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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 stimulates angiogenesis by corneal pocket assay.(A) Representative images of corneal angiogenesis. Small pieces of filter papers presoaked in HRP-3 (1 μg/μl), VEGF (100 ng/μl) or PBS were implanted in corneal pockets to induce vascular sprouting into the cornea for 6 days. # indicates filter paper position. Arrowheads indicate corneal blood vessels. (B) Representative images of corneal blood vessels labeled with fluorescent DiI dye. (C) The number of new sprouting vessels into the cornea was quantified and compared among HRP-3 (n = 5), VEGF (n = 5) and PBS (n = 10). (D) The number of branching points of corneal vessels and (E) total new vessel score were also quantified for the latest cases of HRP-3 (n = 4), VEGF (n = 4) and PBS (n = 5). Data are mean ± s.e.m. *P<0.05, **P<0.01, ***P<0.001, vs. control PBS.
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pone.0127904.g006: HRP-3 stimulates angiogenesis by corneal pocket assay.(A) Representative images of corneal angiogenesis. Small pieces of filter papers presoaked in HRP-3 (1 μg/μl), VEGF (100 ng/μl) or PBS were implanted in corneal pockets to induce vascular sprouting into the cornea for 6 days. # indicates filter paper position. Arrowheads indicate corneal blood vessels. (B) Representative images of corneal blood vessels labeled with fluorescent DiI dye. (C) The number of new sprouting vessels into the cornea was quantified and compared among HRP-3 (n = 5), VEGF (n = 5) and PBS (n = 10). (D) The number of branching points of corneal vessels and (E) total new vessel score were also quantified for the latest cases of HRP-3 (n = 4), VEGF (n = 4) and PBS (n = 5). Data are mean ± s.e.m. *P<0.05, **P<0.01, ***P<0.001, vs. control PBS.

Mentions: Regardless of its angiogenic activities quantified by various in vitro assays, HRP-3 as an angiogenic factor should be independently verified in vivo. We characterized the angiogenic activity of HRP-3 in vivo by corneal pocket angiogenesis assay and Matrigel plug assay. The cornea is an avascular tissue with convenience to visualize and quantify neovascularization. For corneal pocket assay, small pieces of filter paper were presoaked in the solution of HRP-3, VEGF or PBS and implanted into mouse corneal pockets. After 6 days, corneal blood vessels were detected by slit-lamp microscopy and verified by staining with lipophilic fluorescent DiI dye (Fig 6A and 6B). Quantification of newly-formed corneal blood vessels and their branching points indicated that HRP-3 and VEGF significantly stimulated angiogenesis (Fig 6C and 6D). Moreover, a semiquantitative scoring method by combining the number, density and length of corneal blood vessels (S1 Table) showed that HRP-3 and VEGF induced significant corneal angiogenesis (Fig 6E).


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 stimulates angiogenesis by corneal pocket assay.(A) Representative images of corneal angiogenesis. Small pieces of filter papers presoaked in HRP-3 (1 μg/μl), VEGF (100 ng/μl) or PBS were implanted in corneal pockets to induce vascular sprouting into the cornea for 6 days. # indicates filter paper position. Arrowheads indicate corneal blood vessels. (B) Representative images of corneal blood vessels labeled with fluorescent DiI dye. (C) The number of new sprouting vessels into the cornea was quantified and compared among HRP-3 (n = 5), VEGF (n = 5) and PBS (n = 10). (D) The number of branching points of corneal vessels and (E) total new vessel score were also quantified for the latest cases of HRP-3 (n = 4), VEGF (n = 4) and PBS (n = 5). Data are mean ± s.e.m. *P<0.05, **P<0.01, ***P<0.001, vs. control PBS.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4440747&req=5

pone.0127904.g006: HRP-3 stimulates angiogenesis by corneal pocket assay.(A) Representative images of corneal angiogenesis. Small pieces of filter papers presoaked in HRP-3 (1 μg/μl), VEGF (100 ng/μl) or PBS were implanted in corneal pockets to induce vascular sprouting into the cornea for 6 days. # indicates filter paper position. Arrowheads indicate corneal blood vessels. (B) Representative images of corneal blood vessels labeled with fluorescent DiI dye. (C) The number of new sprouting vessels into the cornea was quantified and compared among HRP-3 (n = 5), VEGF (n = 5) and PBS (n = 10). (D) The number of branching points of corneal vessels and (E) total new vessel score were also quantified for the latest cases of HRP-3 (n = 4), VEGF (n = 4) and PBS (n = 5). Data are mean ± s.e.m. *P<0.05, **P<0.01, ***P<0.001, vs. control PBS.
Mentions: Regardless of its angiogenic activities quantified by various in vitro assays, HRP-3 as an angiogenic factor should be independently verified in vivo. We characterized the angiogenic activity of HRP-3 in vivo by corneal pocket angiogenesis assay and Matrigel plug assay. The cornea is an avascular tissue with convenience to visualize and quantify neovascularization. For corneal pocket assay, small pieces of filter paper were presoaked in the solution of HRP-3, VEGF or PBS and implanted into mouse corneal pockets. After 6 days, corneal blood vessels were detected by slit-lamp microscopy and verified by staining with lipophilic fluorescent DiI dye (Fig 6A and 6B). Quantification of newly-formed corneal blood vessels and their branching points indicated that HRP-3 and VEGF significantly stimulated angiogenesis (Fig 6C and 6D). Moreover, a semiquantitative scoring method by combining the number, density and length of corneal blood vessels (S1 Table) showed that HRP-3 and VEGF induced significant corneal angiogenesis (Fig 6E).

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