Abstract 5806: SRF Protects Brain Endothelial Cells Against Hypoxic Damage
Ischemia resulting from brain vascular disorders is the major cause of stroke. Hypoxia triggers both angiogenesis and apoptosis through the induction of HIF1α-activated VEGF and the alteration of mitochondrial permeability, respectively. The balance between these two events is crucial to both the progression of vascular disorders and the outcome of injury healing. Our previous study showed that SRF (Serum response factor) is essential to VEGF-induced angiogenesis. This study aimed to determine the roles of SRF in hypoxia-induced brain endothelial cell angiogenesis and apoptosis.
Methods: Mouse brain endothelial cells (bEND3) were transfected with either an SRF expressing plasmid, SRF shRNA, or the vehicle (control), and cultured under hypoxic condition (5% CO2: 94% N2: 1% O2) for 0, 0.5, 2, 6 and 24 hours. Angiogenesis and apoptosis were assessed by 3-D in vitro angiogenesis assay and Annexin V binding assay, respectively.
After 30-min hypoxic treatment, SRF mRNA expression was dramatically increased by 54 fold and then gradually declined and returned to normal at 24 hours. SRF protein expression and phosphorylation were also increased by 111±24 % and 74±7 % (both P < 0.01), respectively, after 6-hour hypoxia. Hypoxia also enhanced SRF binding activity to DNA.
Forced overexpression of SRF increased hypoxia-induced angiogenesis by 232±15 % (P < 0.01); and knockdown of SRF completely abolished it.
After 24-hour hypoxia, 29±4 % of the cells underwent apoptosis; while overexpression of SRF reduced this percentage to 5.7±1 %; and knockdown of SRF increased it to 85±4 % (all P < 0.001).
Overexpression of SRF increased Bcl-2/Bax ratio by 23±4 % at baseline and by 167±20 % after 24-hour hypoxia; knockdown of SRF decreased these ratios by 69±3 % and 75±8 %, respectively (all P < 0.01). Knockdown of SRF also increased Bax translocation to mitochondria and Cytochrome C release.
Hypoxia triggers SRF activation.
Up-regulation of SRF promotes angiogenesis.
Down-regulation of SRF sensitizes cells to hypoxia-induced apoptosis.
SRF protects against hypoxia-induced apoptosis likely through both direct up-regulation of Bcl-2 and indirect down-regulation of Bax.
This research has received full or partial funding support from the American Heart Association, Western States Affiliate (California, Nevada & Utah).