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

Abstract

Abstract 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.