1. Fluid shear stress stimulates phosphorylation-dependent nuclear export of HDAC5 and mediates expression of KLF2 and eNOS
by Weiye Wang, Chang Hoon Ha, Bong Sook Jhun, Chelsea Wong, Mukesh K. Jain, and Zheng-Gen Jin
Blood
Volume 115(14):
April 8, 2010
©2010 by American Society of Hematology

2. Fluid shear stress stimulated HDAC5 phosphorylation through a Ca2+/calmodulin–dependent pathway in endothelial cells.
Fluid shear stress stimulated HDAC5 phosphorylation through a Ca2+/calmodulin–dependent pathway in endothelial cells. (A) HUVECs were exposed to flow for 0, 15, 30, 60, 120, and 240 minutes. (B-C) HUVECs were exposed to either flow or static condition for 1 hour with the pretreatment of dimethyl sulfoxide (DMSO), KN-62 (30μM), CaM inhibitory peptide (60nM), W-13 (100μM), and BAPTA/AM (30μM) + EGTA (2μM) for 30 minutes. (D) HUVECs were infected with Ad-PKD-KN or Ad-LacZ for 24 hours before the 1-hour flow or static treatment. HDAC5 phosphorylation (Ser259 and Ser498) and HDAC5 were probed by phospho-specific HDAC5 antibody and HDAC5 antibody. *P < .05 compared with control. #P < .05 compared with cells treated with flow + DMSO/Ad-LacZ. Error bars represent ±SEM.
Weiye Wang et al. Blood 2010;115:
©2010 by American Society of Hematology

3. Fluid shear stress induced phosphorylation-dependent HDAC5 nuclear export.
Fluid shear stress induced phosphorylation-dependent HDAC5 nuclear export. (A-B) HUVECs were infected with Ad-GFP-HDAC5 or Ad-GFP-HDAC5-S/A. Twenty-four hours later, cells were conditioned with flow (left lane) or static (right lane) for the time indicated in each image. Images were captured at a magnification of ×60, using a fluorescence microscope.
Weiye Wang et al. Blood 2010;115:
©2010 by American Society of Hematology

4. HDAC5 down-regulated flow-mediated KLF2 expression.
HDAC5 down-regulated flow-mediated KLF2 expression. (A-B) HUVECs were exposed to 1-hour flow or to static conditions after 24-hour infection with Ad-HDAC5-S/A or Ad-LacZ (control). KLF2 mRNA levels were assayed by real-time reverse-transcription (RT)–PCR (A) and protein levels were detected by KLF2 antibody (B). (C-D) HUVECs were exposed to flow or to static conditions after pretreatment with DMSO (control), W-13 (100μM), or PD (another control; 30μM) for 30 minutes. KLF2 mRNA level were assayed by RT-PCR. Vertical lines have been inserted to indicate repositioned gel lanes. *P < .05 compared with control. #P < .05 compared with that treated with flow + Ad-LacZ or DMSO. Error bars represent ±SEM
Weiye Wang et al. Blood 2010;115:
©2010 by American Society of Hematology

5. HDAC5 negatively regulated MEF2 transcriptional activation and KFL2 expression.
HDAC5 negatively regulated MEF2 transcriptional activation and KFL2 expression. (A) HUVECs were infected with Ad-Flag-HDAC5. Twenty-four hours later, cells were conditioned under static conditions (first lane) or flow (second lane) for 1 hour. Immunoprecipitation was performed with Flag antibody–conjugated agarose. MEF2C and Flag-HDAC5 were detected by probing with anti-MEF2C and anti-Flag antibodies. (B-C) HUVECs were transfected with 3xMEF2-luciferase or −221/+1 KLF2 promoter or −221/+1 MEF2 binding site–deleted KLF2 promoter reporter gene together with HDAC5-S/A or pcDNA as control. Twenty-four hours later, cells were conditioned with static or flow for 8 hours. Luciferase activities were assayed by Promega Luciferase assay kit. *P < .05 compared with control (static). #P < .05 compared with that treated with flow + pcDNA. Error bars represent ±SEM.
Weiye Wang et al. Blood 2010;115:
©2010 by American Society of Hematology

6. HDAC5 down-regulated flow-mediated eNOS expression.
HDAC5 down-regulated flow-mediated eNOS expression. (A-B) HUVECs were exposed to 8 or 24 hours of flow or to static conditions 24 hours after infection with Ad-HDAC5-S/A or Ad-LacZ (control). eNOS mRNA levels were assayed by real-time RT-PCR (A) and protein levels were detected by eNOS antibody (B). Vertical lines have been inserted to indicate repositioned gel lanes. *P < .05 compared with control (static). #P < .05 compared with that treated with flow + Ad-LacZ. Error bars represent ±SEM.
Weiye Wang et al. Blood 2010;115:
©2010 by American Society of Hematology

7. HDAC5-dependent pathway modulated flow anti-inflammation in endothelial cells.
HDAC5-dependent pathway modulated flow anti-inflammation in endothelial cells. (A-B) Overexpression of Ad-HDAC5-S/A attenuated flow anti-inflammation. HUVECs were infected with Ad-HDAC5-S/A or Ad-LacZ as control. Twenty-four hours later, cells were subjected to flow or to static conditions for 24 hours followed by the treatment of TNFα or vehicle for 6 hours. U937 monocytes were added to HUVECs and incubated for 30 minutes. HUVECs were washed 3 times. The adherent monocytes were counted in 5 randomly optical fields in each dish. *P < .05, flow + TNFα + Ad-HDAC5-S/A vs flow + TNFα + Ad-LacZ. Error bars represent ±SEM.
Weiye Wang et al. Blood 2010;115:
©2010 by American Society of Hematology

8. Model for HDAC5 modulated flow-induced KLF2 and eNOS expression.
Model for HDAC5 modulated flow-induced KLF2 and eNOS expression. Fluid shear stress stimulates HDAC5 phosphorylation and nuclear export through a Ca2+/calmodulin-dependent pathway, freeing MEF2 to activate KLF2 expression and its downstream gene expression.
Weiye Wang et al. Blood 2010;115:
©2010 by American Society of Hematology