Human antimicrobial peptide LL-37 is present in atherosclerotic plaques and induces death of vascular smooth muscle cells: a laboratory study

Abstract

Background: Death 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.

Methods: We 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.

Results: The neointima of atherosclerotic plaques was found to contain LL-37-like immunoreactivity, mainly in macrophages. In cultured smooth muscle cells, LL-37 at 30 mug/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.

Conclusion: This 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.

Figure 1

Detection of hCAP-18/LL-37 by immunohistochemistry in sections of human aorta. (A) Low power view. Binding of the specific antibody was detected by peroxidase, which yields a brown color reaction against a pale background. Dense binding was found in the neointima (N) of an athreosclerotic lesion. The vascular lumen (L) and the tunica muscularis (TM) are indicated. (bar, 100 μm). (B) Portion of the neointima indicated in (A). hCAP-18/LL-37-like immunoreactivity is found (bar, 100 μm). (C) Control, where the specific antibody has been replaced by nonimmune serum, resulting in loss of labeling (bar, 100 μm). (D) High power image of a slide double stained for hCAP-18/LL-37 (brown) and the macrophage marker CB68 (red). HCAP-18/LL-37 and CD68 immunoreactivity are mainly co-localized to the same cells (bar, 10 μm).

Figure 2

Microscopic images of cultured vascular smooth muscle cells. Cells were incubated in the absence (control, A, B, C) or presence of LL-37 (30 μg/ml) for 2 (D, E, F) or 5 (G, H, I) hours. The images show cells viewed with differential interface contrast (Nomarski) microscopy (A, D, G), stained with 4'6-diamino-2-phenyliondole dihydrochloride (DAPI), (B, E, H), or FITC-annexin V (C, F, I). Control cells have a smooth surface (A) with large evenly stained nuclei (B) and no FITC-annexin V staining of the cell membrane (C). After 2 hours' incubation with LL-37, some cells appear shrunken with a rough surface (D), smaller nuclei with fragmented chromatin (E), and a cell membrane staining with FITC-annexin V (F) indicating apoptosis. After 5 h the apoptotic changes are more marked (G, H, I; bar, 10 μm).

Figure 3

Caspase-3 activity in cultured vascular smooth muscle cells. Cells were incubated for 1, 3 or 7 hours in the presence of LL-37 at 10 (●) or 30 (■) μg/ml. A significant increase in caspase-3 activity was observed in cells incubated for 1, 3 and 7 hours with LL-37 at 30 μg/ml (n = 6, *P < 0.05, one way repeated measures ANOVA followed by Holm-Sidak test). Values are means + or - S.E.M.

Figure 4

Effect of LL-37 on DNA fragmentation in cultured rat and human aortic smooth muscle cells. Rat (filled bars) and human (open bars) cells were analyzed after 16 h incubation. LL-37 at 10 and 30 μg/ml induced a statistically significant DNA fragmentation in rat cells compared to controls and LL-37 at 30 μg/ml induced a statistically significant DNA fragmentation in human cells compared to controls (n = 7, * P < 0.05, Kruskal-Wallis one way ANOVA on ranks followed by Dunnett's test). Values are means + S.E.M.

Figure 5

Release of LDH from cultured vascular smooth muscle cells. Cells were incubated for 1, 3 or 7 hours without (Control, ○) or with LL-37 at 10 (●) or 30 (■) μg/ml. The release of LDH was significantly increased from cells incubated for 1, 3 and 7 hours with LL-37 at 30 μg/ml (n = 7, *P < 0.05, One way repeated measures ANOVA followed by Holm-Sidak test). Values are means ± S.E.M.

Figure 6

Flow cytometry of cultured vascular smooth muscle cells. Dot-plots (left) of FITC-annexin V (x-axis)/propidium iodide (y-axis) fluorescence and corresponding histograms for FITC-annexin V fluorescence (right) of vascular smooth muscle cells incubated in the absence (A, B, control) or presence of LL-37 (30 μg/ml) for 10 min (C, D), 30 min (E, F), 60 min (G, H) or 180 min (I, J). The diagrams on the left are divided into four areas and the percentages of the total number of stained cells within each area are given. Viable control cells are found in the lower left part if the diagrams and non-viable cells, heavily stained with FITC-annexin V and propidium iodide, are found in the upper right part of the diagrams. Apoptotic cells, stained with FITC-annexin V but not propidium iodide, are found in the lower right part of the diagrams. Incubation with LL-37 resulted, within 10 min, in a moderate increase in propidium iodide staining (C) of the viable cells followed by a gradually developing increase in FITC annexin V staining from 5.1 % at 10 minutes to 28 % at 180 minutes indicating the development of apoptosis (E-J). The fraction of non-viable cells increased from 17 to 54 % during the whole time course studied.