Fig 2: Disruption of the putative CD40L/CD40 autocrine signalling loop decreases the axon growth response to high neurotrophin concentrations. (A,B) Graphs of axon length of E11 (A) and E12 (B) DRG neurons of Cd40−/− and Cd40+/+ littermates cultured for 24 h with and without BDNF (A) or NGF (B) at concentrations ranging from 0 to 10 ng/ml. (C,D) Graphs of axon length of E11 (C) and E12 (D) DRG neurons cultured for 24 h with a range of BDNF or NGF concentrations plus either function-blocking anti-CD40L and function-blocking anti-CD40 antibodies or isotype controls (each at 2 μg/ml). All cultures also received 25 µM Boc-D-FMK. (E) Axon lengths of E12 DRG BAX-deficient neurons cultured with a range of NGF concentrations together with either function-blocking anti-CD40L and function-blocking anti-CD40 antibodies or isotype controls (each at 2 μg/ml). (F) Axon lengths of E12 DRG neurons cultured with 10 ng/ml NGF and function-blocking anti-CD40L and anti-CD40 antibodies alone or in combination or with isotype controls (each at 2 μg/ml) plus 25 µM Boc-D-FMK. Data are mean±s.e.m. of >50 neurons per condition combined from three experiments of each type. **P<0.01, ***P<0.001; one-way ANOVA, statistical comparison at each BDNF or NGF concentration.

Fig 3: Blood pressure and heart rate of animals after 2K1C (2‐kidney 1‐clip) surgery. A, Systolic blood pressure (SBP) and diastolic blood pressure (DBP), (B) heart rate (HR), and (C) pulse pressure (PP) measurements of S rats and Cd40 mutant rats 4 weeks after 2K1C surgery. Data plotted are the recordings obtained every 5 minutes continuously for 24 hours and averaged for 4‐hour intervals over 3 days. Data presented as mean±SEM, n=8. *P<0.05; **P<0.01.

Fig 4: Summary of the in vitro and in vivo experiments that documented the relevance of the CD40/CD40L pathway in proliferative diabetic retinopathy. TNF-α = tumor necrosis factor-α; VEGF = vascular endothelial growth factor; HRMEC = human retinal microvascular endothelial cell; ERK = extracellular-signal regulated kinase; ICAM-1 = intercellular adhesion molecule-1; VCAM-1 = vascular cell adhesion molecule-1; NF-ĸB = nuclear factor-Kappa B; MCP-1 = monocyte chemotactic protein-1; HMGB1 = high mobility group box-1; MMP-9 = matrix metalloproteinase-9; BRB = blood–retinal barrier; ↑ = enhanced.

Fig 5: Quantitative analysis of gene expression of (A) CD40 and (B) PAI‐1 in renal cortex tissue of Dahl S and Cd40 mutant rats after 2K1C surgery.Gene expression was normalized to respective sham group (2‐way ANOVA; genotype×surgery: CD40: P<0.001; PAI‐1: P=NS). Data presented as mean±SEM, n=8 per group. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. 2K1C indicates 2‐kidney 1‐clip; ΔΔCT, delta delta threshold cycle; NS, not significant; PAI‐1, plasminogen activator inhibitor 1; and TBP, TATA‐box binding protein.