of extracellular vesicles like microparticles (MPs). However, the mechanism by which MPs modulate the pathology of ARDS is poorly understood yet may pave the way to develop cell‐free cell therapeutics.

Objective

To identify the mechanism(s) by which MSC derived MPs enhance pulmonary microvascular barrier function.

Methods

MPs were purified from conditioned medium produced by MSCs either stimulated with Ca2+ ionophore or unstimulated. Monolayers of primary human pulmonary microvascular endothelial cells (HPMECs) were injured with thrombin to induce barrier disruption, measured by transendothelial electrical resistance (TEER), mimicking lung microvascular leak as observed in ARDS. MPs were administered to treat thrombin‐injured HPMECs and changes in intracellular Ca2+ concentration ([Ca2+]i) were determined by ratiometric imaging of Fura‐2.

Results

Thrombin administered to HPMECs caused an initial, brief spike in [Ca2+]i followed by a second phase characterized as a prolonged, gradual increase in [Ca2+]i. Following the thrombin‐induced [Ca2+]i spike, treatment with MPs from unstimulated MSCs eliminated the second phase and caused a sustained, decrease in [Ca2+]i below baseline. In parallel, MPs from unstimulated MSCs enhanced TEER recovery and VE‐cadherin integrity in intercellular junctions of HPMECs following thrombin‐induced permeability. Conversely, treatment with MPs from Ca2+ ionophore stimulated MSCs amplified the second phase of the [Ca2+]i response to thrombin and prevented recovery of HPMEC barrier function. HPLC‐MS revealed MPs from unstimulated MSCs had lower ceramide and higher sphingosine‐1‐phosphate (S1P) content than MPs from Ca2+ ionophore stimulated MSCs. S1P degradation by S1P lyase or blockade of the S1P receptor 1 attenuated the barrier‐protective effect of MPs from unstimulated MSCs, while ceramide degradation by neutral ceramidase improved barrier recovery following treatment with MPs from Ca2+ ionophore stimulated MSCs.

Conclusion

MPs from unstimulated MSCs therapeutically enhance lung capillary barrier function, presumably by attenuating endothelial [Ca2+]i responses by resetting the ceramide/S1P rheostat. These findings provide a mechanistic basis for development and optimization/enrichment of MSC derived MPs as a promising cell‐free cell therapy for ARDS.

Support or Funding Information

This work was supported by the Ontario Research Fund and Canadian Institutes of Health Research.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.