Fig 2: Female syndecan-4-/- hearts exhibit alterations in SCOP phosphatase, fasting C-peptide and GLUT4 levels. (A) Immunoblot analysis of SCOP in LV of WT-SED, WT-ET, KO-SED and KO-ET female mice. (B) Fasting serum glucose levels, (C) insulin and (D) C-peptide levels in female syndecan-4-/- and WT mice (n = 8–14, indicated on the graph bars). Immunoblot analyses of (E) GLUT4, (F) GLUT1 and (G) pSer1177-eNOS and total eNOS in LV of WT-SED, WT-ET, KO-SED and KO-ET female mice. Quantified immunoblot values are presented as mean percentages ± SEM, normalized to the WT-SED (n = 5–10, indicated on the graph bars). Coomassie staining (A,E–G) was used as loading controls for immunoblots. Glucose levels are presented in mmol/L (B) and insulin and C-peptide values are presented in ng/mL (C,D). Comparison between the groups were analyzed using Mann-Whitney U tests (*p < 0.05 and **p < 0.01).

Fig 3: Increased GM1 levels in 1 ng/ml TNFa-treated HAECs reduce insulin signaling(A) At day 0, cells were reseeded with 1 ng/ml TNFa and with or without AMP-dNM in HAEC medium and incubated for 3 days. At day 3, cells were harvested and analyzed (B–F). Control cells were grown in HAEC medium. (B) Real-time PCR analysis of IRS2 and eNOS performed using cDNA derived from control and TNFa-treated HAECs. Results shown were normalized against values obtained for control HAECs (value = 1). (C) Cell surface levels of GM1 in TNFa-treated HAECs with or without AMP-dNM treatment analyzed by flow cytometry. MFIs of three independent experiments relative to control HAECs are shown. (D) Western blot analysis of insulin signaling performed in TNFa-treated HAECs with or without AMP-dNM treatment. (E) Histograms show mean densitometric readings ± SD of phosphorylated or non-phosphorylated proteins in insulin-stimulated cells normalized to loading control (ß-ACTIN). All values were obtained from three independent experiments. (F) Immunocytochemical staining performed in TNFa-treated HAECs with or without AMP-dNM treatment. Representative images are shown (GM1, green; IRa, red; DAPI, blue; GM1 and IRa co-localization, yellow). *P < 0.05. Control (Ctr): untreated cells.

Fig 4: NOS3 mRNA correlates with placental perfusion indices and does not depend on NOS3 intronic 27 nt-ncRNA polymorphism. (A) NOS3 mRNA levels increased >15-fold in patients suffering from severe impairment of placental perfusion (group 3). (B) Protein levels of eNOS and eNOS(Ser1177ph) correlated with NOS3 mRNA levels. NOS3 mRNA levels were as follows: 12.9 (lane 1), 22.6 (lane 2), 157.7 (lane 3), 56.8 (lane 4), 74.7 (lane 5), 136.9 (lane 6). (C) No correlation between NOS3 mRNA levels and genotype at the 27 nt-ncRNA locus in intron 5 of the NOS3 gene was found. In human alleles, two tandem-repeated homologous motifs were found in typical allele-specific order (top right). The number of patients analysed for each group is stated (n).

Fig 5: ATV-Exos activated angiogenesis-related AKT/eNOS pathway. HUVECs were treated with LG, HG, HG + Exos, and HG + ATV-Exos, and the protein levels of p-AKT, AKT, p-eNOS, eNOS and PTEN and HSP90 were explored by Western blotting with HSP90 being used as a control. *P < 0.05