Human Cathelicidin Peptide LL-37 Induces Cell Death in Autophagy-Dysfunctional Endothelial Cells

Cathelicidins are a family of antimicrobial peptides that are expressed in several mammalian species. They are characterized by highly conserved cathelin-like prosequences and variable C-terminal sequences that correspond to the mature antibacterial peptides (1). LL-37 is the sole mature peptide of human cathelicidin; it comprises 37 aa and contains the first two leucine residues and an amphipathic α-helical conformation, which is cleaved from the proform of a human cationic antibacterial protein of 18 kDa (2). In addition to its membrane-disrupting antimicrobial properties, LL-37 binds to LPS (3), an outer membrane component of Gram-negative bacteria, and exerts an LPS-neutralizing action, as well as membrane-disrupting antimicrobial properties (1). Moreover, it has been shown that LL-37 exerts multidirectional actions on host cells (4). LL-37 is directly chemotactic for neutrophils, monocytes, and T cells, whereas LL-37 promotes the migration of dendritic cells through the ability to degranulate mast cells. LL-37 activates epithelial cells to induce cytokine and chemokine production. Furthermore, LL-37 acts on endothelial cells to promote cell proliferation (angiogenesis) and induce inflammatory responses (such as the upregulation of ICAM-1) (5).

Atherosclerosis is a chronic inflammatory and immune disease of vascular walls involving multiple cell types (monocytes, macrophages, T cells, endothelial cells, and vascular smooth muscle cells) (6). Endothelial dysfunction, such as the decrease of NO production, is a critical early event of the pathogenesis of atherosclerosis and contributes to the initiation and progression of plaque formation. In the advanced stage, damaged or apoptotic endothelial cells become procoagulant in the plaque lesions, thus promoting the formation of thrombi. Notably, recent studies demonstrated the involvement of cathelicidins (human LL-37 and its mouse ortholog CRAMP [cathelin-related antimicrobial peptide]) in the pathogenesis of atherosclerosis; it has been shown that LL-37 is deposited in the atherosclerotic lesions of human aortas (5), and that the deletion of CRAMP results in the reduction of plaque lesions and macrophage accumulation in apolipoprotein E (ApoE)^−/− atherosclerotic mice (7).

Autophagy is a lysosome-dependent cellular degradation process that is triggered by cellular stresses. In endothelial cells, the lack of autophagy results in the intracellular accumulation of the low-density lipoprotein and oxidized low-density lipoprotein (8), the impaired secretion of the von Willebrand factor (9), and decreased production of NO (10). The lack of autophagy also induces the inflammatory activation in endothelial cells (such as the upregulation of ICAM-1) (11). Importantly, the autophagy of endothelial cells is modulated in the pathogenesis of atherosclerosis; in its early stage, autophagy is activated in endothelial cells; however, autophagy becomes dysfunctional in the advanced stage of the disease, leading to apoptotic cell death and plaque instability (12).

The observations described earlier led to the hypothesis that the upregulation of LL-37 in atherosclerotic aortas and the dysregulated autophagy of endothelial cells are involved in the pathogenesis of atherosclerosis. However, it remains to be elucidated how LL-37 modulates the autophagy of endothelial cells in the pathogenesis of this disease. Therefore, in this study, we evaluated the effect of LL-37 on the autophagy of endothelial cells using HUVECs and human coronary artery endothelial cells (HCAECs). Our results indicated that LL-37 enhances autophagy in endothelial cells but induces cell death in autophagy-dysfunctional endothelial cells.

