LL-37 induces angiostatic activity through FPRL1 in microvascular endothelial cells

Objectives: Cationic antimicrobial protein 18 (hCAP18) is the only antimicrobial peptide of the cathelichidins family identified from human. hCAP18 is the proprotein and LL-37 is the mature processed form. Cathelicidin antimicrobial peptides seem to play a central role in host defense by direct antimicrobial activity and modulation of inflammation. Recently, LL-37 was found to have angiogenic activity. The purpose of the present study is to examine the effect of LL-37 peptide in angiogenesis. Materials and Methods: LL-37 peptide was synthesized as a C-terminal amide of 37 amino acids (LLGDFFRK SKEKIGKEFK RIVQRIKDFL RNLVPRTES). Human microvascular endothelial cells (HMVECs) were cultured in EGM-2MV growth medium (including 5% fetal bovine serum: FBS) with or without peptide. Angiogenesis was evaluated by tube-formation assay using the full growth factor Matrigel. The cell migration was assessed wound healing assay-using chamber of the cell culture insert (ibidi GmbH). Cell proliferation was determined by MTT assay. Formyl peptide receptor-like 1 (FPRL1) mRNA was estimated by RT-PCR. In addition, FPRL1 protein was analyzed by Western blot using a specific antibody. For inhibition experiments, pertussis toxin (Ptx: 10 ng/ml, Sigma-Aldrich) was applied. The statistical difference was analyzed using Student t-test (n=5). Results: hCAP18/LL-37 peptide (0, 0,1, 0.5, 1, and 10 µg/ml) inhibited the tube formation of HMVECs in dose dependently (No. of brunch points: 91±19.16, 90.33±7.37, 77.33±6.43, 27.67±5.69, and 11.67±2.08, respectively) and decreased cell migration, whereas increased proliferation of HMVECs. RT-PCR and Western Blot showed the expression of FPRL1 in HMVECs. Ptx abolished effect of LL-37- induced inhibition of endothelial tube formation. Conclusions: These results indicate that LL-37 has angiostatic activity through FPRL1. We suggest that LL-37 peptide will potentially be useful in therapeutic agent for the treatment of periodontitis and other inflammatory diseases.