Fig 2: CCL4 inhibition by siRNA recovered functions of EPCs from type 2 DM patients with increased angiogenic protein expressions. Western blotting and statistical analyses of CCL4 ((A); n = 3), p-AKT, p-eNOS, VEGF, and SDF-1a. The angiogenic proteins were reversed after the administration of siCCL4 ((B); n = 3). Tube formation ability and quantitative analysis of EPCs from type 2 DM patients and control subjects; the images were captured using a (×40) microscope. The tube formation ability was improved after the administration of siCCL4 ((C); n = 3). Migration ability and quantitative analysis of EPCs from type 2 DM patients and control subjects; the images were captured using a (×100) microscope. The migration ability was improved after the administration of siCCL4 ((D); n = 3). * p < 0.05, ** p < 0.01.

Fig 3: CCL4 inhibition by knockout improved wound repair in diabetic mice. Representative wound areas (A). The closure rates of 3-millimeter punch biopsies were measured ((B); n = 6). Representative images with H&E staining (C). Representative images with immunostaining of CD31 and Ki67. Both CD31- and Ki67-positive areas were enhanced in the CCL4-knockout mice (D,E). Western blotting and statistical analyses of CCL4, p-AKT, p-eNOS, VEGF, and SDF-1a in the wound area. The angiogenic proteins were enhanced in tissues from the CCL4-knockout mice ((F); n = 3). Western blotting and statistical analyses of TNF-a and IL-6 in the wound area. The inflammatory proteins were decreased in tissues from the CCL4-knockout mice ((G); n = 3). WT, wild-type mice; CCL4KO, CCL4-knockout mice; WT-DM, wild-type diabetic mice; CCL4KO-DM, CCL4-knockout diabetic mice. * p < 0.05, ** p < 0.01 compared with WT, # p < 0.05 compared with WT-DM.

Fig 4: AMPk and eNOS activation is reduced after an acute load of fructose. In high fructose fed rats challenged with an acute load of fructose, there was a decreased activation of AMPk (A) and eNOS (B). Allopurinol treatment prevented all these effects. For western blotting, 4 out 5 randomly selected samples per group were analyzed. **** = p < 0.0001.

Fig 5: Expression of tPA, uPA, ICAM-1, VCAM-1, KLF4, eNOS and BDNF in the corpus striatum of 2- and 7-month-old WT and NZBWF1 mice. Depicted are real-time qPCR analyses of (A) tPA, (B) uPA, (C) ICAM-1, (D) VCAM-1, (E) KLF4, (F) eNOS and (K) BDNF gene expression. Relative changes in mRNA levels were determined using the ?CT method and were normalised to the ribosomal subunit S18. (G,J) Representative Western blots and corresponding densitometry of (H) KLF4, (I) eNOS and (L) BDNF protein expression, including both (L') mature BDNF and (L?) proBDNF. Densitometric analyses were performed using the ImageJ software, and normalised values were determined by dividing the mean optical density of bands over the mean optical density of their corresponding GAPDH bands. Data represent the mean of (n = 5) WT 2 mo, (n = 7) NZBWF1 2 mo, (n = 4) WT 7 mo and (n = 9) NZBWF1 7 mo mice in each experimental group. Results are expressed as mean ± S.D. * p = 0.05, ** p = 0.01, *** p = 0.001, **** p = 0.0001 vs. WT at the corresponding age, as determined by one-way ANOVA followed by Tukey post hoc test. tPA = tissue plasminogen activator; uPA = urokinase plasminogen activator; ICAM-1 = intercellular adhesion molecule 1; VCAM-1 = vascular cell adhesion molecule 1; KLF4 = Krüppel-like factor 4; eNOS = endothelial nitric oxide synthetase; BDNF = brain-derived neurotrophic factor.