Fig 1: DNA-PKcs deletion attenuates endotoxemia-mediated myocardial microvascular dysfunction. (A) DNA-PKcsf/f mice were bred to Tie2Cre mice to generate endothelial-cell-specific DNA-PKcs knockout (DNA-PKcsf/f/Tie2Cre) mice. Endotoxemic cardiomyopathy was induced via LPS (10 mg/kg) injection and heart function was assessed by echocardiography 48 h later. (B to E) Analysis of serum TnT, CK-MB, BNP, and LDH levels in DNA-PKcsf/f/Tie2Cre and control DNA-PKcsf/f mice. (F) Survival data for DNA-PKcsf/f/Tie2Cre and control DNA-PKcsf/f mice in the presence of LPS. (G and H) Cardiac microvascular imaging after gelatin-ink perfusion and syndecan-1 immunofluorescence in heart tissue from DNA-PKcsf/f/Tie2Cre and control DNA-PKcsf/f mice. (I and J) Syndecan-1 immunofluorescence in aortae isolated from DNA-PKcsf/f/Tie2Cre and control DNA-PKcsf/f mice. α-SMA was used to stain smooth muscle. (K) TEM analysis of ultrastructural alterations in microvessels from DNA-PKcsf/f/Tie2Cre and control DNA-PKcsf/f mice. (L and M) Fibrin immunofluorescence in myocardial microvessels from DNA-PKcsf/f/Tie2Cre and control DNA-PKcsf/f mice. (N) Western blot analysis of fibrin expression in cardiac microvessels from DNA-PKcsf/f/Tie2Cre and control DNA-PKcsf/f mice. Experiments were repeated at least 3 times. Data are shown as mean ± SEM (n = 6 mice or 3 independent samples per group). *P < 0.05.
Fig 2: Endotoxemia triggers cardiac endothelial DNA damage through DNA-PKcs. Primary mouse CMECs were transduced with control shRNA (sh-scramble), FANCD2 shRNA (sh-FANCD2), or UBE2T shRNA (sh-UBE2T) before exposure to LPS (0, 1, 5, and 10 μg/ml) for 24 h. (A and B) Representative immunostaining and statistical data for γH2AX-positive CMECs transduced with sh-scramble or sh-FANCD2. (C and D) DNA damage was assessed by comet assays in CMECs transduced with sh-scramble or sh-FANCD2. The arrows indicate nuclei with DNA damage. (E and F) Representative immunostaining and statistical data for γH2AX-positive CMECs transduced with sh-scramble or sh-UBE2T. (G and H) DNA damage was assessed by comet assays in CMECs transduced with sh-scramble or sh-UBE2T. The arrows indicate nuclei with DNA damage. (I and J) MTT assay was used to evaluate cell viability in CMECs infected with sh-FANCD2 or sh-UBE2T. (K and L) ELISA was used to measure LDH levels in culture media from CMECs infected with sh-FANCD2 or sh-UBE2T. (M) Representative western blots and statistical data showing changes in phos-DNA-PKcs expression in LPS-exposed CMECs. (N) ELISA was used to evaluate DNA-PKcs activity in CMECs treated with LPS. (O) MTT assay was used to evaluate cell viability in CMECs transduced with sh-DNA-PKcs or sh-scramble. (P) ELISA was used to measure LDH levels in culture media from CMECs transduced with sh-DNA-PKcs or sh-scramble. (Q) MTT assay was used to evaluate cell viability in CMECs treated with the DNA-PKcs inhibitor NU7441. (R) ELISA was used to measure LDH levels in culture media from CMECs treated with NU7441. (S and T) ELISA was used to evaluate DNA-PKcs activity 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.
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