MDA-5 activation by cytoplasmic double-stranded RNA impairs endothelial function and aggravates atherosclerosis.

Recent studies have highlighted the relevance of viral nucleic acid immunorecognition by pattern recognition receptors in atherogenesis. Melanoma differentiation associated gene 5 (MDA-5) belongs to the intracellular retinoic acid inducible gene-I like receptors and its activation promotes pro-inflammatory mechanisms. Here, we studied the effect of MDA-5 stimulation in vascular biology. To gain insights into MDA-5 dependent effects on endothelial function, cultured human coronary artery endothelial cells (HCAEC) were transfected with the synthetic MDA-5 agonist polyIC (long double-stranded RNA). Human coronary endothelial cell expressed MDA-5 and reacted with receptor up-regulation upon stimulation. Reactive oxygen species formation, apoptosis and the release of pro-inflammatory cytokines was enhanced, whereas migration was significantly reduced in response to MDA-5 stimulation. To test these effects in vivo, wild-type mice were transfected with 32.5 µg polyIC/JetPEI or polyA/JetPEI as control every other day for 7 days. In polyIC-treated wild-type mice, endothelium-dependent vasodilation and re-endothelialization was significantly impaired, vascular oxidative stress significantly increased and circulating endothelial microparticles and circulating endothelial progenitor cells significantly elevated compared to controls. Importantly, these effects could be abrogated by MDA-5 deficiency in vivo. Finally, chronic MDA-5 stimulation in Apolipoprotein E/toll-like receptor 3 (TLR3) double(-) deficient (ApoE(-/-) /TLR3(-/-) ) mice-enhanced atherosclerotic plaque formation. This study demonstrates that MDA-5 stimulation leads to endothelial dysfunction, and has the potential to aggravate atherosclerotic plaque burden in murine atherosclerosis. Thus, the spectrum of relevant innate immune receptors in vascular diseases and atherogenesis might not be restricted to TLRs but also encompasses the group of RLRs including MDA-5.

Activation of endothelial toll-like receptor 3 impairs endothelial function.

RATIONALE: Endothelial dysfunction and atherosclerosis are chronic inflammatory diseases characterized by activation of the innate and acquired immune system. Specialized protein receptors of the innate immune system recognize products of microorganisms and endogenous ligands such as nucleic acids. Toll-like receptor 3 (TLR3), for example, detects long double-stranded RNA and is abundantly expressed in endothelial cells. Whether innate immunity contributes to atherogenic mechanisms in endothelial cells is poorly understood. OBJECTIVE: We sought to determine the effects of TLR3 activation in endothelial cells. METHODS AND RESULTS: We first investigated whether stimulation of TLR3 influences endothelial biology in mice. Intravenous injection of polyinosine polycytidylic acid, a synthetic double-stranded RNA analog and TLR3 ligand, impaired endothelium-dependent vasodilation, increased vascular production of reactive oxygen species, and reduced reendothelialization after carotid artery injury in wild-type mice compared with controls but had no effect in TLR3(-/-) animals. TLR3 stimulation not only induced endothelial dysfunction but also enhanced the formation of atherosclerotic plaques in apolipoprotein E-deficient mice. In vitro incubation of endothelial cells with polyinosine polycytidylic acid induced production of the proinflammatory cytokines interleukin-8 and interferon-γ-induced protein 10, increased formation of reactive oxygen species, diminished proliferation, and increased apoptosis, which suggests that endothelial cells are able to directly detect and respond to TLR3 ligands. Neutralization of interleukin-8 and interferon-γ-induced protein 10 antagonizes the observed negative effects of polyinosine polycytidylic acid. We found elevated levels of circulating endothelial progenitor cells in polyinosine polycytidylic acid-treated mice, although they displayed increased endothelial dysfunction. Stimulation of TLR3 in cultured endothelial progenitor cells, however, led to increased formation of reactive oxygen species, increased apoptosis, and reduced migration. Injection of endothelial progenitor cells that had been incubated with polyinosine polycytidylic acid ex vivo hindered reendothelialization after carotid artery injury. Therefore, endothelial progenitor cell function was affected by TLR3 stimulation. Finally, apolipoprotein E-deficient/TLR3-deficient mice exhibited improved endothelial function compared with apolipoprotein E-deficient/TLR3(+/+) littermates. CONCLUSIONS: Immunorecognition of long double-stranded RNA by endothelial cells may be an important mechanism involved in endothelial cell activation and development of endothelial dysfunction.

Endothelial RIG-I activation impairs endothelial function.

BACKGROUND: Endothelial dysfunction is a crucial part of the chronic inflammatory atherosclerotic process and is mediated by innate and acquired immune mechanisms. Recent studies suggest that pattern recognition receptors (PRR) specialized in immunorecognition of nucleic acids may play an important role in endothelial biology in a proatherogenic manner. Here, we analyzed the impact of endothelial retinoic acid inducible gene I (RIG-I) activation upon vascular endothelial biology. METHODS AND RESULTS: Wild type mice were injected intravenously with 32.5 µg of the RIG-ligand 3pRNA (RNA with triphosphate at the 5'end) or polyA control every other day for 7 days. In 3pRNA-treated mice, endothelium-depended vasodilation was significantly impaired, vascular oxidative stress significantly increased and circulating endothelial microparticle (EMP) numbers significantly elevated compared to controls. To gain further insight in RIG-I dependent endothelial biology, cultured human coronary endothelial cells (HCAEC) and endothelial progenitor cells (EPC) were stimulated in vitro with 3pRNA. Both cells types express RIG-I and react with receptor upregulation upon stimulation. Reactive oxygen species (ROS) formation is enhanced in both cell types, whereas apoptosis and proliferation is not significantly affected in HCAEC. Importantly, HCAEC release significant amounts of proinflammatory cytokines in response to RIG-I stimulation. CONCLUSION: This study shows that activation of the cytoplasmatic nucleic acid receptor RIG-I leads to endothelial dysfunction. RIG-I induced endothelial damage could therefore be an important pathway in atherogenesis.