Fig 1: Anti-VEGF-A treatment modulates endothelial protein expression in APP/PS1 mice. ELISA measurements of VEGF-A (A) and eNOS (B) concentrations after 2 weeks of anti-VEGF-A treatment or saline control injections in APP/PS1 and wild-type (WT) mice (APP/PS1–saline: n = 6, APP/PS1–anti-VEGF-A: n = 6 (7), WT–saline: n = 9, WT–anti-VEGF-A: n = 5 (6); one-way ANOVA with Tukey’s post hoc multiple comparison correction to compare across groups: APP/PS1–saline (VEGF-A) versus APP/PS1–anti-VEGF-A (VEGF-A) P < 0.075, WT–saline (VEGF-A) versus WT–anti-VEGF-A (VEGF-A) P = 0.81, APP/PS1–saline (VEGF-A) versus WT–saline (VEGF-A) P < 0.05, APP/PS1–anti-VEGF-A (VEGF-A) versus WT–saline (VEGF-A) P = 0.93, APP/PS1–saline (eNOS) versus APP/PS1–anti-VEGF-A (eNOS) P < 0.01, WT–saline (eNOS) versus WT–anti-VEGF-A (eNOS) P < 0.01, APP/PS1–saline (eNOS) versus WT–saline (eNOS) P < 0.05). (C) Z-projection of confocal microscopy image stacks from representative cortical areas from mice of all four groups, revealing increased occludin density in anti-VEGF-A-treated APP/PS1 mice as compared to saline-injected APP/PS1 mice. Integrated density of occludin fluorescence as a function of the integrated density of the endothelial cell marker Glut-1 in the cortex (C) and hippocampus (D) (APP/PS1–saline: n = 3, APP/PS1–anti-VEGF-A: n = 3, WT–saline: n = 3, WT–anti-VEGF-A: n = 3; one-way ANOVA with Tukey’s post hoc multiple comparison correction to compare across groups: APP/PS1–saline cortex versus APP/PS1–anti-VEGF-A cortex P = 0.0006, saline WT cortex versus WT–anti-VEGF-A cortex P = 0.53, APP/PS1–saline cortex versus WT–saline cortex P = 0.0005, APP/PS1–anti-VEGF-A cortex versus WT–saline cortex P > 0.99, APP/PS1–saline hippocampus versus APP/PS1–anti-VEGF-A hippocampus P = 0.0004, WT–saline hippocampus versus WT–anti-VEGF-A hippocampus P > 0.99, APP/PS1–saline hippocampus versus WT–saline hippocampus P = 0.0002, APP/PS1–anti-VEGF-A hippocampus versus WT–saline hippocampus P > 0.99). Each point represents one mouse and the red horizontal represents the median. Sex differences are indicated by colour, with black data-points representing females and blue representing males.
Fig 2: KYP-2047 treatment on VEGF/α-SMA expression and eNOS levels. VEGF/α-SMA ratio expression was analyzed by Western blot, suggesting an increment of this marker in CVI group, compared to control animals (A), see the densitometric units score (B); treatment with KYP-2047 significantly reduced VEGF/α-SMA expression (A), see the densitometric units score (B). ELISA kit for eNOS expression on saphene vein samples was performed; treatment with KYP-2047 (10 mg/kg, i.p.) significantly reduced IL-8 quantification (C), compared to the high amount of eNOS released in the CVI-damaged groups (C). Data represent the means of at least three independent experiments. One-way ANOVA followed by Bonferroni post-hoc. *** p < 0.001 versus Sham; ### p < 0.001 versus CVI.
Fig 3: DNA-PKcs mediates endotoxemia-related endothelial dysfunction through disruption of the actin cytoskeleton. CMECs were transduced with sh-DNA-PKcs or sh-Scramble before being exposed to LPS (10 μg/ml) for 24 h. (A and B) F-actin immunofluorescence. Cytochalasin D (CYD) and jasplakinolide were applied to CMECs cultures to induce and prevent, respectively, F-actin depolymerization. Scale bar, 90 μm. (C to E) Western blot analysis of VE-cadherin and claudin-5 expression. (F and G) Evaluation of endothelial barrier function by FITC clearance (F) and TER (G) assays. (H to J) Western blot analysis of ICAM1 and VCAM1 expression. (K to M) RT-qPCR-based analysis of Il-6, Mcp-1, and Tnfα transcription. (N) ELISA-based analysis of eNOS activity. Experiments were repeated at least 3 times. Data are shown as mean ± SEM (n = 3 cell isolations per group). *P < 0.05.
