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

Area of Science:

• Endothelial cell biology
• Vascular inflammation
• Biophysics

Background:

• Inflammation causes vascular leakage via gaps between endothelial cells (ECs).
• EC contraction is implicated in paracellular gap formation but difficult to measure.
• Existing measurement methods are slow and complex.

Purpose of the Study:

• Develop a high-throughput screen to quantify paracellular gaps, EC contractile forces, and F-actin stress fibers.
• Investigate barrier-protective mechanisms of Y-27632 and Angiopoietin-1 (Angpt-1).

Main Methods:

• Multiplexed, high-throughput screening platform.
• Simultaneous quantification of paracellular gaps, EC contractile forces, and F-actin stress fibers.
• Assessment of Rho-associated kinase inhibitor Y-27632 and Angpt-1 effects.

Main Results:

• Y-27632 reduced EC contraction and actin stress fibers.
• Angpt-1 did not reduce EC contraction.
• Both Y-27632 and Angpt-1 reduced thrombin-, LPS-, and TNFα-induced paracellular gap formation.
• Angpt-1 demonstrated barrier defense independent of EC contraction.

Conclusions:

• A novel platform enables simultaneous measurement of endothelial barrier parameters.
• Angpt-1 protects vascular integrity through a previously undescribed mechanism independent of EC contraction.
• The high-throughput screen will accelerate research into endothelial barrier function and drug discovery.