Abstract
Hepatoma-derived growth factor-related protein-3 (Hdgfrp3 or HRP-3) was recently reported as a neurotrophic factor and is upregulated in hepatocellular carcinoma to promote cancer cell survival. Here we identified HRP-3 as a new endothelial ligand and characterized its in vitro and in vivo functional roles and molecular signaling. We combined open reading frame phage display with multi-round in vivo binding selection to enrich retinal endothelial ligands, which were systematically identified by next generation DNA sequencing. One of the identified endothelial ligands was HRP-3. HRP-3 expression in the retina and brain was characterized by Western blot and immunohistochemistry. Cell proliferation assay showed that HRP-3 stimulated the growth of human umbilical vein endothelial cells (HUVECs). HRP-3 induced tube formation of HUVECs in culture. Wound healing assay indicated that HRP-3 promoted endothelial cell migration. HRP-3 was further confirmed for its in vitro angiogenic activity by spheroid sprouting assay. HRP-3 extrinsically activated the extracellular-signal-regulated kinase ½ (ERK1/2) pathway in endothelial cells. The angiogenic activity of HRP-3 was independently verified by mouse cornea pocket assay. Furthermore, in vivo Matrigel plug assay corroborated HRP-3 activity to promote new blood vessel formation. These results demonstrated that HRP-3 is a novel angiogenic factor.
Highlights
Angiogenic factors regulate retinal blood vessels in physiological and pathological conditions
No other hepatoma-derived growth factor (HDGF) family member was detected by open reading frame phage display (OPD)-next generation DNA sequencing (NGS). These data indicated that HRP-3 had the highest binding activity to retinal endothelium among the three identified family members, implicating that HRP-3 may play an important role in endothelial regulation
The results showed that HRP-3 at 500 ng/ml significantly induced human umbilical vein endothelial cells (HUVECs) proliferation at 24 and 48 h (P
Summary
Angiogenic factors regulate retinal blood vessels in physiological and pathological conditions. Vascular endothelial growth factor (VEGF) plays an important role in the pathogenesis of retinal vascular diseases, including exudative (i.e., vascular) age-related macular degeneration (AMD), diabetic macular edema (DME), proliferative diabetic retinopathy and retinopathy of prematurity [1]. Anti-VEGF therapies have been approved for DME and PLOS ONE | DOI:10.1371/journal.pone.0127904. Anti-VEGF therapies have been approved for DME and PLOS ONE | DOI:10.1371/journal.pone.0127904 May 21, 2015
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