Abstract
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.
Highlights
It is increasingly accepted that large artery stiffening, which commonly occurs with aging, hypertension, diabetes, etc., contributes to the microvascular abnormalities of the kidney, brain, and eyes that characterize these pathophysiologic conditions [1,2,3,4,5]
Development of the mimetic flow circulatory system To determine the effects of upstream compliance and flow pulsatility on proinflammatory responses and underlying molecular signaling in downstream pulmonary artery endothelial cells (PAECs), we stimulated PAECs with pulsatile flows downstream to ‘‘soft’’ or ‘‘stiff’’ tubes which, by virtue of differences in tube deformation, dampened the pulsatile flow from the pump to various degrees (Figure 1)
Because TLR4 has more complicated role and relatively minor effects on high pulsatility flow (HPF)-mediated responses compared to TLR2, we have focused on illuminating TLR2-mediated signaling underlying endothelial mechanotransduction of flow pulsatility
Summary
It is increasingly accepted that large artery stiffening, which commonly occurs with aging, hypertension, diabetes, etc., contributes to the microvascular abnormalities of the kidney, brain, and eyes that characterize these pathophysiologic conditions [1,2,3,4,5]. A group of progressive and fatal diseases, it has become evident that stiffening of large proximal pulmonary arteries occurs, often early, in the course of this spectrum of diseases that have been conventionally characterized by dysfunction and obliteration of small distal pulmonary arteries [6]. While both clinical and animal studies convincingly demonstrate an association between proximal artery stiffening and distal artery dysfunction, few studies have examined the underlying cellular and molecular mechanisms through which these pathologic features might be inherently linked. It is clear that a better understanding of the contribution of pulsatility (the kinetic component) of unidirectional physiologic flow to molecular changes in the downstream vascular endothelium is necessary for a better understanding of the effects of artery stiffening on cardiovascular health
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