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Hepatoma-derived growth factor-related protein-3 is a novel angiogenic factor.

LeBlanc ME, Wang W, Caberoy NB, Chen X, Guo F, Alvarado G, Shen C, Wang F, Wang H, Chen R, Liu ZJ, Webster K, Li W - PLoS ONE (2015)

Bottom Line: One of the identified endothelial ligands was HRP-3.HRP-3 extrinsically activated the extracellular-signal-regulated kinase ½ (ERK1/2) pathway in endothelial cells.These results demonstrated that HRP-3 is a novel angiogenic factor.

View Article: PubMed Central - PubMed

Affiliation: Bascom Palmer Eye Institute, Department of Ophthalmology, University of Miami School of Medicine, Miami, Florida, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus

Identification of HRP-3 as an endothelial ligand.(A) OPD-based in vivo binding selection. OPD cDNA libraries were purified and intravenously injected into mice. After circulating for 20 min, unbound phages were removed by intracardial perfusion. The retinas were isolated and homogenized to release endothelium-bound phages, which were amplified in bacteria and used as in put for the next round of selection. After 3 rounds of selection, the cDNA inserts of enriched phage clones were amplified and identified by NGS with simultaneous quantification of the copy numbers of cDNA inserts for individual clones. (B) Total phages bound to retinal endothelium. At the end of each round of selection, total endothelium-bound phages in the retinal homogenate were quantified by plaque assay and expressed as plaque forming units (pfu)/retina. (C) The copy number of the cDNA inserts for HDGF, HRP-2 and HRP-3 quantified by NGS.
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pone.0127904.g001: Identification of HRP-3 as an endothelial ligand.(A) OPD-based in vivo binding selection. OPD cDNA libraries were purified and intravenously injected into mice. After circulating for 20 min, unbound phages were removed by intracardial perfusion. The retinas were isolated and homogenized to release endothelium-bound phages, which were amplified in bacteria and used as in put for the next round of selection. After 3 rounds of selection, the cDNA inserts of enriched phage clones were amplified and identified by NGS with simultaneous quantification of the copy numbers of cDNA inserts for individual clones. (B) Total phages bound to retinal endothelium. At the end of each round of selection, total endothelium-bound phages in the retinal homogenate were quantified by plaque assay and expressed as plaque forming units (pfu)/retina. (C) The copy number of the cDNA inserts for HDGF, HRP-2 and HRP-3 quantified by NGS.

Mentions: We performed in vivo binding selection with OPD cDNA libraries (Fig 1A, top panel). Three rounds of selection resulted in 90-fold increase in total phage binding activity to retinal endothelium (Fig 1B). Instead of manually isolating and identifying individual enriched phage clones, we amplified the cDNA inserts of all enriched phages and sequenced them by NGS (Fig 1A, bottom panel). All identified sequences were aligned against NCBI CCDS database to identify enriched endothelial ligands. A total of 489,126 valid sequences were identified and matched to the database. Among identified putative ligands was HRP-3 with 11,140 copies of its cDNA insert detected by NGS (Fig 1C). In addition, HDGF and HRP-2 were identified with 80 and 3 copies of their cDNA inserts, respectively. No other HDGF family member was detected by OPD-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.


Hepatoma-derived growth factor-related protein-3 is a novel angiogenic factor.

LeBlanc ME, Wang W, Caberoy NB, Chen X, Guo F, Alvarado G, Shen C, Wang F, Wang H, Chen R, Liu ZJ, Webster K, Li W - PLoS ONE (2015)

Identification of HRP-3 as an endothelial ligand.(A) OPD-based in vivo binding selection. OPD cDNA libraries were purified and intravenously injected into mice. After circulating for 20 min, unbound phages were removed by intracardial perfusion. The retinas were isolated and homogenized to release endothelium-bound phages, which were amplified in bacteria and used as in put for the next round of selection. After 3 rounds of selection, the cDNA inserts of enriched phage clones were amplified and identified by NGS with simultaneous quantification of the copy numbers of cDNA inserts for individual clones. (B) Total phages bound to retinal endothelium. At the end of each round of selection, total endothelium-bound phages in the retinal homogenate were quantified by plaque assay and expressed as plaque forming units (pfu)/retina. (C) The copy number of the cDNA inserts for HDGF, HRP-2 and HRP-3 quantified by NGS.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4440747&req=5

pone.0127904.g001: Identification of HRP-3 as an endothelial ligand.(A) OPD-based in vivo binding selection. OPD cDNA libraries were purified and intravenously injected into mice. After circulating for 20 min, unbound phages were removed by intracardial perfusion. The retinas were isolated and homogenized to release endothelium-bound phages, which were amplified in bacteria and used as in put for the next round of selection. After 3 rounds of selection, the cDNA inserts of enriched phage clones were amplified and identified by NGS with simultaneous quantification of the copy numbers of cDNA inserts for individual clones. (B) Total phages bound to retinal endothelium. At the end of each round of selection, total endothelium-bound phages in the retinal homogenate were quantified by plaque assay and expressed as plaque forming units (pfu)/retina. (C) The copy number of the cDNA inserts for HDGF, HRP-2 and HRP-3 quantified by NGS.
Mentions: We performed in vivo binding selection with OPD cDNA libraries (Fig 1A, top panel). Three rounds of selection resulted in 90-fold increase in total phage binding activity to retinal endothelium (Fig 1B). Instead of manually isolating and identifying individual enriched phage clones, we amplified the cDNA inserts of all enriched phages and sequenced them by NGS (Fig 1A, bottom panel). All identified sequences were aligned against NCBI CCDS database to identify enriched endothelial ligands. A total of 489,126 valid sequences were identified and matched to the database. Among identified putative ligands was HRP-3 with 11,140 copies of its cDNA insert detected by NGS (Fig 1C). In addition, HDGF and HRP-2 were identified with 80 and 3 copies of their cDNA inserts, respectively. No other HDGF family member was detected by OPD-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.

Bottom Line: One of the identified endothelial ligands was HRP-3.HRP-3 extrinsically activated the extracellular-signal-regulated kinase ½ (ERK1/2) pathway in endothelial cells.These results demonstrated that HRP-3 is a novel angiogenic factor.

View Article: PubMed Central - PubMed

Affiliation: Bascom Palmer Eye Institute, Department of Ophthalmology, University of Miami School of Medicine, Miami, Florida, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus