Atherosclerosis, a hyperlipidemia-induced chronic inflammatory process of the arterial wall, develops preferentially at sites where disturbed laminar flow compromises endothelial cell (EC) function. Here we show that endothelial miR-126-5p maintains a proliferative reserve in ECs through suppression of the Notch1 inhibitor delta-like 1 homolog (Dlk1) and thereby prevents atherosclerotic lesion formation. Endothelial recovery after denudation was impaired in Mir126−/− mice because lack of miR-126-5p, but not miR-126-3p, reduced EC proliferation by derepressing Dlk1. At nonpredilection sites, high miR-126-5p levels in endothelial cells confer a proliferative reserve that compensates for the antiproliferative effects of hyperlipidemia, such that atherosclerosis was exacerbated in Mir126−/− mice. In contrast, downregulation of miR-126-5p by disturbed flow abrogated EC proliferation at predilection sites in response to hyperlipidemic stress through upregulation of Dlk1 expression. Administration of miR-126-5p rescued EC proliferation at predilection sites and limited atherosclerosis, introducing a potential therapeutic approach.

Onset of cardiovascular complications as a consequence of atherosclerosis exhibits a circadian incidence with a peak in the morning hours. Although development of atherosclerosis extends for long periods of time through arterial leukocyte recruitment, we hypothesized that discrete diurnal invasion of the arterial wall could sustain atherogenic growth. Here, we show that myeloid cell recruitment to atherosclerotic lesions oscillates with a peak during the transition from the activity to the resting phase. This diurnal phenotype is regulated by rhythmic release of myeloid cell-derived CCL2, and blockade of its signaling abolished oscillatory leukocyte adhesion. In contrast, we show that myeloid cell adhesion to microvascular beds peaks during the early activity phase. Consequently, timed pharmacological CCR2 neutralization during the activity phase caused inhibition of atherosclerosis without disturbing microvascular recruitment. These findings demonstrate that chronic inflammation of large vessels feeds on rhythmic myeloid cell recruitment, and lay the foundation for chrono-pharmacology-based therapy.

MicroRNAs (miRNAs) are versatile regulators of gene expression with profound implications for human disease including atherosclerosis, but whether they can exert posttranslational functions to control cell adaptation and whether such noncanonical features harbor pathophysiological relevance is unknown. Here, we show that miR-126-5p sustains endothelial integrity in the context of high shear stress and autophagy. Bound to argonaute-2 (Ago2), miR-126-5p forms a complex with Mex3a, which occurs on the surface of autophagic vesicles and guides its transport into the nucleus. Mutational studies and biophysical measurements demonstrate that Mex3a binds to the central U- and G-rich regions of miR-126-5p with nanomolar affinity via its two K homology domains. In the nucleus, miR-126-5p dissociates from Ago2 and binds to caspase-3 in an aptamer-like fashion with its seed sequence, preventing dimerization of the caspase and inhibiting its activity to limit apoptosis. The antiapoptotic effect of miR-126-5p outside of the RNA-induced silencing complex is important for endothelial integrity under conditions of high shear stress promoting autophagy: ablation of Mex3a or ATG5 in vivo attenuates nuclear import of miR-126-5p, aggravates endothelial apoptosis, and exacerbates atherosclerosis. In human plaques, we found reduced nuclear miR-126-5p and active caspase-3 in areas of disturbed flow. The direct inhibition of caspase-3 by nuclear miR-126-5p reveals a noncanonical mechanism by which miRNAs can modulate protein function.

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The efficacy of adoptive cell therapy for solid tumours is hampered by the poor accumulation of the transferred T cells in tumour tissue. Here, we show that the forced expression of the C-X-C chemokine receptor type 6 (CXCR6, whose ligand is highly expressed by human and murine pancreatic cancer cells and by tumour-infiltrating immune cells) in antigen-specific T cells enhanced the recognition and lysis of pancreatic cancer cells and the efficacy of adoptive cell therapy for pancreatic cancer. In mice with subcutaneous pancreatic tumours treated with T cells with either a transgenic T-cell receptor or a murine chimeric antigen receptor targeting the tumour-associated antigen epithelial cell-adhesion molecule, and in mice with orthotopic pancreatic tumours or patient-derived xenografts treated with T cells expressing a chimeric antigen receptor targeting mesothelin, the T cells exhibited enhanced intratumoral accumulation, exerted sustained antitumoral activity and prolonged animal survival only when co-expressing CXCR6. Arming tumour-specific T cells with tumour-specific chemokine receptors may represent a promising strategy for the realization of adoptive cell therapy for solid tumours.