Involvement of the excretory/secretory and surface-associated antigens of Dirofilaria immitis adult worms in the angiogenic response in an in-vitro endothelial cell model

Background: Angiogenesis is a process by which new vessels are formed from pre-existing ones when the physiological conditions of the vascular endothelium are altered. Heartworm disease ( Dirofilaria immitis) causes changes in the vascular endothelium of the pulmonary arteries due to obstruction, friction and hypoxia. The aim of this study was to analyze whether the excretory/secretory antigen of adult worms interacts and modulates the angiogenic mechanism, viable cell number and cell migration, as well as the formation of pseudo-capillaries. Methods: : Cultures of human vascular endothelial cells (HUVECs) stimulated with excretory/secretory antigens (DiES), surface-associated antigens (Cut) from D. immitis adult worms, VEGF, as well as DiES+VEGF and Cut+VEGF were used. The production of VEGF-A and other proangiogenic [soluble VEGFR-2 (sVEGFR-2), membrane Endoglin (mEndoglin)] and antiangiogenic [VEGFR-1/soluble Flt (sFlt), soluble Endoglin (sEndoglin)] molecules was assessed using commercial ELISA kits. Cell viability was analyzed by live cell count and cytotoxicity assays by a commercial kit. In addition, viable cell number by MTT-based assay, cell migration by wound-healing assay carrying out scratched wounds, and the capacity of pseudo-capillary formation to analyze cell connections and cell groups in Matrigel cell cultures, were evaluated. In all cases, non‑stimulated cultures were used as controls. Results: : DiES+VEGF and Cut+VEGF significantly increased the production of VEGF and VEGFR2, and only Cut+VEGF significantly increased the production of VEGFR1/sFlt compared to other groups and non-stimulated cultures. Moreover, only DiES+VEGF produced a significant increase in viable cell number and cell migration, as well as in the organization and number of cell connections. Conclusions: : Excretory/secretory and surface-associated antigens of adult D. immitis activated the angiogenic mechanism by mainly stimulating the synthesis of proangiogenic factors, and only excretory/secretory antigens increased viable cell number, activated cell migration and the formation of pseudo-capillaries. These processes could lead to vascular endothelial remodeling of the infected host and favor the long-term survival of the parasite.