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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

HRP-3 promotes endothelial cell growth.(A) Activity of HRP-3, HDGF and VEGF to stimulate HUVEC growth at different times. (B) Dose-dependent activity of HRP-3 to induce HUVEC growth at 48 h. (C) Dose-dependent activity of HDGF to facilitate HUVEC growth at 48 h. Data are mean ± s.e.m. in one representative experiment. n = 4 (4 wells/group). These results were validated three times with similar outcomes. *P<0.05, **P<0.01, ***P<0.001, vs. control.
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pone.0127904.g003: HRP-3 promotes endothelial cell growth.(A) Activity of HRP-3, HDGF and VEGF to stimulate HUVEC growth at different times. (B) Dose-dependent activity of HRP-3 to induce HUVEC growth at 48 h. (C) Dose-dependent activity of HDGF to facilitate HUVEC growth at 48 h. Data are mean ± s.e.m. in one representative experiment. n = 4 (4 wells/group). These results were validated three times with similar outcomes. *P<0.05, **P<0.01, ***P<0.001, vs. control.

Mentions: Given that HDGF was previously reported as an endothelial mitogen [14], we characterized the activity of HRP-3 to stimulate the proliferation of endothelial cells. We expressed HRP-3 and HDGF with a C-terminal polyhistidine tag in bacteria and purified the recombinant proteins (S2 Fig). Purified proteins were analyzed for their abilities to stimulate the proliferation of HUVECs and HAECs. The results showed that HRP-3 at 500 ng/ml significantly induced HUVEC proliferation at 24 and 48 h (P<0.01) (Fig 3A). In contrast, HDGF (500 ng/ml) and VEGF (50 ng/ml) stimulated significant endothelial proliferation only at 48 h (P<0.05). Dose curve study showed that HRP-3 at 50, 150, 500 and 1,500 ng/ml significantly induced endothelial cell proliferation at 48 h (Fig 3B and 3C). Similarly, HDGF at 150, 500 and 1,500 significantly promoted endothelial cell growth (Fig 3D). HRP-3 was also capable of stimulating the proliferation of HAECs (S3 Fig). These results suggest that HRP-3 is an endothelial mitogen.


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)

HRP-3 promotes endothelial cell growth.(A) Activity of HRP-3, HDGF and VEGF to stimulate HUVEC growth at different times. (B) Dose-dependent activity of HRP-3 to induce HUVEC growth at 48 h. (C) Dose-dependent activity of HDGF to facilitate HUVEC growth at 48 h. Data are mean ± s.e.m. in one representative experiment. n = 4 (4 wells/group). These results were validated three times with similar outcomes. *P<0.05, **P<0.01, ***P<0.001, vs. control.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0127904.g003: HRP-3 promotes endothelial cell growth.(A) Activity of HRP-3, HDGF and VEGF to stimulate HUVEC growth at different times. (B) Dose-dependent activity of HRP-3 to induce HUVEC growth at 48 h. (C) Dose-dependent activity of HDGF to facilitate HUVEC growth at 48 h. Data are mean ± s.e.m. in one representative experiment. n = 4 (4 wells/group). These results were validated three times with similar outcomes. *P<0.05, **P<0.01, ***P<0.001, vs. control.
Mentions: Given that HDGF was previously reported as an endothelial mitogen [14], we characterized the activity of HRP-3 to stimulate the proliferation of endothelial cells. We expressed HRP-3 and HDGF with a C-terminal polyhistidine tag in bacteria and purified the recombinant proteins (S2 Fig). Purified proteins were analyzed for their abilities to stimulate the proliferation of HUVECs and HAECs. The results showed that HRP-3 at 500 ng/ml significantly induced HUVEC proliferation at 24 and 48 h (P<0.01) (Fig 3A). In contrast, HDGF (500 ng/ml) and VEGF (50 ng/ml) stimulated significant endothelial proliferation only at 48 h (P<0.05). Dose curve study showed that HRP-3 at 50, 150, 500 and 1,500 ng/ml significantly induced endothelial cell proliferation at 48 h (Fig 3B and 3C). Similarly, HDGF at 150, 500 and 1,500 significantly promoted endothelial cell growth (Fig 3D). HRP-3 was also capable of stimulating the proliferation of HAECs (S3 Fig). These results suggest that HRP-3 is an endothelial mitogen.

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