Fig 2: IL-6 induces LRG1-dependent defective angiogenesis. Mouse metatarsal bones (a) and aortic rings (b) from wild type C57Bl6 and Lrg1-/- mice were cultured and treated with 50 ng/ml IL-6 and analysed for angiogenesis and sprouting respectively. (c) Representative images of metatarsal bones treated with PBS as control (Ctl) or 50 ng/ml IL-6 and stained with antibodies against CD31 (green), NG2 (blue), and aSMA (red) for mural cell coverage and analysis. Scale bars, 50µm. N = 3 independent experiments. ANOVA with multiple comparisons test (a–c: Dunn’s, b: Tukey’s), Mean ± SEM, * = 0.05, ** = 0.01, **** = 0.0001.

Fig 3: Leucine-rich a-2-glycoprotein (LRG1) protein expression were upregulated in activated EC. Putatively atherogenic flow at the centre of swirled wells upregulated the expression of (A) LRG1 and (B) phospho-IkBa in HUVECs, compared to putatively atheroprotective flow at the edge of the wells. Expression of (C) LRG1 and (D) phospho-IkBa was upregulated in HUVECs by TNF-a. (E) Summary of the motif analysis conducted using FIMO. The schematic diagram shows the 2 kb sequence near the transcription start site (bent arrow) of the LRG1 promoter region containing two putative NF-?B (RelA) binding sites (red box) predicted by FIMO. (F) ChIP-qPCR analysis of TNF-a-induced association between NF-?B/p65 and the LRG1 promoter in HDMECs. [(A,B,E), Unpaired two-tailed Student’s t-test; n = 3. (C), One-way ANOVA followed by Bonferroni post hoc test; n = 3. *p < 0.05; **p < 0.01].

Fig 4: Soluble TNFR1 (sTNFR1) was upregulated in the serum of CLI patients and was correlated with LRG1 expression. (A) ELISA measurement of sTNFR1 concentration in healthy controls and CLI patients (with and without diabetes). sTNFR1 was significantly higher in the serum of CLI patients (with and without diabetes) compared to healthy controls. (B) Receiver Operating Characteristic (ROC) curve analysis of the ability of serum sTNFR1 to differentiate between healthy controls and CLI patients, and between healthy controls and CLI patients with diabetes. (C) The concentrations of sTNFR1 and LRG1 in healthy controls and CLI patients were significantly correlated. (D) ELISA measurement revealed that the concentration of sTNFR1 in the serum of 37-week-old Lrg1–/– mice was significantly lower than that in the serum of wild-type counterparts. [(A), One-way ANOVA followed by Bonferroni post hoc test; n = 11/group. (C), Linear regression analysis, n = 33; Dashed lines indicate the 95% confidence intervals for the regression line. (D), Unpaired two-tailed Student’s t-test; n = 6. **p < 0.01; ***p < 0.001].

Fig 5: Tocilizumab antibody blocks LRG1 gene expression. HPMEC cells treated with PBS as control (Ctl), 50 ng/ml IL-6, and 20 µg/ml Tocilizumab (Toc) antibody in combination with IL-6 as indicated. RNA and protein were extracted and subjected to RT-qPCR and western blotting. (a) Human LRG1 gene expression normalised to HPRT housekeeping genes. Protein expression of phosphorylated and total STAT3 (b), SMAD1/5 (c) and SMAD2 (d). Quantification was performed by densitometric analysis of normalised phosphorylated to normalised total protein using ImageJ. Representative images of cropped blots (original blots are provided in the supplementary information). N = 3 independent experiments. ANOVA with multiple comparisons test (Tukey’s), Mean ± SEM, * = 0.05, ** = 0.01, *** = 0.001.