Abstract
BackgroundDeath of smooth muscle cells in the atherosclerotic plaques makes the plaques more prone to rupture, which can initiate an acute ischemic event. The development of atherosclerosis includes the migration of immune cells e.g. monocytes/macrophages and T lymphocytes into the lesions. Immune cells can release antimicrobial peptides. One of these, human cathelicidin antimicrobial peptide hCAP-18, is cleaved by proteinase 3 generating a 4.5 kDa C-terminal fragment named LL-37, which has been shown to be cytotoxic. The aim of the study was to explore a potential role of LL-37 in the pathophysiology of atherosclerosis.MethodsWe investigated the presence of LL-37 in human atherosclerotic lesions obtained at autopsy using immunohistochemistry. The direct effects of LL-37 on cultured vascular smooth muscle cells and isolated neutrophil granulocytes were investigated with morphological, biochemical and flow cytometry analysis.ResultsThe neointima of atherosclerotic plaques was found to contain LL-37-like immunoreactivity, mainly in macrophages. In cultured smooth muscle cells, LL-37 at 30 μg/ml caused cell shrinkage, membrane blebbing, nuclear condensation, DNA fragmentation and an increase in caspase-3 activity as studied by microscopy, ELISA and enzyme activity assay, respectively. Flow cytometry demonstrated that LL-37 in a subset of the cells caused a small but rapidly developing increase in membrane permeability to propidium iodide, followed by a gradual development of FITC-annexin V binding. Another cell population stained heavily with both propidium iodide and FITC-annexin V. Neutrophil granulocytes were resistant to these effects of LL-37.ConclusionThis study shows that LL-37 is present in atherosclerotic lesions and that it induces death of vascular smooth muscle cells. In a subset of cells, the changes indicate the development of apoptosis triggered by an initial mild perturbation of plasma membrane integrity. The findings suggest a role for LL-37 as a mediator of immune cell-induced death of vascular smooth muscle cells in atherosclerosis.
Highlights
Death of smooth muscle cells in the atherosclerotic plaques makes the plaques more prone to rupture, which can initiate an acute ischemic event
In an earlier study, aiming to investigate the effects of LL37 on nitric oxide synthesis in the blood vessel wall, we found that LL-37 non- inhibits the expression of inducible nitric oxide synthase in vascular smooth muscle cells
Since apoptosis of vascular smooth muscle cells has been implicated as an important factor in the pathophysiology of atheromatous disease [22], we investigated the presence of LL-37 in atherosclerotic plaques as well as morphological and biochemical effects of LL-37 on cultured vascular smooth muscle cells
Summary
Death of smooth muscle cells in the atherosclerotic plaques makes the plaques more prone to rupture, which can initiate an acute ischemic event. During the development of the plaques an increasing number of smooth muscle cells die, at least partly due to programmed cell death, apoptosis [2,3] This impairs the collagen synthesis, which weakens the fibrous cap and makes the atherosclerotic plaques more prone to rupture [1]. The 18-kDa protein human cationic antimicrobial protein (hCAP-18) is the only human cathelicidin [8,9,10] It is found in neutrophil granulocytes, lymphocytes and monocytes as well as in tissues e.g. squamous epithelium, the lung and the epididymis and is released extracellularly [11,12,13,14,15]. Apart from having a broad antibacterial activity [17,18], LL-37 exhibits lipopolysaccharide (LPS)-binding and LPS-neutralizing properties [17] as well as toxic effects on leukocytes and erythrocytes [19,20]
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