Stiffening-Induced High Pulsatility Flow Activates Endothelial Inflammation via a TLR2/NF-κB Pathway

<div><p>Stiffening of large arteries is increasingly used as an independent predictor of risk and therapeutic outcome for small artery dysfunction in many diseases including pulmonary hypertension. The molecular mechanisms mediating downstream vascular cell responses to large artery stiffening remain unclear. We hypothesize that high pulsatility flow, induced by large artery stiffening, causes inflammatory responses in downstream pulmonary artery endothelial cells (PAECs) through toll-like receptor (TLR) pathways. To recapitulate the stiffening effect of large pulmonary arteries that occurs in pulmonary hypertension, ultrathin silicone tubes of variable mechanical stiffness were formulated and were placed in a flow circulatory system. These tubes modulated the simulated cardiac output into pulsatile flows with different pulsatility indices, 0.5 (normal) or 1.5 (high). PAECs placed downstream of the tubes were evaluated for their expression of proinflammatory molecules (ICAM-1, VCAM-1, E-selectin and MCP-1), TLR receptors and intracellular NF-κB following flow exposure. Results showed that compared to flow with normal pulsatility, high pulsatility flow induced proinflammatory responses in PAECs, enhanced TLR2 expression but not TLR4, and caused NF-κB activation. Pharmacologic (OxPAPC) and siRNA inhibition of TLR2 attenuated high pulsatility flow-induced pro-inflammatory responses and NF-κB activation in PAECs. We also observed that PAECs isolated from small pulmonary arteries of hypertensive animals exhibiting proximal vascular stiffening demonstrated a durable ex-vivo proinflammatory phenotype (increased TLR2, TLR4 and MCP-1 expression). Intralobar PAECs isolated from vessels of IPAH patients also showed increased TLR2. In conclusion, this study demonstrates for the first time that TLR2/NF-κB signaling mediates endothelial inflammation under high pulsatility flow caused by upstream stiffening, but the role of TLR4 in flow pulsatility-mediated endothelial mechanotransduction remains unclear.</p></div

The circulating media from the cells exposed to HPF upregulate the inflammatory responses in normal PAECs through the TLR2/NF-κB pathway.

<p>The conditioned circulating media collected from cell cultures under the HPF and LPF conditions, respectively labeled as LPF-FM and HPF-FM, were used to culture normal PAECs for 24 h. (A) The mRNA expressions of ICAM-1, VCAM-1, MCP-1 and E-selectin were upregulated by HPF-FM condition, which were then reduced by OxPAPC and siTLR2. (B) Similar changes were shown in the MCP-1 protein expression. (C) NF-κB intranuclear translocation increased in PAECs stimulated with HPF-FM, which was reduced by OxPAPC and siTLR2. *: p<0.05 versus LPF-FM, †: p<0.05 versus HPF-FM.</p

Activation of TLR2/NF-κB pathway mediated pro-inflammatory responses of PAECs exposed to stiffening-induced HPF.

<p>Pulse flow modulates TLR2-induced NF-κB activation in BPAECs. (A) Representative fluorescent images and quantitative measures of NF-kBp65 staining (red) in PAECs show HPF stimulation of PAECs led to increased intranuclear translocation or activation of NF-κB, which was reduced by TLR2/4 inhibitor OxPAPC and TLR2 siRNA. Blue stains show the nuclei. The scale bar shows 50 µm. (B) HPF stimulation of PAECs increased the mRNA levels of IKKα and IKKβ, both of which were attenuated in siRNA-transfected cells with knockdown of TLR2. (C) NF-κB inhibitor (BAY 11–7082) decreased the MCP-1 expression by PAECs exposed to HPF. *: p<0.05 versus LPF, †: p<0.05 versus HPF.</p

Pharmacological or siRNA inhibition of TLR2 results in suppression of PAEC proinflammatory responses caused by stiffening-induced HPF.

<p>(A, C) At the mRNA level, TLR2/4 inhibitor OxPAPC or TLR2 siRNA but not TLR4 inhibitor CLI-095, decreased PAEC expression of ICAM-1, VCAM-1, MCP-1 and E-selectin mRNAs under HPF; TLR4 siRNA decreased ICAM-1 and E-selectin but not VCAM-1 and MCP-1 mRNAs. “*”: p<0.05 versus LPF, “†”: p<0.05 versus HPF. (B, D) At the protein level, the MCP-1 expression in PAECs exposed to HPF was inhibited by OxPAPC or TLR2 siRNA treatment but not CLI-095 or TLR4 siRNA. The black line in the blot images (D, right) shows separated lanes obtained on the same gel.</p

Enhanced TLR and MCP-1 expression in the distal pulmonary artery endothelium <i>in vivo</i>.

<p>(A) PAECs from calves with hypoxia-induced pulmonary hypertension (PH) show elevated expression of TLR2 and TLR4, compared to control (CO). *:p<0.05. (B) Both immunostaining and western blotting results show elevated MCP-1 expression by PH-ECs compared to CO-ECs from calves. “PA” indicates the lumen of a pulmonary artery. *:p<0.05. (C) Enhanced TLR2 expression in the pulmonary arterial endothelium of human with pulmonary arterial hypertension (PAH). Cryosections of human intra-lobar pulmonary arteries were immunostained with TLR2 (red fluorescence) and counterstained with DAPI (cell nuclei, blue). Elastic lamellae showed green auto-fluorescence.</p

Bovine primer sequences for real-time RT-PCR.

<p>Bovine primer sequences for real-time RT-PCR.</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

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

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

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