Fig 4: DNA-PKcs binds to cofilin2 by recognizing a TQ motif. (A and B) Western blot analysis of Cofilin1/2 in CMECs isolated from DNA-PKcsf/f/Tie2Cre and control DNA-PKcsf/f mice in the presence of LPS. (C and D) Co-IP assays were conducted to evaluate binding of DNA-PKcs to cofilin1/2 and F-actin using extracts from LPS-treated CMECs isolated from DNA-PKcsf/f/Tie2Cre and control DNA-PKcsf/f mice. (E) HCAECs were transfected with HA-DNA-PKCs before LPS treatment. Then, the interaction between DNA-PKcs and F-actin as well as the interplay between DNA-PKcs and cofilin1/2 were measured through Co-IP. (F) Immunoblots of DNA-PKcs and cofilin1 immunoprecipitates (Ips) in LPS-treated HCAECs. (G) Immunoblots of DNA-PKcs and cofilin2 Ips in LPS-treated HCAECs. (H) DNA-PKcs and ADF Ips from LPS-treated HCAECs were immunoblotted as indicated. (I) Docking analysis of the interaction between DNA-PKcs and cofilin2. (J and K) Putative hydrogen and hydrophobic bonds between DNA-PKcs and cofilin2 are indicated. (L) Amino acid sequences of cofilin2 in various species. (M) HCAECs were transfected with His-tagged cofilin2 constructs, including cofilin2 without Ser24 (His-cofilin2ΔS24), without Thr25 (His-cofilin2ΔT25), without Gln26 (His-cofilin2ΔQ26), or without Thr25 and Gln26 (His-cofilin2ΔT25Q26). After cells were exposed to LPS, His immunoprecipitates were collected and immunoblotted to determine their interaction with DNA-PKcs. (N and O) Immunofluorescence analysis of F-actin expression in HCAECs transfected with different mutant cofilin2 constructs. (P and Q) Endothelial barrier function was determined by FITC clearance assay and TER detection in HCAECs transfected with different cofilin2 constructs. (R) qPCR analysis of Icam1 expression in HCAECs transfected with different cofilin2 constructs. (S) ELISA assay of eNOS activity in HCAECs transfected with different cofilin2 constructs. Experiments were repeated at least 3 times and the data are shown as mean ± SEM (n = 6 mice per group). *P < 0.05.
Fig 5: Repression of cofilin2Thr25 phosphorylation confers protection against endotoxemia-induced myocardial microvascular injury. WT, heterozygous cofilin2T25A/+, and homozygous cofilin2T25A/A mice were injected with LPS to model endotoxemic cardiomyopathy (n = 6 mice/group). (A and B) Western blot analysis of cofilin2 phosphorylation in CMECs isolated from mice. (C and D) Western blot analysis of ICAM1 in WT, heterozygous cofilin2T25A/+, and homozygous cofilin2T25A/A mice. (E and F) Immunofluorescence of VE-cadherin in myocardial microvessels. (G and H) Immunofluorescence of Gr-1+ neutrophils in WT, cofilin2T25A/+, and cofilin2T25A/A mice. DAPI was used to stain nuclei and TnT to stain cardiomyocytes. Scale bar, 65 μm. (I to K) Western blot analysis of p-eNOS and ET-1 expression in cardiac microvessels from WT, cofilin2T25A/+, and cofilin2T25A/A mice. (L to N) Western blots was used to evaluate cofilin2 phosphroyaltion in human circulating CD34+ ECs and EPCs. Experiments were repeated at least 3 times. Data are shown as mean ± SEM (n = 6 mice or 3 independent cell isolations per group). *P < 0.05. (O) LPS activates DNA-PKcs which ecognizes a TQ motif in cofilin2 and consequently induces cofilin2 phosphorylation at Thr25. Phosphorylated cofilin2 shows increased affinity for F-actin and promotes F-actin depolymerization, leading to disruption of the endothelial barrier integrity, microvascular inflammation, and defective eNOS-dependent vasodilation.
Supplier Page from Abcam for Mouse eNOS ELISA Kit