An angiogenic role for the human peptide antibiotic LL-37/hCAP-18

Abstract

Antimicrobial peptides are effector molecules of the innate immune system and contribute to host defense and regulation of inflammation. The human cathelicidin antimicrobial peptide LL-37/hCAP-18 is expressed in leukocytes and epithelial cells and secreted into wound and airway surface fluid. Here we show that LL-37 induces angiogenesis mediated by formyl peptide receptor-like 1 expressed on endothelial cells. Application of LL-37 resulted in neovascularization in the chorioallantoic membrane assay and in a rabbit model of hind-limb ischemia. The peptide directly activates endothelial cells, resulting in increased proliferation and formation of vessel-like structures in cultivated endothelial cells. Decreased vascularization during wound repair in mice deficient for CRAMP, the murine homologue of LL-37/hCAP-18, shows that cathelicidin-mediated angiogenesis is important for cutaneous wound neovascularization in vivo. Taken together, these findings demonstrate that LL-37/hCAP-18 is a multifunctional antimicrobial peptide with a central role in innate immunity by linking host defense and inflammation with angiogenesis and arteriogenesis.

Figure 1

LL-37 induces physiologic angiogenesis in the CAM assay. (a) LL-37 (5 μg/pellet) induces the formation of wheel spoke–like vessel formation in comparison with the scrambled control peptide sLL-37 or the solvent. bFGF was used as positive control. Bars: 1 mm. (b) Cross sections of CAMs. LL-37 increases the number of erythrocyte-filled vessels (Masson-Goldner stain of erythrocyte-filled vessels). Bars: 250 μm or 62 μm in the ×10 and ×40 micrographs, respectively. (c) The numbers of erythrocyte-filled vessels were counted in hot spots. *P < 0.05 as compared with the solvent group (n = 6/group; three sections per CAM).

Figure 2

LL-37 induces angiogenesis and arteriogenesis in the rabbit hind-limb model. (a–d) Application of the peptide in a rabbit hind-limb model resulted in increased collateral growth in the LL-37–treated animals (a) as compared with the sLL-37–treated animals (b) or the buffer control group (c). bFGF/VEGF was used as positive control (d). (e) Collateral growth was significantly increased in the LL-37 group. (f) Tissue specimens from the calf (gastrocnemius muscle) revealed higher capillary density in the treated group as compared with the control group. (g) Blood flow velocity as assessed by cinedensitometry of the passage time of the contrast agent between the internal iliac artery and the anterior tibial artery was significantly augmented in the LL-37 group. *P < 0.05 as compared with the buffer control (n = 4/group).

Figure 3

Mice with disrupted Cnlp gene show decreased wound revascularization. (a and b) Vascular structures are identified in microsections by CD31 immunostaining at the wound edge 3 days after full-thickness aseptic injury. An asterisk denotes the wound site and location of crust. Wild-type 129/SvJ mice show multiple vascular structures in granulation tissue at the margin of the repairing wound (a). Mice with homozygous deletion of Cnlp, therefore lacking CRAMP, have fewer vessels at an identical location relative to the wound (b). The bar = 12.5 μm in both micrographs. (c) Numbers of vessels in the skin near the site of injury are significantly decreased in Cnlp^–/– compared with wild-type mice, but are similar distal to the wound in each group (*P < 0.05; n = 3/group, three sections per animal).

Figure 4

Effects of LL-37 on endothelial cells in vitro. (a) In vitro proliferation assays. Different concentrations of LL-37 and controls were added to cultivated HUVECs. Numbers at the left of the graph indicate the numbers of cells per microliter of volume after 72 hours. VEGF was used as positive control. Additionally, we tested sLL-37, HNP-1, -2, and -3, hBD-3, and the propeptides hCAP-18. In vitro proliferation was not inhibited by human serum. Application of the FPRL1 agonist peptide WKYMVm (W peptide) results in increased growth of cell numbers. Addition of antiserum to FPRL1 blunted increased cellular proliferation induced by LL-37. *P < 0.05 as compared with the control group (n = 10/group). (b and c) Hamster aortic ring–sprouting assay. The addition of LL-37 to the culture medium resulted in increased sprouting from the aortic rings as compared with control groups that received bFGF or no peptide (b). *P < 0.05 in the LL-37 and bFGF groups as compared with the controls (n = 7/group) (c). aFPRL1, antiserum plus FPRL1.

Figure 5

Endothelial cells express FPRL1 in vitro and in vivo. (a) Detection of FPRL1 transcripts in cultivated HUVECs by RT-PCR. Control is the BEAS-2B cell line. (b) Detection of FPRL1 protein in HUVECs by Western blot analysis using a specific antiserum. (c) Immunohistochemistry revealed expression in endothelial cells (arrow) of sections of lung tissue. (d) Control serum revealed no positive staining. Bar: 60 μm.

Figure 6

LL-37 binds to endothelial FPRL1 and induces cellular signaling. (a and b) LL-37 induces Ca²⁺ flux in HUVECs. Fura-2–loaded HUVECs were stimulated with LL-37, and relative levels of intracellular Ca²⁺ were monitored using a calcium-imaging system. Local application of LL-37 led to a concentration-dependent increase of intracellular calcium. Preincubation of the cells with pertussis toxin (Ptx) resulted in partial inhibition of the Ca²⁺ flux (a). Preincubation with fMLP cross-desensitized the LL-37–induced Ca²⁺ mobilization; sLL-37 had no effect (b). (c) LL-37 activates NF-κB in endothelial cells. This response can be inhibited by GF109203X and the addition of N-acetylcystein. *P < 0.05 as compared with the control group (n = 8). NAC, N-acetylcystein.