Comparative proteomic analysis of the angiogenic response in an in vitro model of vascular endothelial cells to excretory/secretory and somatic antigens of Dirofilaria immitis

BACKGROUND: Dirofilaria immitis infection induces endothelial inflammation and vascular remodeling, contributing to heartworm disease pathology. While excretory/secretory antigens (DiES) are known to promote angiogenesis, somatic antigens (DiSA) exhibit different modulatory effects. This study aims to characterize the differential angiogenic response of endothelial cells to these antigenic fractions using a proteomic approach. METHODS: Human umbilical vein endothelial cells were cultured and stimulated for 24 h with DiES or DiSA, either alone or supplemented with vascular endothelial growth factor (VEGF)-A to simulate active angiogenesis. Proteomic profiles of cell supernatants and lysates were analyzed using data-independent acquisition-label-free quantification (DIA-LFQ) liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Bioinformatic analyses, including Gene Ontology and pathway enrichment, were performed to identify dysregulated proteins and molecular mechanisms related to angiogenesis. RESULTS: Stimulation with DiES, particularly in combination with VEGF-A, produced the most significant enrichment of angiogenic processes, characterized by the downregulated anti-angiogenic proteins tryptophanyl-tRNA synthetase 1 (WARS1) and the modulation of the phosphatidylinositol-3 kinase (PI3K)/protein kinase B (AKT) pathway [upregulated 3-phosphoinositide-dependent protein kinase 1 (PDPK1) and downregulated AKT3]. Conversely, DiSA induced a limited angiogenic response with fewer dysregulated proteins. Notably, angiopoietin-2 (ANGPT2) was upregulated in all treatments, suggesting universal vascular destabilization. However, in the absence of VEGF-A, both antigens induced markers of vascular regression and cell lysis. CONCLUSIONS: DiSA and DiES exert distinct immunomodulatory effects on the endothelium. DiES acts synergistically with VEGF-A to drive angiogenesis via the ANGPT/tyrosine kinase receptor (TIE) and PI3K/AKT axes, whereas DiSA fails to support this process effectively. These proteomic insights contribute to elucidate the molecular mechanisms driving vascular remodeling and pathology in heartworm disease.