Heartworm disease caused by Dirofilaria immitis is a vector-borne zoonotic disease responsible for the infection of mainly domestic dogs and cats, or these are those for which the most data are known. Humans are an accidental host where a benign, asymptomatic pulmonary nodule may originate. Dirofilaria immitis also harbours the endosymbiont bacteria of the genus Wolbachia, which play a role in moulting, embryogenesis, inflammatory pathology, and immune response. When Wolbachia sp. is released into the bloodstream, endothelial and pulmonary damage is exacerbated, further encouraging thrombus formation and pulmonary hypertension, facilitating congestive heart failure and death of the animal. Previous studies have shown that parasite excretory/secretory products are able to activate the pro-angiogenic pathway (formation of new vessels) to facilitate parasite survival. The aim of this study was to analyse the role of Wolbachia sp. and its relationship with the cellular processes and the angiogenic pathway in a model of human endothelial cells in vitro. The use of recombinant Wolbachia Surface Protein (rWSP) showed that its stimulation exerted an anti-angiogenic effect by detecting an increase in the production of VEGFR-1/sFlt1 and sEndoglin and did not affect the production of VEGFR-2 and mEndoglin (pro-angiogenic molecules). Furthermore, it did not stimulate cell proliferation or migration, although it did negatively stimulate the formation of pseudocapillaries, slowing down this process. These cellular processes are directly related to the angiogenic pathway so, with these results, we can conclude that Wolbachia sp. is related to the stimulation of the anti-angiogenic pathway, not facilitating the survival of D. immitis in vascular endothelium.

Heartworm disease, caused by Dirofilaria immitis, is a vector-borne zoonotic disease, (mainly affecting canids and felids) causing chronic vascular and pulmonary pathology in its early stages, which worsens with parasite load and/or death of adult worms in the pulmonary artery or right heart cavity, and can be fatal to the host. Angiogenesis is a mechanism by which new blood vessels are formed from existing ones. The aim of this work was to study the effect of two molecules of the D. immitis excretory/secretory antigen (DiES) on the angiogenic process, taking into account that this antigen is able to interact with this process and use it as a survival mechanism. For this purpose, an in vitro model of endothelial cells was used and treated with two recombinant proteins, i.e., actin (Act) and fructose-bisphosphate aldolase (FBAL) proteins belonging to DiES, and both pro- and antiangiogenic molecules were analyzed, as well as the cellular processes of cell proliferation, migration, and pseudocapillary formation. Act and FBAL proteins, together with vascular endothelial growth factor (VEGF-A), as an angiogenic precursor, are able to stimulate the production of proangiogenic factors as well as cellular processes of proliferation, migration, and pseudocapillary formation. This implies that these molecules could be produced by D. immitis to facilitate its survival, and the relationship between parasite and canine host would be further elaborated.

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.