Maintenance of HDACs and H3K9me3 Prevents Arterial Flow-Induced Venous Endothelial Damage

Figures

• 

  FIGURE 1. ALS increased the expression of H3K9me3 and promoted the cell quiescence in arterial ECs. (A) Western blotting analysis for the expression of H3K9me3, cyclin A1, and cyclin B1 in HUVECs, which were subjected to ST, ALS, or OS treatment for 12 and 24 h. ALS increased the H3K9me3 expression and decreased the cyclin A1 expression, whereas OS did not change the H3K9me3 at 12 and 24 h. (B) The time-lapse FRET ratio images of arterial ECs expressing the H3K9me3 biosensors were recorded to track the status of H3K9me3 every 2 h under ST, ALS, or OS over the time course of 12 h. The results were normalized to 0 h for each experiment. Scale bar in the phase image: 20 μm. (C) Western blotting analysis for the expression of H3K9me3 and cyclin A1 in synchronized HUVECs at 0, 3, 6, 12, and 24 h under ALS stimulation. The low expression of cyclin A1 was accompanied by the high level of H3K9me3. Data were represented as mean ± SEM, n = 3. p-values were calculated using one-way ANOVA in (A) and (C) and using two-way ANOVA in (B) with Tukey’s multiple comparisons post-tests. In (A) and (B), *p < 0.05, ALS-treated HUVECs compared to the ST group; #p < 0.05, ALS-treated HUVECs compared to the OS…

• 

  FIGURE 2. The different effect of arterial and venous ECs under ALS. (A) Morphology observations of arterial ECs (HUVECs) and venous ECs (HSVECs) after being subjected to ALS for 0, 1, 3, and 6 h. (B) The Western blot analysis for the expression of H3K9me3 and β-actin in total cell lysates from (A). The expression of H3K9me3 was time-dependent increase in arterial ECs but not in vECs. (C) The time-lapse FRET ratio images of HUVECs and HSVECs expressing the H3K9me3 biosensors were recorded to track the status of H3K9me3 every 2 h under ALS for 12 h. Compared to HUVECs, no FRET ratio of activated H3K9me3 was observed in vECs upon ALS stimulation at different time points (0, 1, 3, and 6 h). (D) Protein expressions were examined by using Western blotting analysis. HUVECs and HSVECs were subjected to ST or ALS condition for 24 h. Lower expression of H3K9me3 in ALS-treated HSVECs compared to ALS-treated HUVECs was shown. ALS decreased the HDAC4 and HDAC5 and increased VCAM-1 in ALS-treated HSVECs. The increases of HDAC3, HDAC4, HDAC5, p-HDAC3, and p-HDAC4,5,7 were observed in ALS-treated HUVSCs at 24 h. Scale bar in (A) = 200 μm. Arrow indicated the flow direction of ALS (left to right). Scale…

• 

  FIGURE 3. PBA inhibited the expression of H3K9me3 in arterial ECs. (A) The images were presented the cell morphology of PBA (150 μM) or vehicle-treated arterial ECs (HUVECs) under ALS at 3, 6, and 24 h. PBA-induced arterial ECs had gradually increased the number of round-up damage morphology after subjected to ALS at 3, 6, and 24 h. (B) Protein expressions were examined by using Western blotting analysis. The decreases of H3K9me3, p-HDAC3, p-HDAC4,5,7, HDAC3, HDAC4, and HDAC5 and the increase of VCAM-1 were observed after treating PBA under ALS in HUVECs compared to vehicle group. (C) By utilizing the H3K9me3 FRET biosensor to examine the H3K9me3 status, the FRET ratio of H3K9me3 was declined in PBA-treated HUVECs under ALS condition compared to the vehicle group (C). Scale bar in (A) = 200 μm. Scale bar in (C) = 20 μm. Data were represented as mean ± SEM. Arrow indicated the flow direction of ALS (left to right).

• 

  FIGURE 4. Reverse the ALS-induced inflammation by ITSA-1 in vEC. (A) The cell images of 150 μM ITSA-1- or vehicle-treated vECs (HSVECs) were shown under ALS for 0, 3, 6, and 24 h. The ITSA-1-treated HSVECs were significantly aligned parallel to the flow direction of ALS at 24 h. (B) Protein expression was analyzed by using Western blotting assay against H3K9me3, p-HDAC3, p-HDAC4,5,7, HDAC3, HDAC4, HDAC5, and VCAM-1. ITSA-1-treated HSVECs reduced the VCAM-1 expression by increasing the HDAC3, p-HDAC4, 5, 7, and H3K9me3 as compared to the vehicle group after exposed to ALS at 3 and 6 h. (C) The level of H3K9me3 was raised in the ITSA-1 group but not in vehicle group under ALS in H3K9me3 FRET biosensor-transfected HSVECs. Scale bar in (A) = 200 μm. Scale bar in (C) = 20 μm. Data were represented as mean ± SEM. Arrow indicated the flow direction of ALS (left to right).

• 

  FIGURE 5. Inhibition of FAK decreased the ALS-induced H3K9 methylation in arterial ECs. (A) The expression of p-FAK was decreased in PBA (150 μM)-treated arterial ECs (HUVECs) by Western blotting assay after being subjected to ALS at 0, 3, 6, and 24 h. (B) ALS significantly triggered the cell peel-off in FI14-treated HUVECs at 24 h. (C) Protein expression was analyzed by using Western blotting assay against H3K9me3, p-FAK, and t-FAK. Blocking the phosphorylation of FAK by FI14 treatment decreased the expression of H3K9me3 in ALS-stimulated HUVECs. (D) The normalized H3K9me3 FRET ratio was declined in FI14-treated HUVECs after being subjected to ALS by transfecting the H3K9me3 FRET biosensor. Scale bar in (B) = 200 μm. Scale bar in (D) = 20 μm. Data were represented as mean ± SEM. Arrow indicated the flow direction of ALS (left to right).

• 

  FIGURE 6. Protective effect of EGF on preventing ALS-induced EC inflammation. (A) Western blotting analysis for activating H3K9me3 by treating ITSA-1 in vEC (HSVECs); the expression of p-FAK was increased after ALS stimulation. (B) Treating EGF (100 ng/ml) in HSUVEs prevented the cell peel-off and the cells were parallel to the flow direction under ALS. (C) Protein expression was analyzed by using conventional Western blotting assay against H3K9me3, p-FAK, and t-FAK. The expression of H3K9me3 was gradually increased by raising the expression of p-FAK in EGF-treated HSVECs under ALS. (D) To validate the flow regulation of H3K9me3 in EGF-treated HUVECs, the results also confirmed that EGF induced H3K9me3 over the time course of 4 h in H3K9me3 biosensor-transfected HSVECs under ALS. (E) Alexa fluor 546-Phalloidin staining was used to determine the expression of F-actin on vECs with or without EGF treatment under ALS for 24 h. Nuclei were stained with DAPI (blue). Scale bar in cell phase images = 200 μm. Scale bar in FRET images = 20 μm. Data were represented as mean ± SEM. Arrow indicated the flow direction of ALS (left to right).

• 

  FIGURE 7. Schematic diagram depicting the mechanism of HDACs and H3K9me3-mediated inflammation in triggering vEC damage under ALS stimulation. The blue and yellow capsule symbol means that the ALS-induced damages were rescued by treating with ITSA-1 or EGF.