LL-37 (L¹LGDFFRKSKEKIGKEFKRIVQRIKDFLRMLVPRTES³⁷) was synthesized on a peptide synthesizer (model PSSM-8; Shimadzu, Kyoto, Japan) using the solid-phase method and F-moc chemistry and was then purified as described previously (13). The lysosomal protease inhibitors E-64d and pepstatin A were purchased from Sigma-Aldrich (St. Louis, MO). Human recombinant IL-1β was purchased from PeproTech (Cranbury, NJ). The anti–LL-37 antiserum was raised in rabbits using LL-37 covalently coupled to keyhole limpet hemocyanin, as described previously (14). Abs to LC3 (rabbit PM036 and mouse M152-3), Atg7 (rabbit PM039), p62 (rabbit PM045), and normal rabbit IgG (PM035) were from MBL (Nagoya, Japan); anti-ubiquitin Abs (rabbit E4I2J and mouse FK2) were from Cell Signaling Technology (Danvers, MA); the anti-CRAMP Ab (PA-CRPL-100) was from Innovagen (Lund, Sweden); and the anti-LL-37 Ab (D-5) was from Santa Cruz (Dallas, TX).

HUVECs and HCAECs were purchased from Lonza (Basel, Switzerland) and maintained in endothelial cell growth medium (EGM)-2 (containing 2% FBS and the endothelial cell growth supplement EGM-2 Bullet kit) and EGM-2MV (containing 5% FBS and the EGM-2MV Bullet kit), respectively, in the presence of antibiotics at 37°C in 5% CO₂. The cells of passages 5–12 were used in all experiments. Tissue culture–treated dishes and plates were obtained from Iwaki (Tokyo, Japan).

HUVECs were seeded in six-well plates and cultured overnight. Cells were transfected with 5 nM of an atg7 small interfering RNA (siRNA; s20650) or a nontargeting control siRNA (Silencer Select; Ambion, Thermo Fisher Scientific, Waltham, MA) using the Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific) for 6 h. Thereafter, cells were washed with EGM-2 and further incubated for up to 72 h. Subsequently, cell lysates were prepared and the expression of Atg7, p62, and GAPDH was detected by Western blotting, as described later. The expression of Atg7 was substantially suppressed in the atg7-siRNA–transfected cells compared with nontargeting control siRNA-transfected cells at 48 and 72 h after transfection (Fig. 4B). Thus, in this study, 48 h after transfection, the siRNA-transfected HUVECs were incubated with LL-37 for the indicated periods and subjected to the following experiments.

HUVECs or HCAECs were incubated with LL-37 (2, 5, and 10 µg/ml) for 24 h in the absence or presence of lysosomal protease inhibitors (E-64d and pepstatin A, 10 µg/ml each) to detect LC3. Alternatively, to evaluate the time-dependent degradation of LL-37, we incubated HUVECs (atg7 knockdown [KD] and control cells) with LL-37 (5 µg/ml) for 15 min, extensively washed them with HBSS (to remove unbound LL-37), and incubated them in EGM-2 for 1–12 h. Cells were lysed in radioimmunoprecipitation buffer (50 mM Tris–HCl [pH 8.0], 150 mM NaCl, 0.5% sodium deoxycholate, 1% Nonidet P-40, and 0.1% SDS; Fujifilm Wako Pure Chemical, Osaka, Japan) containing the Complete protease inhibitor mixture (Sigma-Aldrich). Cell lysates (10 µg of protein/lane) were mixed with Laemmli sample buffer containing 2-ME, subjected to SDS-PAGE, and transferred to polyvinylidene fluoride membranes (Immobilon-P; Millipore, Billerica, MA) using a semidry transfer system (Trans-Blot; Bio-Rad, Hercules, CA). Membranes were blocked with BlockAce (DS Pharma Biomedical, Osaka, Japan) or skim milk (Nacalai Tesque, Kyoto, Japan), then probed with rabbit anti-LC3 (PM036), anti-Atg7, anti-p62, anti-ubiquitin (E4I2J), or anti–LL-37 (D-5) Abs, followed by incubation with HRP-conjugated goat anti-rabbit IgG (Chemicon International, Temecula, CA) or goat anti-mouse IgG/IgM (Jackson ImmunoResearch Laboratories, West Grove, PA). Signals were developed using the SuperSignal West Pico/Dura Chemiluminescent Substrate (Pierce Biotechnology, Rockford, IL) and detected using a FUSION FX luminescent image analyzer (Vilber Lourmat, Collégien, France). GAPDH was detected with an HRP-conjugated anti-GAPDH Ab (5A12; Fujifilm Wako Pure Chemical) as a loading control.

HUVECs or HCAECs were seeded in Nunc Lab Tek II CC2 chamber slides (Thermo Fisher Scientific) and cultured overnight. Cells were incubated with LL-37 (5 µg/ml) for 24 h in the presence of E-64d and pepstatin A. Alternatively, atg7 KD and nontargeting control cells were incubated with LL-37 (5 µg/ml) for 24 h. Cells were then fixed with 2% paraformaldehyde (PFA), permeabilized with 0.2% saponin, blocked with 5% skim milk, and incubated with the mouse anti-LC3 Ab (M152-3) or rabbit LL-37 antiserum. After washing with HBSS, cells were further incubated with Alexa Fluor 594–labeled goat anti-mouse IgG or Alexa Fluor 488–labeled goat anti-rabbit IgG, followed by mounting with Vectashield Hardset with DAPI (Vector Laboratories, San Diego, CA). Fluorescent images were captured with a BZ-X710 microscope (Keyence, Itasca, IL) or an FV1000-D confocal microscope (Olympus, Tokyo, Japan). The rate of LC3-positive puncta was calculated as a percentage by dividing by the number of total cells (DAPI staining). The colocalization analysis of LL-37 and LC3 was performed on a pixel-by-pixel basis using the FLUOVIEW software (Olympus).

HUVECs (atg7 KD and nontargeting control cells) or HCAECs (in the presence/absence of E-64d and pepstatin A) were incubated with LL-37 (2, 5, and 10 µg/ml) for 24 h. Cells were detached with 0.05% trypsin-EDTA, collected in test tubes, and suspended in HBSS. Viable trypan blue–excluded cells were counted with a hemocytometer. Alternatively, cells were incubated with Annexin V–FITC and propidium iodide (PI) solutions (Annexin V-FITC Apoptosis Detection Kit; BioVision, Mountain View, CA) for 5 min, then analyzed by flow cytometry (FACSCalibur, Becton Dickinson, Franklin Lakes, NJ). Annexin V⁺/PI⁺ cells were classified as dead cells with apoptosis, whereas Annexin V⁻/PI⁺ cells were classified as dead cells with necrosis.

HUVECs were incubated with LL-37 (5 µg/ml) in the absence or presence of E-64d and pepstatin A for 4 h, and cell lysates were prepared using immunoprecipitation (IP) lysis buffer (25 mM Tris–HCl [pH 7.4], 150 mM NaCl, 1% Nonidet P-40, 1 mM EDTA, and 5% glycerol; Pierce, Thermo Fisher Scientific) containing the Halt protease and phosphatase inhibitor mixture (Thermo Fisher Scientific). IP was performed using the Dynabeads protein A IP kit (Thermo Fisher Scientific), according to the manufacturer’s instructions. Briefly, protein A–conjugated magnetic beads were incubated with anti-p62 or anti-ubiquitin (FK2) Abs or normal IgG, washed, and then incubated with the cell lysates overnight. Proteins were eluted with Laemmli sample buffer containing 2-ME and subjected to Western blotting using anti-p62, anti–LL-37 (D-5), or anti-ubiquitin (E4I2J) Abs, as described earlier.

HUVECs (atg7 KD and nontargeting control cells) were incubated with LL-37 (5 µg/ml) for 4 h, detached with 0.05% trypsin-EDTA, and collected in test tubes. Cells were fixed with 2% PFA, permeabilized with 0.5% Triton X-100, washed with HBSS, incubated with the PROTEOSTAT aggresome detection dye (ENZO Life Sciences, Farmingdale, NY) (15), and analyzed by flow cytometry (FACSCalibur). To analyze the colocalization of LL-37 with PROTEOSTAT dye–positive aggregates, we incubated atg7 KD cells in chamber slides with LL-37 (5 µg/ml), fixed them with 2% PFA, permeabilized with 0.5% Triton X-100, washed with HBSS, and incubated with the anti–LL-37 rabbit antiserum; after washing with HBSS, cells were further incubated with Alexa Fluor 488–labeled goat anti-rabbit IgG and the PROTEOSTAT dye, followed by mounting in Vectashield Hardset with DAPI. Fluorescent images were captured using a BZ-X710 microscope. The PROTEOSTAT-positive and LL-37–positive cells were counted among dead cells with condensed nuclei or live cells with larger round nuclei (DAPI staining), and the percentages of LL-37⁺ and LL-37⁻ cells among the PROTEOSTAT⁺ dead or live cells were calculated.

All animal experimental procedures were approved by the Ethics Committee for the Use of Laboratory Animals of Juntendo University (2021291, June 2021). Ten-week-old male C57BL/6 mice and spontaneously hyperlipidemic male C57BL/6.KOR-ApoE^shl mice were obtained from Japan SLC (Shizuoka, Japan). Mice were transcardially perfused with cold heparin/PBS followed by 4% PFA under anesthesia. The aortic roots were isolated, postfixed with 4% PFA, cryoprotected with 30% sucrose/PBS, and then embedded in optimal cutting temperature compound (Sakura Finetechnical, Tokyo, Japan). Frozen sections (thickness, 8 µm) were prepared using a CM3050S cryostat (Leica Biosystems, Wetzlar, Germany) and incubated with 0.5% Triton X-100/PBS at 80°C for 20 min (for Ag retrieval), followed by incubation with an anti-CRAMP Ab (1:1000) and Alexa Fluor 594–labeled goat anti-rabbit IgG. Sections were further incubated with the TUNEL reaction buffer containing fluorescein-dUTP (in situ cell death detection kit, Fluorescein; Roche, Darmstadt, Germany) at 37°C for 1 h, followed by mounting in mounting media. Fluorescent images were captured using a BZ-X710 microscope.

Data are presented as the mean ± SD. Statistical significance was determined by two-way ANOVA, followed by Dunnett’s multiple comparisons test (GraphPad Prism 8, GraphPad Software, San Diego, CA). Differences were considered statistically significant at p < 0.05.

A previous study indicated that LL-37 upregulates autophagy-related gene expression in macrophages and induces autophagosome formation, to promote the killing of intracellular bacteria (16). To examine whether LL-37 induces autophagy in endothelial cells, we incubated HUVECs with LL-37 (2, 5, and 10 µg/ml) for 24 h in the absence or presence of lysosomal protease inhibitors (E-64d and pepstatin A); subsequently, LC3-I (cytosolic LC3) and LC3-II (autophagosomal membrane–bound LC3) were detected by Western blotting. LC3-I (upper band) was faintly detected in LL-37–treated cells in the absence of inhibitors, and LL-37 weakly but substantially increased the level of LC3-II (lower band) shifted from LC3-I in the absence of protease inhibitors (Fig. 1A, left panel). However, LL-37 apparently increased the level of LC3-II (expressed not only as the LC3-II/GAPDH ratio but also as the LC3-II/LC3-I ratio) in a dose-dependent manner in the presence of protease inhibitors (Fig. 1A, right panel, 1B, 1C). Furthermore, an immunocytochemical analysis was performed using an anti-LC3 Ab. LL-37 (5 µg/ml) obviously enhanced the formation of LC3-positive puncta and increased the number of cells containing these puncta in the presence of protease inhibitors (Fig. 1D, 1E). In addition, using HCAECs, we confirmed that LL-37 upregulates LC3-II and increases the number of cells containing LC3-positive puncta (Fig. 2A–C). These observations suggest that LL-37 induces autophagy in endothelial cells.

