Adenosine A1 Receptors Promote Vasa Vasorum Endothelial Cell Barrier Integrity via Gi_{i} and Akt-Dependent Actin Cytoskeleton Remodeling

Background: In a neonatal model of hypoxic pulmonary hypertension, a dramatic pulmonary artery adventitial thickening, accumulation of inflammatory cells in the adventitial compartment, and angiogenic expansion of the vasa vasorum microcirculatory network are observed. These pathophysiological responses suggest that rapidly proliferating vasa vasorum endothelial cells (VVEC) may exhibit increased permeability for circulating blood cells and macromolecules. However, the molecular mechanisms underlying these observations remain unexplored. Some reports implicated extracellular adenosine in the regulation of vascular permeability under hypoxic and inflammatory conditions. Thus, we aimed to determine the role of adenosine in barrier regulation of VVEC isolated from the pulmonary arteries of normoxic (VVEC-Co) or chronically hypoxic (VVEC-Hyp) neonatal calves. Principal Findings We demonstrate via a transendothelial electrical resistance measurement that exogenous adenosine significantly enhanced the barrier function in VVEC-Co and, to a lesser extent, in VVEC-Hyp. Our data from a quantitative reverse transcription polymerase chain reaction show that both VVEC-Co and VVEC-Hyp express all four adenosine receptors (A1, A2A, A2B, and A3), with the highest expression level of A1 receptors (A1Rs). However, A1R expression was significantly lower in VVEC-Hyp compared to VVEC-Co. By using an A1R-specific agonist/antagonist and siRNA, we demonstrate that A1Rs are mostly responsible for adenosine-induced enhancement in barrier function. Adenosine-induced barrier integrity enhancement was attenuated by pretreatment of VVEC with pertussis toxin and GSK690693 or LY294002, suggesting the involvement of G$_{i}$ proteins and the PI3K-Akt pathway. Moreover, we reveal a critical role of actin cytoskeleton in VVEC barrier regulation by using specific inhibitors of actin and microtubule polymerization. Further, we show that adenosine pretreatment blocked the tumor necrosis factor alpha (TNF-α)-induced permeability in VVEC-Co, validating its anti-inflammatory effects. Conclusions: We demonstrate for the first time that stimulation of A1Rs enhances the barrier function in VVEC by activation of the G$_{i}$/PI3K/Akt pathway and remodeling of actin microfilament

Effects of adenosine receptor agonists on the VVEC barrier function.

<p>Activation of A1R improves VVEC barrier function. VVE-Co (<b>A</b>) and VVEC-Hyp (<b>B</b>) were stimulated with various agonists of adenosine receptors (CCPA, 1 nM; CGS21680, 30 nM; BAY 60-5683 10 nM; IB-MECA, 1 nM) and barrier function was analyzed by TER. VVE-Co (<b>C</b>) and VVEC-Hyp (<b>D</b>) were stimulated with adenosine (Ado, 100 μM) with and without A1R specific antagonist (PSB 36, 1 nM, 30 min), and barrier function was analyzed by TER. VVE-Co (E) and VVEC-Hyp (F) were stimulated with A1R specific agonist (CCPA, 1 nM) with and without A1R antagonist (PSB 36, 1 nM, 30 min), and barrier function was analyzed by TER.</p

Adenosine-induced AKT phosphorylation in VVEC is mediated via Gαi.

<p>To dissect a role of Gi proteins in Akt activation, VVEC-Co (<b>A</b>) and VVEC-Hyp (<b>C</b>) were pre-treated with PTx (100 ng/ml, 18 h) and stimulated with 100 μM adenosine (Ado) or 10 nM CCPA for the indicated periods of time. To determine the role of adenosine A1R in Akt activation, VVEC-Co (<b>B</b>) and VVEC-Hyp (<b>D</b>) were pre-treated with 10 nM PSB 36 (30 min), a specific A1R antagonist, followed by stimulation with 100 µM adenosine (Ado) or 10 nM CCPA for the indicated periods of time. Data are representative from at least three independent experiments.</p

The qRT-PCR analysis of adenosine receptors expression in VVEC.

<p>The fold change in mRNA expression of each adenosine receptor relative to internal housekeeping gene (ß-actin) was calculated. (<b>A</b>) Adenosine receptor mRNA levels were normalized versus VVEC-Co A3R, whose expression level was arbitrarily established as 1. The results are shown as mean ± SEM from at least three distinct VVEC populations. Data were analyzed by one-way ANOVA followed by Tukey's multiple comparison test for VVEC-Co and VVEC-Hyp separately. (<b>B</b>) Adenosine receptor mRNA levels in VVEC-Hyp were normalized to VVEC-Co for each gene. Data were analyzed by Student's t-test. **p<0.01, ***p<0.001.</p

Proliferation of VVEC-Co and VVEC-Hyp in ECIS arrays.

<p>Equal numbers of VVEC-Co and VVEC-Hyp (100,000 cells/well) were seeded in ECIS arrays and the TER was measured for 36 h. Results are presented as mean ± SEM and derived from three independent experiments.</p

Schematic representation of the proposed signaling pathway of adenosine-induced enhancement of barrier function in VVEC.

<p>Schematic representation of the proposed signaling pathway of adenosine-induced enhancement of barrier function in VVEC.</p

PI3K/Akt pathway mediates adenosine-induced increase in TER in VVEC.

<p>VVEC-Co (<b>A</b>) and VVEC-Hyp (<b>B</b>) were pre-incubated with LY294002 (5 µM; PI3K inhibitor) or GSK690693 (10 nM; Akt inhibitor) for 30 min and then exposed to adenosine. Barrier function was measured by TER assay. Results were obtained from three independent experiments and are presented as mean ± SE. * p<0.05.</p

Adenosine enhances the VVEC barrier function.

<p>VVEC monolayers in ECIS arrays were incubated in serum free medium for 1 h. Adenosine (50–500 µM) was added to VVEC-Co (<b>A</b>) or VVEC-Hyp (<b>B</b>) after a steady baseline was established, and the TER measurements continued for 6 h. Data are representative of multiple independent experiments (minimum of three).</p

A1R is involved in adenosine-induced VVEC barrier function. Effect of A1R siRNA on CCPA-induced increase in TER in VVEC.

<p>(<b>A, B</b>) VVEC were incubated with A1R specific siRNA or non-specific siRNA for 48 h and then cells were stimulated with CCPA (1 nM) in TER measurement assay. The depletion of A1R mRNA and protein was confirmed by RT-PCR (<b>C</b>) and the Western blot analysis with anti-A1R antibody. (<b>D</b>). Results are presented as mean ± SE from three independent experiments.</p