Fig 2: 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.

Fig 3: DNA-PKcs promotes endothelial barrier dysfunction, inflammation, and vasoconstriction in myocardial microvessels. (A to C) Western blot analysis of claudin-5 and VE-cadherin expression in DNA-PKcsf/f/Tie2Cre and control DNA-PKcsf/f mice. (D and E) Immunohistochemistry was used to observe albumin leakage into myocardium in DNA-PKcsf/f/Tie2Cre and control DNA-PKcsf/f mice. (F) FITC-dextran clearance assays were performed following 24-h LPS treatment (10 μg/ml) in CMECs isolated from DNA-PKcsf/f/Tie2Cre and control DNA-PKcsf/f mice. FITC-dextran permeation was measured to assess alterations in endothelial barrier function. (G) TER assays were performed in CMECs isolated from DNA-PKcsf/f/Tie2Cre and control DNA-PKcsf/f mice to evaluate changes in endothelial barrier integrity following LPS exposure. (H to J) Western blot analysis of VCAM1 and VCAM1 expression in cardiac microvessels from DNA-PKcsf/f/Tie2Cre and control DNA-PKcsf/f mice treated with LPS. (K and L) Immunofluorescence of Gr-1+ neutrophils in heart samples from DNA-PKcsf/f/Tie2Cre and control DNA-PKcsf/f mice treated with LPS. DAPI was used to stain nuclei and TnT was used to stain cardiomyocytes. (M to O). RT-qPCR was used to analyze the transcription of Mmp-9, IL-6, and Tnf-α in cardiac tissue from DNA-PKcsf/f/Tie2Cre and control DNA-PKcsf/f mice after LPS exposure. (P to R) Western blot analysis of p-eNOS and ET-1 expression in heart tissues from DNA-PKcsf/f/Tie2Cre and control DNA-PKcsf/f mice treated with LPS. (S) Endothelial-dependent and endothelial-independent relaxation responses were assessed in aortic rings by applying Ach (10−9–10−5 M) or SNP (10−10–10−6 M). 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.