Next, we investigated the association between LL-37 and the autophagosomal membrane and autophagosome-related molecules in endothelial cells. Double immunostaining of LL-37–treated HUVECs using an anti–LL-37 antiserum and an anti-LC3 Ab revealed the colocalization of LL-37 and LC3 puncta around the nuclei in the presence of E-64d and pepstatin A (Fig. 3A). We also investigated the localization of LL-37 and LC3 using confocal microscopy, which indicated that LL-37 partially but substantially colocalized with LC3 around nuclei (Fig. 3B). In fact, a pixel-based colocalization analysis indicated that, in the perinuclear area of the cells, pixels that were positive for LL-37 and LC3 were more frequent than those positive for LC3 or LL-37 alone (Fig. 3B, 3C), although these differences were not statistically significant. In addition, we confirmed the colocalization of LL-37 and LC3 puncta around nuclei in the presence of E-64d and pepstatin A in HCAECs (Fig. 2D). Because LC3 is localized at the autophagosomal membrane, the interaction between LL-37 and p62, which is an autophagosome membrane–associated molecule, was further examined.

p62 recognizes ubiquitinated substrates (such as misfolded or aggregated proteins) via its ubiquitin-associated domain and shuttles the ubiquitinated substrates to autophagosomes for degradation (17). Thus, we first evaluated LL-37, p62, and ubiquitinated proteins without IP in the lysates of cells treated with or without LL-37 in the absence or presence of E-64d and pepstatin A (Fig. 3D). LL-37 was detected exclusively in the LL-37–treated cell lysates, whereas p62 and ubiquitinated proteins were detected in both LL-37–treated and untreated cells. Interestingly, smear bands of LL-37 were detected in long exposure blots of the LL-37–treated cell lysates without inhibitors and were further increased by the presence of inhibitors. Second, the lysates of LL-37–treated cells were immunoprecipitated with the anti-p62 Ab, followed by the detection of the LL-37, p62, and ubiquitinated proteins (Fig. 3E). LL-37 and ubiquitinated proteins were recovered with p62. Importantly, LL-37 and p62 were apparently increased by the presence of inhibitors. Third, the lysates of LL-37–treated cells were immunoprecipitated with the anti-ubiquitin Ab, followed by the detection of LL-37, p62, and ubiquitinated proteins (Fig. 3F). LL-37, p62, and ubiquitinated proteins were increased by the presence of inhibitors; notably, smear bands of LL-37 were detected in the cell lysates without inhibitors and were further increased by the presence of inhibitors.

Based on these observations, it is reasonable to speculate that: (1) LL-37 is ubiquitinated, recognized by p62 to form an LL-37/p62 complex, and colocalizes with LC3 at the autophagosomal membrane; and (2) the ubiquitination is enhanced in the presence of lysosomal protease inhibitors.

The degradation of LL-37 by HUVECs was examined by incubating the cells with LL-37 (5 µg/ml) for 24 h in the absence or presence of E-64d and pepstatin A, followed by immunocytochemistry using the anti–LL-37 antiserum. Interestingly, a small amount of LL-37 was detected in the cells without E-64d and pepstatin A; in contrast, LL-37 accumulated in the cells as puncta in the perinuclear area in the presence of inhibitors at 24 h after LL-37 treatment (Fig. 4A). Subsequently, the degradation of LL-37 was examined using atg7 KD cells. Atg7 is essential for autophagosome formation, and autophagy is dysfunctional in atg7 KD endothelial cells (8, 9). As shown in (Fig. 4B, the expression of Atg7 was successfully suppressed in atg7-siRNA–transfected cells compared with nontargeting control siRNA-transfected cells; in contrast, the expression of p62 was increased in atg7 KD cells, in which autophagy is dysregulated and the degradation of p62 is suppressed (18). Interestingly, LL-37 accumulated as puncta in the perinuclear area in atg7 KD cells compared with nontargeting control cells (Fig. 4C).

