# Glyceraldehyde 3-Phosphate Dehydrogenase and Galectin from Dirofilaria immitis Excretory/Secretory Antigens Activate Proangiogenic Pathway in In Vitro Vascular Endothelial Cell Model

## TL;DR

This study shows that two proteins from Dirofilaria immitis, when combined with VEGF-A, activate proangiogenic pathways in endothelial cells, potentially aiding the parasite's survival.

## Contribution

The study identifies the role of D. immitis excretory/secretory proteins in activating proangiogenic pathways in vascular endothelial cells.

## Key findings

- rDiGAPDH+VEGF-A and rDiGAL+VEGF-A significantly increased sVEGFR-2, mEndoglin, and VEGF-A expression.
- These combinations also enhanced cell proliferation, migration, and pseudocapillary formation in endothelial cells.
- The proteins may help D. immitis survive in the circulatory system by stimulating proangiogenic pathways.

## Abstract

Dirofilaria immitis is the causative agent of heartworm disease and mainly affects domestic dogs, among others, causing an acute inflammatory pathology. In this study, the effects of two D. immitis proteins, which are secreted into the bloodstream, on the vascular endothelium and parasite survival were analyzed. Our results highlight the significant increase in the production of molecules and cellular processes related to the formation of D. immitis.

Heartworm disease is caused by Dirofilaria immitis, which mainly affects canids and felids. Adult D. immitis worms are located between the heart’s right ventricle and the pulmonary artery. These parasites produce an inflammatory and hypoxic process in the vascular endothelium. It has been demonstrated that D. immitis excretory/secretory antigens are able to stimulate the angiogenic process as a survival mechanism of D. immitis in the vascular endothelium, stimulating the proangiogenic pathway and related cellular processes. Our goal was to study the role of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and galectin (GAL) (proteins of D. immitis excretory/secretory antigens) plus vascular endothelial growth factor isoform A (VEGF-A) in the angiogenic process and their relationship with three cellular processes (cell proliferation, cell migration, and pseudocapillary formation) in an in vitro model of vascular endothelial cells. Cell viability and cytotoxicity were analyzed by live cell analysis and a commercial kit, respectively. VEGF-A, sVEGFR-2, VEGFR-1/sFlt, soluble endoglin, and membrane endoglin were analyzed by commercial ELISA kits. Cell proliferation, cell migration, and pseudocapillary formation were analyzed by MTT-based assay, the wound healing technique, and counting cell connections and cell clusters, respectively. rDiGAPDH+VEGF-A and rDiGAL+VEGF-A significantly increased the expression of sVEGFR-2, mEndoglin, and VEGF-A compared to cultures treated with only the proteins (rDiGAPDH and rDiGAL), VEGF-A, or unstimulated cultures. In addition, they also produced a significant increase in cell proliferation, cell migration, and pseudocapillary formation. Therefore, these proteins together with VEGF-A can activate the proangiogenic pathway and could be related to D. immitis survival in the circulatory system.