LL37 promotes angiogenesis: a potential therapeutic strategy for lower limb ischemic diseases

Abstract

Purpose: To study the angiogenic capacity of antimicrobial peptide LL37 (cathelicidin antimicrobial peptide), explore its molecular mechanisms, and provide new ideas for treating lower limb ischemic diseases.

Methods: LL37 was applied exogenously to human umbilical vein endothelial cells (HUVECs), and its effects on cell proliferation, migration, and angiogenesis were assessed using Cell Counting Kit-8 (CCK-8), plate cloning, scratch, and angiogenesis assays. A mouse lower limb ischemia model was established, with LL37 injected intramuscularly on days 0, 4, and 8. Blood flow recovery was evaluated by laser Doppler flowmetry. Immunofluorescence staining detected cluster of differentiation 31 (CD31) and cluster of differentiation 34 (CD34) expression, while Hematoxylin and Eosin (H&E) staining assessed muscle cell morphology. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting analyzed gene and protein expression changes in HUVECs.

Results: LL37 enhanced the proliferative, migratory, and pro-angiogenic abilities of HUVECs. It significantly improved blood flow recovery in ischemic limbs, with higher CD31/CD34 expression and more intact muscle morphology. qRT-PCR analysis demonstrated elevated expression of angiogenesis-related genes in LL37-treated HUVECs. Western blotting revealed increased vascular endothelial growth factor A (VEGFA) expression and enhanced phosphorylation levels of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway in LL37-treated cells.

Conclusion: LL37 promotes angiogenesis via the VEGFA-PI3K/AKT/mTOR pathway, showing potential for treating lower limb ischemia by improving perfusion.

Keywords: LL37; VEGFA-PI3K/AKT/mTOR pathway; angiogenesis; antimicrobial peptide; lower limb ischemia.

FIGURE 1

(a) The CCK8 assay examined the growth curves of control cells and LL37-treated HUVECs. (b) The plate cloning assay examined the proliferation of control cells and LL37-treated HUVECs. (c) The angiogenesis assay examined the angiogenesis of control cells and LL37-treated HUVECs. (d) The migration ability of control cells and LL37-treated HUVECs was detected by wound healing assay. (e) The capacity for migration and invasion of control cells and LL37-treated HUVECs was detected by transwell assay. Note: All experiments were independently repeated at least three times. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 indicate statistical significance.

FIGURE 2

(a) Laser Doppler detection of lower limb blood perfusion in mice. Warmer colors (red or yellow) indicate higher perfusion, while cooler colors (blue) indicate lower perfusion. The perfusion values of the affected and healthy limbs were measured, and the ratio of the two was calculated for quantitative analysis. (b) Immunofluorescence detection of the expression of CD31 and CD34 in the lower limb muscles of mice. Blue fluorescence indicates nuclei, red fluorescence represents CD31, and green fluorescence represents CD34 (×200 magnification). (c) Observation of the morphology of mouse lower limb muscle cells using H&E staining. Note: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 indicate statistical significance.

FIGURE 3

(a) qRT-PCR was used to detect the expression of angiogenesis-related genes in HUVECs treated with LL37 for 24 h (b) qRT-PCR was used to detect the expression of angiogenesis-related genes in HUVECs treated with LL37 for 48 h. (c) The changes in protein levels in HUVECs after treatment with LL37 were detected by Western blot. (d) Quantitative analysis of changes in protein expression levels in HUVECs after LL37 treatment. (e) Quantitative analysis of changes in protein phosphorylation levels in HUVECs after LL37 treatment.

FIGURE 4

(a–c) The angiogenesis assay examined the angiogenesis of control cells and LL37-treated HUVECs. (d) The changes in protein levels in HUVECs after treatment with LL37 and VEGF165 were detected by Western blot.