I. Halaweish1, C. Tafatia1, M. Mohamed1, B. Liu1, W. He1, Z. Chang1, Y. Li1, H. B. Alam1 1University Of Michigan,Surgery,Ann Arbor, MI, USA
Introduction: Treatment with histone deacetylase inhibitors, such as valproic acid (VPA), has been shown to be protective in models of hemorrhagic shock; however, the precise mechanism remains unclear. We simulated the cellular effects of shock by subjecting human umbilical vein endothelial cells (HUVEC) to hypoxia-induced injury in-vitro, to better characterize the mechanistic actions of VPA.
Methods: HUVECs (ATCC HUVEC-EC-C CRL-1730) were grown to 90% confluency in F12K medium supplemented with 20% fetal bovine serum and 5 mg/mL endothelial cell growth factor. After addition of either phosphate buffered saline (control) or 4 mmol/L VPA (treatment), cells were incubated in either normoxic or hypoxic conditions for 48 hours at 37°C. Cellular viability was evaluated by redox dye assay (n=4). Cellular morphology was assessed by light microscopy. Levels of acetylated histone H3, hypoxia inducible factor 1α (HIF-1 α), cleaved caspase 3, vascular adhesion molecule 1 (VCAM1), and vascular endothelial growth factor receptor 1 (VEGFR1) were measured by Western blotting.
Results: High levels of acetylated histone H3 were detected in the VPA-treated cells. As expected, the hypoxic cells demonstrated increased levels of HIF-1α compared to normoxic cells. Redox dye assay showed that the number of viable cells increased 26% with VPA treatment in normoxic conditions and 22% in hypoxic conditions (n=4, p=0.002, and p=0.001 respectively). Hypoxia caused structural alteration in the morphology of HUVECs, which was prevented by VPA treatment (Figure). There was a significant decrease in apoptosis in VPA-treated cells as evidenced by decrease in cleaved-caspase 3 expression in both normoxic and hypoxic conditions (58%, p=0.05 and 48% p=0.002, respectively). Hypoxia increased the levels of VCAM1 by 50% compared to normoxic control (n=3, p=0.003); which was normalized by VPA treatment. Finally, VPA treatment decreased the expression of VEGFR1 by 18% compared to hypoxic control (n=3, p = 0.02).
Conclusion: This is the first study to demonstrate that VPA treatment increases the viability and stabilizes the structural morphology of endothelial cells in a hypoxic-injury model. In addition, it decreases the expression of adhesion molecules (VCAM1 and VEGFR1) responsible for leukocyte recruitment and inflammatory response.
