AM966, an Antagonist of Lysophosphatidic Acid Receptor 1, Increases Lung Microvascular Endothelial Permeability through Activation of Rho Signaling Pathway and Phosphorylation of VE-Cadherin

Figure 4

LPA1 is required for AM966-induced phosphorylation of VE-cadherin. (a) HLMVECs (~70% confluent) were transfected with LPA1 siRNA (siLPA1) or control (sicont) for 72 h, and then cells were treated with AM966 (1.0 μM) for 30 min. Cell lysates were immunoblotted with P-VE-cadherin, total VE-cadherin, and LPA1 antibodies. (b) Analysis of P-VE-cadherin by densitometry of the results in (a) was performed by Image J software (), and statistical analysis was shown. (c) HLMVECs (~70% confluent) were transfected with minigenes encoding Gα12 or Gα13 peptide for 24 h, and then cells were treated with AM966 (1.0 μM) for 30 min. Cell lysates were immunoblotted with P-VE-cadherin and total VE-cadherin antibodies. (d) Analysis of P-VE-cadherin by densitometry of the results in (c) was performed by Image J software (), and statistical analysis was shown. Shown are representative blots from three independent experiments. (e) HLMVECs (~70% confluent) transfected with LPA1 siRNA (siLPA1) or control (sicont) were plated on gold microelectrodes and then cells were treated with AM966 (1.0 μM) or DMSO. The TEER tracing represents pooled data (±SEM) from 3 independent experiments. (f) The resistance in response to AM966 treatments during indicated time period (e) was quantified and statistical analysis was performed.

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