Vascular Behçet's disease (VBD) is characterized by systemic inflammation involving various immune cell types. Emerging evidence supports that macrophage metabolic dysfunction contributes to VBD pathogenesis. Our previous study has discovered a novel macrophage subset, THBS1^high macrophage, which was significantly increased in aortas from VBD patients. This subset exhibits pro-inflammatory property, hypermetabolic phenotype and is closely associated with disease activity. Based on these findings, we hypothesized that THBS1 overexpression in VBD macrophages may be implicated in metabolic reprogramming.

Mass spectrometry-based untargeted metabolomics and metabolic flux analysis (MFA) based on ¹³C tracers were used to explore metabolic reprogramming of macrophages. Protein-protein interaction was validated using co-immunoprecipitation assays. Recombinant protein and pharmacological intervention were performed for functional studies in macrophages.

THBS1 overexpression (THBS1-OE) macrophages exhibited increased glycolysis and impaired oxidative phosphorylation compared to control macrophages. Using proteomic and metabolomic analyses, we identified increased glucose-6-phosphate dehydrogenase (G6PD), a metabolic enzyme of the pentose phosphate pathway (PPP), in THBS1-OE macrophages. MFA assay also elucidated elevated intermediates of PPP in THBS1-OE macrophages. VBD serum promoted the interaction of THBS1 and G6PD, facilitating the enzymic activity of G6PD to maintain PPP activation. Mechanistically, molecular docking further confirmed the interaction between THBS1 and G6PD, and simultaneously provided the candidate binding peptide segments. G6PD inhibition attenuated cytokine release, M1 polarization and glycolytic activity in THBS1-OE macrophages, whereas recombinant G6PD protein restored these effects in THBS1-knockdown macrophages. In addition, THBS1-OE induced PPP activation aggravated phenotypic transition and apoptosis of smooth muscle cells and T cell activation, indicating that robust THBS1-G6PD interaction contributes to vascular injury.

Aberrant THBS1 overexpression in VBD macrophages triggers metabolic reprogramming to sustain its pro-inflammatory state, and ultimately contributes to vascular inflammation and injury. These findings advance our understanding of the mechanisms underlying pro-inflammatory macrophage activation in VBD and offer a promising theoretical basis for the development of novel targeted therapeutic strategies.