Crotoxin promotes macrophage reprogramming towards an antiangiogenic phenotype

Luciana De Araújo Pimenta,Sandra Coccuzzo Sampaio,Maria Cristina Cirillo,Rui Curi,Marisa Langeani Bretones,Maíra Estanislau S De Almeida

doi:10.1038/s41598-019-40903-0

Luciana De Araújo Pimenta, Sandra Coccuzzo Sampaio + Show 4 more

Open Access

https://doi.org/10.1038/s41598-019-40903-0

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Abstract

Crotoxin (CTX) is the primary toxin of South American rattlesnake Crotalus durissus terrificus venom. CTX reduces tumour mass, and tumour cell proliferation and these effects seem to involve the formation of new vessels. Angiogenesis has a key role in tumour growth and progression and is regulated by macrophage secretory activity. Herein, the effect of CTX on macrophage secretory activity associated with angiogenesis was investigated in vitro. Thymic endothelial cells (EC) were incubated in the presence of macrophages treated with CTX (12.5 nM) or supernatants of CTX-treated macrophages and endothelial cell proliferation, migration and adhesion activities, and the capillary-like tube formation in the matrigel-3D matrix was measured. Angiogenic mediators (MMP-2, VEGF and TNF-α) were measured in the cell culture medium. Macrophages pre-treated with CTX and supernatant of CTX-treated macrophages inhibited EC proliferation, adhesion to its natural ligands, and migration (as evaluated in a wound-healing model and Time Lapse assay) activities. Decreased capillary-like tube formation and MMP-2, VEGF and TNF-α levels in the supernatant of macrophages treated with CTX was also described. CTX promotes macrophage reprogramming towards an antiangiogenic phenotype.

Highlights

Crotoxin (CTX) is a β-heterodimeric neurotoxin formed by the noncovalent association of two subunits, one acidic termed crotapotin and one basic known as phospholipase A2 (PLA2)[1]
We investigated whether the inhibitory activity of macrophages treated with CTX would be dependent on cell-cell contact or the supernatant of cultured macrophages could have the same effect
The CTX antiangiogenic effect resulted of a decrease in the secretion of the pro-angiogenic factors Macrophages also release metalloproteases (MMPs)-2, tumour necrosis factor-α (TNF-α) and VEGF

Summary

Introduction

Crotoxin (CTX) is a β-heterodimeric neurotoxin formed by the noncovalent association of two subunits, one acidic termed crotapotin (subunit CA~9.5 kDa) and one basic (subunit CB~14.5 kDa) known as phospholipase A2 (PLA2)[1]. CTX raises glucose and glutamine utilization and oxidation inhibits spreading and phagocytosis activities[15] and increases production of hydrogen peroxide and nitric oxide by macrophages[10] In this sense, it is important to point out the immunomodulatory effects of CTX, accompanied by tumor regression, observed in vivo experimental models, occurs after administration of low concentration (μg), with rapid onset and long duration and are observed for up to 14 days after a single dose[10]. Macrophages secrete LXA4 and its stable analogue (15-epi-LXA4) with antiangiogenesis properties These lipid mediators are generated through lipoxygenase and exert specific biological effects upon binding to membrane G-protein coupled formyl peptide receptors-FPRs ( known as ALXR) that have been reported in several cell types including macrophages[40,41]. We performed the measurements and tube-forming assay by human endothelial cells (HUVEC-CS) on the matrigel-3D matrix using the supernatant of human macrophages (THP-1 macrophage differentiated) pretreated with CTX

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