To further examine the uptake and degradation of LL-37 by endothelial cells, we incubated HUVECs (atg7 KD and nontargeting control cells) with LL-37 (5 µg/ml) for 15 min, extensively washed them (to remove unbound LL-37), and incubated them for 1–12 h, followed by the detection of LL-37 using Western blotting. The level of LL-37 was time dependently decreased in the nontargeting control cells (Fig. 4D, 4E). In contrast, the degradation of LL-37 was delayed, and the LL-37 level remained constant in the atg7 KD cells compared with the control cells; in fact, the half-life of LL-37 was 3–6 h in the control cells, whereas it was >12 h in the atg7 KD cells (Fig. 4E). In addition, we confirmed that virtually the same amounts of LL-37 were detected early (within 15 min) after LL-37 treatment both in atg7 KD and control cells; thus, the uptake of LL-37 by endothelial cells was not different between atg7 KD and control cells (Fig. 4D). Taken together, these observations suggest that LL-37 is rapidly internalized by endothelial cells and is subsequently degraded by lysosomal proteases; however, in autophagy-dysfunctional endothelial cells, LL-37 is internalized by endothelial cells, but its degradation is disturbed, resulting in the accumulation of LL-37 in these cells.

It has been reported that the intracellular accumulation of proteins leads to cell death in autophagy-dysfunctional conditions (19, 20). To determine whether LL-37 induces cell death in autophagy-dysfunctional endothelial cells, we evaluated the effect of LL-37 on the viability of atg7 KD and nontargeting control HUVECs via trypan blue staining. Viability was slightly decreased in the atg7 KD cells compared with nontargeting control cells in the absence of LL-37, although this difference was not statistically significant (Fig. 5A). LL-37 (2, 5, and 10 µg/ml) did not affect the viability of control cells. In contrast, LL-37 significantly reduced the number of viable cells in the atg7 KD cells at 5 and 10 µg/ml. Furthermore, Annexin V/PI staining revealed that LL-37 (2, 5, and 10 µg/ml) did not essentially increase the number of apoptotic (Annexin V⁺/PI⁺), necrotic (Annexin V⁻/PI⁺), and dead (Annexin V⁺/PI⁺ and Annexin V⁻/PI⁺) cells in the control group. In contrast, in the atg7 KD cells, LL-37 significantly increased the number of apoptotic and necrotic cells (at 10 µg/ml; (Fig. 5B, 5C), as well as the dead cell number (at 5 and 10 µg/ml; (Fig. 5D). In addition, we confirmed that LL-37 decreased the cell viability and increased the cell death of HCAECs in the presence of protease inhibitors, as assessed by trypan blue staining and PI staining, respectively (Fig. 2E, 2F). These observations suggest that LL-37 enhances cell death (apoptosis and necrosis) in autophagy-dysfunctional endothelial cells.

It has been reported that the formation of intracellular protein aggregates leads to cell death in autophagy-dysfunctional conditions (19, 20). Because LL-37 enhanced cell death in autophagy-dysfunctional endothelial cells (Fig. 5), we hypothesized that LL-37 increases protein aggregate formation in autophagy-dysfunctional HUVECs. To examine whether LL-37 increases aggregate formation in HUVECs (atg7 KD and nontargeting control cells), these cells were incubated with LL-37 (5 µg/ml) for 24 h, and the formation of intracellular aggregates was examined by immunocytochemical staining and flow cytometry using the PROTEOSTAT aggregate detection dye, which specifically intercalates into aggregated proteins (15). An immunocytochemical analysis indicated that aggregates were weakly detected in LL-37–treated nontargeting control cells; in contrast, aggregates were apparently increased in LL-37–treated atg7 KD cells (Fig. 6A). Moreover, a flow cytometrical analysis indicated that the level of aggregates was slightly but significantly increased by LL-37 in the nontargeting control cells. Notably, the aggregate level was further increased by LL-37 in the atg7 KD cells (Fig. 6B, 6C). These observations indicate that LL-37 enhances aggregate formation in autophagy-dysfunctional endothelial cells.

