Endothelial cells suppress monocyte activation through secretion of extracellular vesicles containing antiinflammatory microRNAs.

The blood contains high concentrations of circulating extracellular vesicles (EVs), and their levels and contents are altered in several disease states, including cardiovascular disease. However, the function of circulating EVs, especially the microRNAs (miRNAs) that they contain, are poorly understood. We sought to determine the effect of secreted vesicles produced by quiescent endothelial cells (ECs) on monocyte inflammatory responses and to assess whether transfer of microRNAs occurs between these cells. We observed that monocytic cells cocultured (but not in contact) with ECs were refractory to inflammatory activation. Further characterization revealed that endothelium-derived EVs (EC-EVs) suppressed monocyte activation by enhancing immunomodulatory responses and diminishing proinflammatory responses. EVs isolated from mouse plasma also suppressed monocyte activation. Importantly, injection of EC-EVs in vivo repressed monocyte/macrophage activation, confirming our in vitro findings. We found that several antiinflammatory microRNAs were elevated in EC-EV-treated monocytes. In particular, miR-10a was transferred to monocytic cells from EC-EVs and could repress inflammatory signaling through the targeting of several components of the NF-κB pathway, including IRAK4. Our findings reveal that ECs secrete EVs that can modulate monocyte activation and suggest that altered EV secretion and/or microRNA content may affect vascular inflammation in the setting of cardiovascular disease.

Differential role of an NF-κB transcriptional response element in endothelial versus intimal cell VCAM-1 expression.

RATIONALE: Human and murine Vcam1 promoters contain 2 adjacent nuclear factor-κB (NF-κB)-binding elements. Both are essential for cytokine-induced transcription of transiently transfected promoter-reporter constructs. However, the relevance of these insights to regulation of the endogenous Vcam1 gene and to pathophysiological processes in vivo remained unknown. OBJECTIVE: Determine the role of the 5' NF-κB-binding element in expression of the endogenous Vcam1 gene. METHODS AND RESULTS: Homologous recombination in embryonic stem cells was used to inactivate the 5' NF-κB element in the Vcam1 promoter and alter 3 nucleotides in the 5' untranslated region to allow direct comparison of wild-type versus mutant allele RNA expression and chromatin configuration in heterozygous mice. Systemic treatment with inflammatory cytokines or endotoxin (lipopolysaccharide) induced lower expression of the mutant allele relative to wild-type by endothelial cells in the aorta, heart, and lungs. The mutant allele also showed lower endothelial expression in 2-week atherosclerotic lesions in Vcam1 heterozygous/low-density lipoprotein receptor-deficient mice fed a cholesterol-rich diet. In vivo chromatin immunoprecipitation assays of heart showed diminished lipopolysaccharide-induced association of RNA polymerase 2 and NF-κB p65 with the mutant promoter. In contrast, expression of mutant and wild-type alleles was comparable in intimal cells of wire-injured carotid artery and 4- to 12-week atherosclerotic lesions. CONCLUSIONS: This study highlights differences between in vivo and in vitro promoter analyses, and reveals a differential role for a NF-κB transcriptional response element in endothelial vascular cell adhesion molecule-1 expression induced by inflammatory cytokines or a cholesterol-rich diet versus intimal cell expression in atherosclerotic lesions and injured arteries.

Intravenous immunoglobulin and salicylate differentially modulate pathogenic processes leading to vascular damage in a model of Kawasaki disease.

