Abstract
Hypoxia-inducible factor (HIF)-1α is a crucial transcription factor that regulates the expression of target genes involved in angiogenesis. Prolyl hydroxylase 2 (PHD2) dominantly hydroxylates two highly conserved proline residues of HIF-1α to promote its degradation. This study was designed to construct an intrabody against PHD2 that can inhibit PHD2 activity and promote angiogenesis. Single-chain variable fragment (scFv) against PHD2, INP, was isolated by phage display technique and was modified with an endoplasmic reticulum (ER) sequence to obtain ER-retained intrabody against PHD2 (ER-INP). ER-INP was efficiently expressed and bound to PHD2 in cells, significantly increased the levels of HIF-1α, and decreased hydroxylated HIF-1α in human embryonic kidney cell line (HEK293) cells and mouse mononuclear macrophage leukaemia cell line (RAW264.7) cells. ER-INP has shown distinct angiogenic activity both in vitro and in vivo, as ER-INP expression significantly promoted the migration and tube formation of human umbilical vein endothelial cells (HUVECs) and enhanced angiogenesis of chick chorioallantoic membranes (CAMs). Furthermore, ER-INP promoted distinct expression and secretion of a range of angiogenic factors. To the best of our knowledge, this is the first study to report an ER-INP intrabody enhancing angiogenesis by blocking PHD2 activity to increase HIF-1α abundance and activity. These results indicate that ER-INP may play a role in the clinical treatment of tissue injury and ischemic diseases in the future.
Highlights
There are three members of the hypoxia-inducible factor (HIF) family: Hypoxia-inducible factor (HIF)-1, HIF-2, and HIF-3
RT-PCR results demonstrated that endoplasmic reticulum (ER)-INP was expressed successfully in cells transfected with plasmid of ER-INP (pER-INP) compared with that in the control vector (Fig. 1A)
Western blots of cell lysates with the E-tag antibody showed that ER-retained intrabody against PHD2 (ER-INP) fused with the E-tag was expressed with an estimated molecular weight of about 35 kDa in HEK293, HepG2, and RAW264.7 cells; the expression of ER-INP had no effect on the expression of Prolyl hydroxylase 2 (PHD2) in transfected cells (Fig. 1B)
Summary
There are three members of the hypoxia-inducible factor (HIF) family: HIF-1, HIF-2, and HIF-3. HIF-1 is a crucial transcription factor that regulates the expression of target genes involved in angiogenesis and tissue repair after acute injury[4,5,6]. More evidence has demonstrated that the inhibition of PHD2 activity increased the accumulation of HIF-1α, which dimerised with HIF-1β to transactivate target genes[14,15]. Several reports demonstrated that the HIF1-regulated gene vascular endothelial growth factor (VEGF) was identified as an important mediator of tissue repair during APAP hepatotoxicity[17,18], liver graft injury[19], and liver ischemic injury[20]. The present study was designed to construct an ER-retained intrabody against PHD2 (ER-INP), which inhibited PHD2 activity and increased the stability of HIF-1α. The results suggest that ER-INP may provide a new potential strategy for the treatment of tissue injury and angiogenesis-related diseases
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