Endothelial proteomic response to Dirofilaria repens somatic antigens and the Dr20/22 protein reveals parasite-driven modulation of angiogenesis

BACKGROUND: Although Dirofilaria repens induces perivascular proliferation in subcutaneous nodules, the molecular factors driving this angiogenic response remain unknown. The objective of this study is to investigate the activation of the angiogenic mechanism in an in vitro model of human vascular endothelial cells (HUVECs) stimulated with D. repens somatic antigen (DrSA) and Dr20/22 protein, identifying the key pathways underlying the vascular pathology caused by the parasite. METHODS: After 24 h of stimulation with vascular endothelial growth factor A (VEGF-A), DrSA, Dr20/22, DrSA+VEGF-A, and Dr20/22+VEGF-A, cell viability was assessed, and proteins from the cell lysate and supernatant of HUVEC cultures were processed. Proteomic profiles were analysed by data-independent acquisition/label-free quantitation/liquid chromatography-tandem mass spectrometry (DIA-LFQ LC-MS/MS), and bioinformatics analyses (Gene Ontology, enrichment pathway analysis) were performed to identify proteins differentially expressed in relation to the angiogenic process in subcutaneous dirofilariasis. RESULTS: Supernatant analysis showed that DrSA+VEGF-A and Dr20/22+VEGF-A induced the most robust pro-angiogenic response compared to single treatments. Co-stimulation significantly upregulated key secreted proteins, including angiopoietin-2 (ANGPT2), cadherin-5 (CDH5), and multimerin-1 (MMRN1). This synergistic secretome, characterized by enriched angiogenesis pathways, identifies secreted endothelial cell-specific molecule 1 (ESM1) and matrix metalloproteinase-2 (MMP2) as primary drivers of vascular activation and remodelling in the host-parasite interface. CONCLUSIONS: DrSA and Dr20/22 act as key molecular drivers that manipulate host angiogenesis. While their synergy with VEGF-A clarifies the origin of vascular changes in subcutaneous dirofilariasis, further research is essential to fully characterize the specific pathways governing vascular pathology and subcutaneous nodule formation. These investigations will deepen the scientific understanding of host-parasite interactions and the disease's clinical etiopathogenesis.