OBJECTIVE: Kawasaki disease (KD) is a multisystem vasculitis affecting children and is characterized by immune activation in the acute stage of disease. Systemic inflammation eventually subsides, although coronary arteritis persists, resulting in aneurysm formation. KD is the leading cause of acquired heart disease among children in North America. Accepted treatment guidelines include high-dose intravenous immunoglobulin (IVIG) and aspirin in the acute phase. Although this therapy is effective, the cellular and molecular mechanisms involved are not clear. The aim of this study was to examine the effect of IVIG and salicylate at each stage of disease development. METHODS: Using a murine model of KD, we established and validated several in vitro techniques to reflect 3 key steps involved in disease pathogenesis, as follows: thymidine incorporation to evaluate T cell activation, enzyme-linked immunosorbent assay to measure tumor necrosis factor alpha (TNFalpha) production, and real-time polymerase chain reaction to examine TNFalpha-mediated expression of matrix metalloproteinase 9 (MMP-9). RESULTS: At therapeutic concentrations, IVIG, but not salicylate, effectively reduced the immune response leading to TNFalpha expression. Unexpectedly, pharmacologic doses of salicylate were not able to inhibit TNFalpha production and in fact enhanced its production. Neither drug directly regulated MMP-9 expression but did so only indirectly via modulating TNFalpha. TNFalpha activity was a prerequisite for local expression of MMP-9 at the coronary artery. CONCLUSION: Therapeutic concentrations of IVIG and salicylate differentially modulate the expression of TNFalpha and its downstream effects. Further dissection of the biologic effects of aspirin in acute KD is necessary for the rational design of therapy.

Matrix metalloproteinase 9 activity leads to elastin breakdown in an animal model of Kawasaki disease.

OBJECTIVE: Kawasaki disease (KD) is a multisystem vasculitis leading to damage in the coronary circulation and aneurysm formation. Because cardiac tissue from affected children is not available, investigation of the mechanisms responsible for coronary artery damage in KD requires use of a disease model. The present study was undertaken to examine, in an experimental model, the role of matrix metalloproteinase 9 (MMP-9) on coronary artery inflammation and vascular damage. METHODS: C57BL/6 mice were injected with Lactobacillus casei cell wall extract to induce coronary arteritis. Hearts were isolated and assayed for MMP-9 protein expression and enzymatic activity by immunoblotting or confocal microscopy and zymography, respectively. MMP-9-deficient mice were used to examine the necessity of MMP-9 for disease development. RESULTS: Localized inflammation at the coronary artery led to elastin breakdown and aneurysm formation. This occurred in the absence of smooth muscle cell apoptosis. Following disease induction, there was an increase in the amount and enzymatic activity of MMP-9, an elastolytic protease. MMP-9 was up-regulated by tumor necrosis factor alpha (TNFalpha) and produced primarily by vascular smooth muscle cells. In MMP-9-deficient animals, vascular inflammation continued to develop, but the incidence of elastin breakdown was significantly reduced. Elastin breakdown in the coronary artery was virtually eliminated by ablation of MMP-9. CONCLUSION: These findings show that TNFalpha up-regulates expression of MMP-9, an important proteinase responsible for extracellular matrix breakdown, leading to coronary artery damage in this model of KD. These results have important implications regarding treatments for improving coronary outcome in affected children.

Elastolytic matrix metalloproteinases and coronary outcome in children with Kawasaki disease.

Kawasaki disease (KD) is a multisystem vasculitis that leads to coronary artery damage and aneurysm formation. Elastolytic matrix metalloproteinases (MMPs) have been implicated in the pathogenesis of arterial aneurysms. To determine the relationship between circulating levels of elastolytic MMPs and development of coronary artery aneurysms in children with KD, we partnered studies done in affected children with an animal model of disease. In affected children, circulating protein levels and enzymatic activity of MMP-2 and MMP-9 did not have a statistically significant relationship with coronary artery outcome after adjusting for demographic characteristics, and clinical and laboratory features. Although matrix-degrading proteolytic activity was specific for affected mice and localized to inflamed coronary artery segments, the enzymatic activity in the systemic circulation of affected and control mice were not different. Similar to affected children, peripheral blood levels of MMP-9 enzymatic activity did not correlate with coronary artery disease in the animal model. Therefore, circulating levels of MMPs known to act locally may not be useful biomarkers of disease. This is especially relevant to enzymatic activity that is tightly regulated at multiple levels including the local tissue environment.