Profiling of signaling pathways in human dermal blood and lymphatic endothelial cells induced by snake venom cysteine-rich secretory protein (svCRiSP) from North American snakes

Montamas Suntravat,Oscar Sanchez,Emelyn Salazar,Abcde Cirilo,Elda Sanchez,Peter Davies,Jack Ocheltree,Armando Reyes,Jacob Galan

doi:10.3390/iect2021-09141

Abstract

Snake venom Cysteine-Rich Secretory Proteins (svCRiSPs) are important components in the venom of many species of snakes including Viperidae and Elapidae. Although the widespread distribution of svCRiSPs in snake venoms is well known, little is known of the contribution that they make to the local pathophysiology of snakebite. This work aimed to investigate the role of svCRiSPs from the most medically significant species of North American snakes (Crotalus atrox, C. adamanteus, C. scutulatus scutulatus, C. horridus, and Agkistrodon piscivorus), focusing on the cellular and molecular mechanisms underlying vascular biology in snakebite. We evaluated the biological activities of svCRiSPs (Css-CRiSP, Catrox-CRiSP, Cada-CRiSP, Chor-CRiSP, and App-CRiSP) by using both in vitro assays on human dermal lymphatic endothelial cells (HDLECs) and human dermal blood endothelial cells (HDBECs) permeability and in vivo Miles assay of vascular permeability. Of all the CRiSPs tested, Css-CRiSP and App-CRiSP displayed the highest increase in acute vascular permeability compared to other crotaline CRiSPs. To elucidate the main pathway underlying the endothelial permeability induced by svCRiSPs, we initially screened the changes in protein expression and phosphorylation in HDLECs and HDBECs 30 min after treatment with Css-CRiSP and App-CRiSP using reverse phase protein arrays (RPPA). Stimulating HDBECs with Css-CRiSP and App-CRiSP enhanced caveolin-1 expression. In HDLEC cells, Css-CRiPS and App-CRiSP increased the upregulation of the expression of proteins involved in the phosphoinositide 3-kinase/Akt, Src, MAPK/JNK pathways, cell mobility, and cell adhesion molecules. These preliminary observations suggest that Css-CRiSP and App-CRiSP induce the increased endothelial monolayer permeability in HDBECs and HDLECs via different mechanisms. Knowledge gained from these studies provides insights into the molecular mechanisms that underlie the effects of svCRiSPs on vascular function and contributes to a new level of understanding of the pathophysiology of snakebite.

Highlights

Much less is known of the biological activity of svCRiSPs present in the North American snakes’ venom, especially rattlesnakes (Genus Crotalus) and cottonmouths (Genus Agkistrodon)
Natrin from Naja atra has been shown to produce acute activation of MAP kinase signaling in human umbilical vein endothelial cells (HUVEC) [6]
This study aims to determine key singling pathways induced by svCRiSPs on human endothelial cells using reverse-phase protein microarray analysis (RPPA) analysis

Summary

Introduction

Much less is known of the biological activity of svCRiSPs present in the North American snakes’ venom, especially rattlesnakes (Genus Crotalus) and cottonmouths (Genus Agkistrodon). Several of these svCRiSPs have been shown to block the activity of L-type Ca2+ and/or K+ channels [4,5]. Two svCRiSPs, ES-CRiSP, isolated from an Asian pit viper, and natrin, isolated from cobra venom, have been shown to affect vascular endothelial cell activity [6,7]. Natrin from Naja atra has been shown to produce acute activation of MAP kinase signaling in human umbilical vein endothelial cells (HUVEC) [6]. ES-CRiSP from Echis carinatus sochureki, on the other hand, has not been reported to have an effect on MAP kinase signaling on its own but can block VEGF-dependent activation of Erk1/2 in HUVEC cells [7]

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