Fig 2: After adding various protein inhibitors to the RPMI 1640 or low-level glucose culture medium system and performing a 24-h incubation, the expression levels of the glycolysis-regulating enzyme HIF-1a, the key enzymes PFKFB3 and LDHA, lactic acid and PD-L1 in the two cell lines were detected by western blotting.A After the addition of the HIF-1a inhibitor PX-478 under normal RPMI 1640 or low-glucose culture medium conditions, the expression levels of regulatory enzyme, key enzymes and PD-L1 were detected. B Glycolysis-regulating enzyme, key enzyme and PD-L1 expression levels were evaluated in both cell lines after the addition of another HIF-1a inhibitor, YC-1. C After the PFKFB3 inhibitor PFK-015 was added, glycolysis-regulating enzyme, key enzyme and PD-L1 expression levels were assessed in the two cell lines. D The LD level of two cell lines in RPMI 1640 or low-glucose culture medium treated with the three inhibitors mentioned above is shown. The concentrations of PX-478, YC-1 and PFK-015 were 40 µM, 10 µM and 10 µM, respectively. Independent experiments were performed in triplicate. Data are expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.

Fig 3: PFKFB3 deficiency in endothelial cells inhibits intraplaque neovascularization in vein graft lesions but does not alter a-SMA coverage of microvessels. A and B Representative vein graft lesions stained with CD31 antibody. Endothelial cells are detected as CD31 positivity (dark violet) around the vessel lumen. Microvessels are marked by arrows. Scale bar = 200 µm. C and D Representative vein graft lesions stained with anti-a-SMA. Vascular smooth muscle cells in the vessel wall are stained by the a-SMA antibodies (brown). E Quantification of microvessels in vein grafts of ApoE-/-PFKFB3fl/fl and ApoE-/-PFKFB3ECKO mice. *P = 0.035 versus ApoE-/-PFKFB3fl/fl. Independent samples t test; n = 9 (ApoE-/-PFKFB3fl/fl) or n = 11 (ApoE-/-PFKFB3ECKO). F Quantification of a-SMA coverage of microvessels in vein grafts of ApoE-/-PFKFB3fl/fl and ApoE-/-PFKFB3ECKO mice. Independent samples t test did not show statistical significance (n = 8 for both groups)

Fig 4: PFKFB3 deficiency in endothelial cells reduces macrophage infiltration in vein graft lesions. A Immunohistochemical detection of macrophages in representative vein graft lesions of ApoE-/-PFKFB3fl/fl and ApoE-/-PFKFB3ECKO mice using MAC-3 antibody. Scale bar = 200 µm. B Quantification of macrophages in vein graft lesions of ApoE-/-PFKFB3fl/fl and ApoE-/-PFKFB3ECKO mice. *P = 0.047 versus ApoE-/-PFKFB3fl/fl mice. Independent samples t test; n = 8 (ApoE-/-PFKFB3fl/fl) or n = 10 (ApoE-/-PFKFB3ECKO)

Fig 5: PFKFB3 deletion in endothelial cells evokes a more stable vein graft lesion phenotype. A Masson’s Trichrome staining of representative vein grafts. This staining was used for the evaluation of lesion complexity. Nuclei are stained dark blue, erythrocytes are stained bright-red, and collagen is stained blue. The staining allows easy visualization of intraplaque hemorrhages (red areas) as well as necrotic core and cholesterol crystals (white acellular regions). Scale bar = 200 µm. B Boxed area of panel A showing an almost intact endothelium exposed to the lumen (L) (black arrows), a fibrous cap with numerous vascular smooth muscle cells (white arrows), small foam cells (asterisks), an area with extravasated erythrocytes (##), and neovascularization (#). Scale bar = 100 µm. C Quantification of foam cells in vein graft lesions of ApoE-/-PFKFB3fl/fl and ApoE-/-PFKFB3ECKO mice. Independent samples t test; n = 9 (ApoE-/-PFKFB3fl/fl) or n = 12 (ApoE-/-PFKFB3ECKO )