Substrate Stiffness Modulates Migration and Local Intercellular Membrane Motion in Pulmonary Endothelial Cell Monolayers

Authors

Sunita Subedi Paudel, University of South Alabama
Althea deWeever, University of South AlabamaFollow
Sarah Sayner, University of South AlabamaFollow
Troy Stevens, University of South AlabamaFollow
Dhananjay T. Tambe, University of South AlabamaFollow

Document Type

Article

Publication Title

American Journal of Physiology-Cell Physiology

Abstract

The pulmonary artery endothelium forms a semipermeable barrier that limits macromolecular flux through intercellular junctions. This barrier is maintained by an intrinsic forward protrusion of the interacting membranes between adjacent cells. However, the dynamic interactions of these membranes have been incompletely quantified. Here, we present a novel technique to quantify the motion of the peripheral membrane of the cells, called paracellular morphological fluctuations (PMFs), and to assess the impact of substrate stiffness on PMFs. Substrate stiffness impacted large-length scale morphological changes such as cell size and motion. Cell size was larger on stiffer substrates, whereas the speed of cell movement was decreased on hydrogels with stiffness either larger or smaller than 1.25 kPa, consistent with cells approaching a jammed state. Pulmonary artery endothelial cells moved fastest on 1.25 kPa hydrogel, a stiffness consistent with a healthy pulmonary artery. Unlike these large-length scale morphological changes, the baseline of PMFs was largely insensitive to the substrate stiffness on which the cells were cultured. Activation of store-operated calcium channels using thapsigargin treatment triggered a transient increase in PMFs beyond the control treatment. However, in hypocalcemic conditions, such an increase in PMFs was absent on 1.25 kPa hydrogel but was present on 30 kPa hydrogel—a stiffness consistent with that of a hypertensive pulmonary artery. These findings indicate that 1) PMFs occur in cultured endothelial cell clusters, irrespective of the substrate stiffness; 2) PMFs increase in response to calcium influx through store-operated calcium entry channels; and 3) stiffer substrate promotes PMFs through a mechanism that does not require calcium influx.

First Page

C936

Last Page

C949

DOI

10.1152/ajpcell.00339.2021

Publication Date

Fall 9-2022

Department

College of Medicine

Comments

Copyright © 2022 the American Physiological Society. https://doi.org/10.1152/ajpcell.00339.2021

Supplemental tables, figures, and videos, along with copy right and usage permissions can be at the following links:

Graphical Abstract: doi: 10.1152/ajpcell.00339.2021

Supplemental Figs. S1 and S2; see https://doi.org/10.6084/m9.figshare.17098742

Supplemental Figs. S3–S6; see https://doi.org/10.6084/m9.figshare.16567749

Supplemental Figs. S7 and S8; see https://doi.org/10.6084/m9.figshare.19878733

Supplemental Tables S1–S4; see https://doi.org/10.6084/m9.figshare.16584056

Fig. 2, A–E, Supplemental Videos S1–S5; see https://doi.org/10.6084/m9.figshare.16563912

Supplemental Videos S6–S13; see https://doi.org/10.6084/m9.figshare.16563915

Recommended Citation

Paudel, Sunita Subedi, et al. "Substrate stiffness modulates migration and local intercellular membrane motion in pulmonary endothelial cell monolayers." American Journal of Physiology-Cell Physiology 323.3 (2022): C936-C949.