SUMMARY Proinflammatory stimuli elicit rapid transcriptional responses via transduced signals to master regulatory transcription factors. To explore the role of chromatin-dependent signal transduction in the atherogenic inflammatory response, we characterized the dynamics, structure and function of regulatory elements in the activated endothelial cell epigenome. Stimulation with tumor necrosis factor alpha prompted a dramatic and rapid global redistribution of chromatin activators to massive de novo clustered enhancer domains. Inflammatory super enhancers formed by NF-κB accumulate at the expense of immediately decommissioned, basal endothelial super enhancers, despite persistent histone hyperacetylation. Mass action of enhancer factor redistribution causes momentous swings in transcriptional initiation and elongation. A chemical genetic approach reveals a requirement for BET bromodomains in communicating enhancer remodeling to RNA polymerase and orchestrating the transition to the inflammatory cell state, demonstrated in activated endothelium and macrophages. BET bromodomain inhibition abrogates super enhancer mediated inflammatory transcription, atherogenic endothelial responses and atherosclerosis in vivo.
Interleukin 17A (IL-17) is the signature cytokine produced by TH17 cells and has been implicated in host defense against infection and the pathophysiology of autoimmunity and cardiovascular disease. Little is known, however, about the influence of IL-17 on endothelial activation and leukocyte influx to sites of inflammation. We hypothesized that IL-17 would induce a distinct pattern of endothelial activation and leukocyte recruitment when compared to the TH1 cytokine, IFNγ. We found that IL-17 alone had minimal activating effects on cultured endothelium, while the combination of TNFα and IL-17 produced a synergistic increase in the expression of both P-selectin and E-selectin. Using intravital microscopy of the mouse cremaster muscle, we found that TNFα and IL-17 also led to a synergistic increase in E-selectin dependent leukocyte rolling on microvascular endothelium in vivo. In addition, TNFα and IL-17 enhanced endothelial expression of the neutrophilic chemokines CXCL1, CXCL2, and CXCL5, and led to a functional increase in leukocyte transmigration in vivo and CXCR2-dependent neutrophil but not T-cell transmigration in a parallel-plate flow chamber system. By contrast, endothelial activation with TNFα and IFNγ preferentially induced the expression of the integrin-ligands ICAM1 and VCAM1, as well as the T-cell chemokines CXCL9, CXCL10, and CCL5. These effects were further associated with a functional increase in T-cell but not neutrophil transmigration under laminar shear flow. Overall, these data show that IL-17 and TNFα act in a synergistic manner to induce a distinct pattern of endothelial activation that sustains and enhances neutrophil influx to sites of inflammation.
Epsins are a family of ubiquitin-binding, endocytic clathrin adaptors. Mice lacking both epsins 1 and 2 (Epn1/2) die at embryonic day 10 and exhibit an abnormal vascular phenotype. To examine the angiogenic role of endothelial epsins, we generated mice with constitutive or inducible deletion of Epn1/2 in vascular endothelium. These mice exhibited no abnormal phenotypes under normal conditions, suggesting that lack of endothelial epsins 1 and 2 did not affect normal blood vessels. In tumors, however, loss of epsins 1 and 2 resulted in disorganized vasculature, significantly increased vascular permeability, and markedly retarded tumor growth. Mechanistically, we show that VEGF promoted binding of epsin to ubiquitinated VEGFR2. Loss of epsins 1 and 2 specifically impaired endocytosis and degradation of VEGFR2, which resulted in excessive VEGF signaling that compromised tumor vascular function by exacerbating nonproductive leaky angiogenesis. This suggests that tumor vasculature requires a balance in VEGF signaling to provide sufficient productive angiogenesis for tumor development and that endothelial epsins 1 and 2 negatively regulate the output of VEGF signaling. Promotion of excessive VEGF signaling within tumors via a block of epsin 1 and 2 function may represent a strategy to prevent normal angiogenesis in cancer patients who are resistant to anti-VEGF therapies.
Objective- Coronary artery thrombosis can occur in the absence of plaque rupture because of superficial erosion. Erosion-prone atheromata associate with more neutrophil extracellular traps (NETs) than lesions with stable or rupture-prone characteristics. The effects of NETs on endothelial cell (EC) inflammatory and thrombogenic properties remain unknown. We hypothesized that NETs alter EC functions related to erosion-associated thrombosis. Approach and Results- Exposure of human ECs to NETs increased VCAM-1 (vascular cell adhesion molecule 1) and ICAM-1 (intercellular adhesion molecule 1) mRNA and protein expression in a time- and concentration-dependent manner. THP-1 monocytoid cells and primary human monocytes bound more avidly to NET-treated human umbilical vein ECs than to unstimulated cells under flow. Treatment of human ECs with NETs augmented the expression of TF (tissue factor) mRNA, increased EC TF activity, and hastened clotting of recalcified plasma. Anti-TF-neutralizing antibody blocked NET-induced acceleration of clotting by ECs. NETs alone did not exhibit TF activity or acceleration of clotting in cell-free assays. Pretreatment of NETs with anti-interleukin (IL)-1α-neutralizing antibody or IL-1Ra (IL-1 receptor antagonist)-but not with anti-IL-1β-neutralizing antibody or control IgG-blocked NET-induced VCAM-1, ICAM-1, and TF expression. Inhibition of cathepsin G, a serine protease abundant in NETs, also limited the effect of NETs on EC activation. Cathepsin G potentiated the effect of IL-1α on ECs by cleaving the pro-IL-1α precursor and releasing the more potent mature IL-1α form. Conclusions- NETs promote EC activation and increased thrombogenicity through concerted action of IL-1α and cathepsin G. Thus, NETs may amplify and propagate EC dysfunction related to thrombosis because of superficial erosion.
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