Abstract
Crosstalk between molecular regulators miR-126, hypoxia-inducible factor 1-alpha (HIF-1-α), and high-mobility group box-1 (HMGB1) contributes to the regulation of inflammation and angiogenesis in multiple physiological and pathophysiological settings. Here, we present evidence of an overriding role for miR-126 in the regulation of HMGB1 and its downstream proinflammatory effectors in endothelial cells subjected to hypoxia with concurrent acidosis (H/A). Methods. Primary mouse endothelial cells (PMEC) were exposed to hypoxia or H/A to simulate short or chronic low-flow ischemia, respectively. RT-qPCR quantified mRNA transcripts, and proteins were measured by western blot. ROS were quantified by fluorogenic ELISA and luciferase reporter assays employed to confirm an active miR-126 target in the HMGB1 3′UTR. Results. Enhanced expression of miR-126 in PMECs cultured under neutral hypoxia was suppressed under H/A, whereas the HMGB1 expression increased sequentially under both conditions. Enhanced expression of HMGB1 and downstream inflammation markers was blocked by the premiR-126 overexpression and optimized by antagomiR. Compared with neutral hypoxia, H/A suppressed the HIF-1α expression independently of miR-126. The results show that HMGB1 and downstream effectors are optimally induced by H/A relative to neutral hypoxia via crosstalk between hypoxia signaling, miR-126, and HIF-1α, whereas B-cell lymphoma 2(Bcl2), a HIF-1α, and miR-126 regulated gene expressed optimally under neutral hypoxia. Conclusion. Inflammatory responses of ECs to H/A are dynamically regulated by the combined actions of hypoxia, miR-126, and HIF-1α on the master regulator HMGB1. The findings may be relevant to vascular diseases including atherosclerotic occlusion and interiors of plaque where coexisting hypoxia and acidosis promote inflammation as a defining etiology.
Highlights
As integral vasoregulators, endothelial cells (ECs) serve as multifunctional biosensors that coordinate vascular responses to environmental stress of which hypoxia, oxidative stress, acidosis, and inflammation are especially prominent in myocardial disease and cancer [1,2,3,4,5]
Suppression of hypoxia-induced miR-126 and high-mobility group box-1 (HMGB1) by acidosis of Primary mouse endothelial cells (PMEC): cultured PMECs were subjected to 24 h of hypoxia alone or 72 h hypoxia with concurrent acidosis and isolated RNAs and proteins quantified for expression of mir-126, HMGB1, and HIF-1α
When acidosis was present for 72 h with concurrent hypoxia, mir-126 levels were 4:1 ± 0:05 − fold relative to aerobic cells, a decline of 3-fold relative to hypoxia alone, whereas HMGB1 mRNA levels were further increased over pH neutral hypoxia to 3:2 ± 0:15 of aerobic cell, an increase of 50% over neutral hypoxia
Summary
Endothelial cells (ECs) serve as multifunctional biosensors that coordinate vascular responses to environmental stress of which hypoxia, oxidative stress, acidosis, and inflammation are especially prominent in myocardial disease and cancer [1,2,3,4,5]. By regulating EC survival, senescence, growth, invasion, glucose metabolism, and multiple molecular signaling pathways, hypoxia and HIF factor signaling are central to vascular EC responses to conditions of ischemia and downstream consequences of endothelial dysfunction, remodeling, and vascular disease [6,7,8,9,10]. Acidosis when combined with hypoxia regulates and/or accentuates multiple aspects of the responses of ECs to ischemia, including survival, inflammation, and vessel tone and integrity via stress kinase signaling, calcium, and NO pathways [12,13,14,15]. Multiple microRNAs are known to modulate endothelial inflammatory responses [16] and established roles for miR-126 in regulating vascular integrity, angiogenesis, atherogenesis, and vessel functions that have been described [17,18,19,20,21]. Acidosis occurs most frequently in association with sustained ischemia, inflammation, and metabolic disease where under the most severe conditions of ischemia, affected tissue pH can fall below 6.5 [12] and significantly impact basic physiological processes including immune responses, cell viability, angiogenesis, and localized inflammation [34,35,36,37]
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