Cellular senescence is associated with the induction of a proinflammatory phenotype. Notably, senescent endothelial cells are detected at the sites of atherosclerotic lesions, suggesting the involvement of senescent endothelial cells in atherogenesis. Moreover, bacterial infection has been speculated to contribute to the pathogenesis of atherosclerosis. The present study investigated the effects of Gram-negative bacterial lipopolysaccharide (LPS) and LL-37 (a human antimicrobial peptide of the cathelicidin family), on senescent endothelial cells, using serially passaged human endothelial cells. The results indicated that senescent endothelial cells exhibited the basal proinflammatory phenotype, as evidenced by higher intercellular adhesion molecule-1 (ICAM-1) expression and NF-κB p65 phosphorylation, compared with non-senescent cells. Additionally, exposure to LPS and LL-37 further enhanced the expression of ICAM-1 in senescent endothelial cells, compared with non-senescent cells. Of note, the NF-κB p65 pathway was more activated in senescent endothelial cells stimulated with LPS and LL-37. Furthermore, the expression levels of the receptors for LPS and LL-37 [toll-like receptor 4 (TLR4) and purinergic receptor P2X 7 (P2X7), respectively] were upregulated in senescent endothelial cells. These observations indicated that LPS and LL-37 enhanced the ICAM-1 expression and NF-κB p65 activation in senescent endothelial cells, potentially via the upregulated TLR4 and P2X7. Thus, senescent endothelial cells may contribute to the pathogenesis of atherosclerosis via the basal proinflammatory phenotype and the enhanced inflammatory responses against atherogenic factors, including LPS and LL-37.

Human cathelicidin LL-37 is an antimicrobial peptide that has a broad spectrum of antimicrobial activities but also acts on host cells to exert immunomodulatory functions. It has been suggested that the increase of LL-37 in atherosclerotic aortas and the dysregulated autophagy of endothelial cells are involved in the pathogenesis of atherosclerosis. In this study, to elucidate the role of LL-37 in atherosclerosis, we investigated the effect of LL-37 on autophagy in endothelial cells using HUVECs. First, LL-37 upregulated LC3-II (an autophagosomal membrane marker) and enhanced the formation of LC3-positive puncta in the cells, suggesting that LL-37 induces autophagy in endothelial cells. Second, LL-37 was associated with p62, which recognizes ubiquitinated proteins and transfers them to autophagosomes, suggesting that LL-37 is ubiquitinated and recognized by p62. Third, the degradation of LL-37 was delayed, and LL-37 induced cell death in atg7 knockdown cells, which was accompanied by the formation of protein aggregates in the cells. Taken together, these observations suggest that LL-37 induces autophagy in endothelial cells but enhances cell death in autophagy-dysfunctional conditions, in which the intracellular degradation of LL-37 is disturbed. Thus, LL-37 may exert an adverse action on autophagy-dysfunctional endothelial cells to induce cell death in the pathogenesis of atherosclerosis.