TY - JOUR
T1 - Multiplexed, high-throughput measurements of cell contraction and endothelial barrier function
AU - Rokhzan, Ramin
AU - Ghosh, Chandra C.
AU - Schaible, Niccole
AU - Notbohm, Jacob
AU - Yoshie, Haruka
AU - Ehrlicher, Allen J.
AU - Higgins, Sarah J.
AU - Zhang, Ruyuan
AU - Haller, Hermann
AU - Hardin, Corey C.
AU - David, Sascha
AU - Parikh, Samir M.
AU - Krishnan, Ramaswamy
N1 - Publisher Copyright:
© 2018, United States & Canadian Academy of Pathology.
PY - 2019/1/1
Y1 - 2019/1/1
N2 - Vascular leakage, protein exudation, and edema formation are events commonly triggered by inflammation and facilitated by gaps that form between adjacent endothelial cells (ECs) of the vasculature. In such paracellular gap formation, the role of EC contraction is widely implicated, and even therapeutically targeted. However, related measurement approaches remain slow, tedious, and complex to perform. Here, we have developed a multiplexed, high-throughput screen to simultaneously quantify paracellular gaps, EC contractile forces, and to visualize F-actin stress fibers, and VE-cadherin. As proof-of-principle, we examined barrier-protective mechanisms of the Rho-associated kinase inhibitor, Y-27632, and the canonical agonist of the Tie2 receptor, Angiopoietin-1 (Angpt-1). Y-27632 reduced EC contraction and actin stress fiber formation, whereas Angpt-1 did not. Yet both agents reduced thrombin-, LPS-, and TNFα-induced paracellular gap formation. This unexpected result suggests that Angpt-1 can achieve barrier defense without reducing EC contraction, a mechanism that has not been previously described. This insight was enabled by the multiplex nature of the force-based platform. The high-throughput format we describe should accelerate both mechanistic studies and the screening of pharmacological modulators of endothelial barrier function.
AB - Vascular leakage, protein exudation, and edema formation are events commonly triggered by inflammation and facilitated by gaps that form between adjacent endothelial cells (ECs) of the vasculature. In such paracellular gap formation, the role of EC contraction is widely implicated, and even therapeutically targeted. However, related measurement approaches remain slow, tedious, and complex to perform. Here, we have developed a multiplexed, high-throughput screen to simultaneously quantify paracellular gaps, EC contractile forces, and to visualize F-actin stress fibers, and VE-cadherin. As proof-of-principle, we examined barrier-protective mechanisms of the Rho-associated kinase inhibitor, Y-27632, and the canonical agonist of the Tie2 receptor, Angiopoietin-1 (Angpt-1). Y-27632 reduced EC contraction and actin stress fiber formation, whereas Angpt-1 did not. Yet both agents reduced thrombin-, LPS-, and TNFα-induced paracellular gap formation. This unexpected result suggests that Angpt-1 can achieve barrier defense without reducing EC contraction, a mechanism that has not been previously described. This insight was enabled by the multiplex nature of the force-based platform. The high-throughput format we describe should accelerate both mechanistic studies and the screening of pharmacological modulators of endothelial barrier function.
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U2 - 10.1038/s41374-018-0136-2
DO - 10.1038/s41374-018-0136-2
M3 - Article
C2 - 30310180
AN - SCOPUS:85054734320
SN - 0023-6837
VL - 99
SP - 138
EP - 145
JO - Laboratory Investigation
JF - Laboratory Investigation
IS - 1
ER -