The involvement of aggregate formation in the LL-37–induced death of atg7 KD cells was also evaluated. PROTEOSTAT-positive aggregates were localized at the perinuclear area of LL-37–treated atg7 KD cells (Fig. 7A). LL-37 also accumulated as puncta at the perinuclear area in cells and colocalized with PROTEOSTAT-positive aggregates (Fig. 7A). Furthermore, the percentages of aggregate (PROTEOSTAT)-positive cells without and with LL-37 accumulation among live and dead cells were evaluated. The results of this experiment indicated that the percentage of aggregate-positive cells without LL-37 accumulation was higher among dead cells (14%) compared with that detected among live cells (6%), even though this difference was not significant (Fig. 7B). Importantly, the percentage of aggregate-positive cells with LL-37 accumulation was significantly higher among dead cells (59%) compared with live cells (3%) (Fig. 7B). These observations suggest that the LL-37–induced aggregate formation is associated with the death of autophagy-dysfunctional endothelial cells.

The involvement of LL-37, which is an antimicrobial peptide of human cathelicidin, in the pathogenesis of atherosclerosis has been speculated, because LL-37 accumulates in human atherosclerotic aortas (5), and the deletion of CRAMP resulted in the reduction of plaque formation and macrophage accumulation in ApoE^−/− atherosclerotic mice (7). Actually, using ApoE-deficient atherosclerotic mice, we confirmed that CRAMP accumulated in TUNEL⁺ dead endothelial cells at the surface of aortic valves (Supplemental Fig. 2). In turn, the autophagy of endothelial cells is dysregulated in atherosclerosis (12). In this study, to elucidate the role of LL-37 in atherosclerosis, we investigated the effect of LL-37 on autophagy in endothelial cells. First, the level of LC3-II, which is an autophagosomal membrane marker, was evaluated in LL-37–treated HUVECs. LL-37 only weakly upregulated LC3-II in the absence of lysosomal protease inhibitors (E-64d and pepstatin A); in contrast, LL-37 apparently upregulated LC3-II in the presence of inhibitors (Fig. 1A–C). Because LC3-II is degraded by lysosomal proteases via autophagy (21), LC3-II is likely increased in LL-37–treated HUVECs in the presence of lysosomal protease inhibitors. Furthermore, LL-37 enhanced the formation of LC3-containing puncta (representing autophagosomes) in the presence of inhibitors (Fig. 1D, 1E). These observations indicate that LL-37 induces autophagy in endothelial cells.

Previous studies demonstrated that LL-37 is internalized and colocalizes with LC3 puncta in macrophages and plasmacytoid dendritic cells during autophagy (16, 22, 23). Thus, the colocalization of LL-37 with LC3 was examined in HUVECs by immunocytochemistry. The results of this experiment indicated that LL-37 colocalized with LC3 puncta in the perinuclear area in the presence of lysosomal protease inhibitors (Fig. 3A–C), suggesting the association between LL-37 and the autophagosomal membrane. In the autophagic process, ubiquitinated substrate proteins are recognized by p62 (which is an LC3-II–bound autophagosomal membrane-associated molecule) and transferred to autophagosomes. Thus, the association between LL-37 and p62 and the ubiquitination of LL-37 were examined by IP. Interestingly, LL-37 was recovered using not only an anti-p62 Ab but also an anti-ubiquitin Ab (Fig. 3E, 3F). These observations suggest that LL-37 is ubiquitinated, recognized by p62 to form a complex, and possibly transferred to autophagosomes as a substrate.

Furthermore, the degradation of LL-37 was examined. The degradation of LL-37 was delayed in atg7 KD cells (Fig. 4D, 4E) and in the presence of lysosomal protease inhibitors (data not shown) during the incubation with LL-37. These observations suggest that LL-37 is internalized by endothelial cells and degraded by lysosomal proteases via autophagy; in contrast, the degradation of LL-37 is delayed, and LL-37 accumulates in the autophagy-dysfunctional cells.

Several reports have indicated that LL-37 is internalized by various types of cell (including macrophages, mast cells, epithelial cells, and endothelial cells) by direct translocation (across the membrane) or endocytosis, depending on the cell type. Importantly, we previously revealed that LL-37 is internalized by liver sinusoidal endothelial cells via an interaction with cell-surface heparan sulfate proteoglycans by endocytosis, then transferred to lysosomes (24). Thus, in this study, LL-37 was possibly incorporated into endothelial cells by endocytosis and transferred to lysosomes for degradation.

