Multiplexed, high-throughput measurements of cell contraction and endothelial barrier function

Ramin Rokhzan, Chandra C. Ghosh, Niccole Schaible, Jacob Notbohm, Haruka Yoshie, Allen J. Ehrlicher, Sarah J. Higgins, Ruyuan Zhang, Hermann Haller, Corey C. Hardin, Sascha David, Samir M. Parikh, Ramaswamy Krishnan

Research output: Contribution to journalArticlepeer-review

2 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)138-145
Number of pages8
JournalLaboratory Investigation
Volume99
Issue number1
DOIs
StatePublished - Jan 1 2019
Externally publishedYes

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Molecular Biology
  • Cell Biology

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