It has been reported that the intracellular accumulation of proteins leads to cell death under autophagy-dysfunctional conditions (19, 20). Therefore, the effect of LL-37 on the death of atg7 KD and nontargeting control cells was examined. Our results indicated that LL-37 decreased the number of viable cells and increased that of dead cells (apoptotic and necrotic cells) in atg7 KD cells (Fig. 5), suggesting that LL-37 enhances cell death in autophagy-dysfunctional endothelial cells, in which LL-37 is intracellularly accumulated. To elucidate further the mechanism underlying the LL-37–induced death of atg7 KD cells, we evaluated intracellular protein accumulation (aggregate formation) using the PROTEOSTAT dye. The results demonstrated that LL-37 apparently increased aggregate formation in atg7 KD cells (Fig. 6). Moreover, LL-37 colocalized with PROTEOSTAT⁺ aggregates in atg7 KD cells (Fig. 7A), and the number of aggregate-positive cells with LL-37 accumulation was significantly increased among dead cells compared with live cells (Fig. 7B). These observations suggest that LL-37 increases protein aggregates intracellularly, thereby inducing cell death in autophagy-dysfunctional endothelial cells.

It has been reported that ubiquitinated protein aggregates are accumulated in autophagy-dysfunctional cells (17). The results of this study indicated that LL-37 is possibly ubiquitinated (Fig. 3F) and that it colocalized with the PROTEOSTAT dye in atg7 KD cells (Fig. 7A). The PROTEOSTAT dye specifically intercalates into the β-sheet structures that are typically found in misfolded and aggregated proteins (15). Notably, LL-37 forms β-sheets and β-fibrils in the presence of lipids or the lipid membrane (25). Taken together, these observations suggest that the ubiquitinated LL-37 accumulates intracellularly and forms protein aggregates with the PROTEOSTAT dye in autophagy-dysfunctional HUVECs, which is accompanied by cell death.

The human cathelicidin peptide LL-37 acts on endothelial cells to promote cell proliferation (angiogenesis) (26) and maintains endothelial barrier permeability (27). However, in this study, we revealed that LL-37 induced autophagy in endothelial cells but enhanced cell death in autophagy-dysfunctional conditions, in which the intracellular degradation of LL-37 is disturbed, and protein aggregates accumulate. Interestingly, we confirmed that IL-1β, which is an atherosclerosis-associated inflammatory cytokine (28), upregulated LC3-II, and LL-37 further increased the IL-1β–induced expression of LC3-II (Supplemental Fig. 1A–C). Moreover, cell death was increased by IL-1β and further increased by LL-37, which colocalized with LC3 around nuclei (Supplemental Fig. 1D, 1E). Importantly, it has been reported that the autophagy of endothelial cells is dysregulated in atherosclerosis (12), and that LL-37 is accumulated in atherosclerotic lesions (5). Taken together, these findings suggest that not only IL-1β but also LL-37 plays a role in atherosclerosis, and LL-37 exerts an adverse action on autophagy-dysfunctional endothelial cells to induce cell death in the pathogenesis of atherosclerosis, a chronic inflammatory disease of vascular walls.

We thank members of the Laboratory of Proteomics and Biomolecular Science (Drs. Yuka Hiraoka, Mika Kikkawa, and Yoshiki Miura), the Laboratory of Morphology and Image Analysis (Dr. Shinji Nakamura), the Laboratory of Molecular and Biochemical Research, Biomedical Research Core Facilities (Dr. Tomomi Ikeda and Prof. Hiroshi Koide), and the Division of Cell Biology (Drs. Akemi Koyanagi and Tamami Sakanishi), Research Support Center, Juntendo University Graduate School of Medicine for synthesizing LL-37 peptide or technical assistance.

The authors have no financial conflicts of interest.