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Exosomal transfer of vasorin expressed in hepatocellular carcinoma cells promotes migration of human umbilical vein endothelial cells.

Huang A, Dong J, Li S, Wang C, Ding H, Li H, Su X, Ge X, Sun L, Bai C, Shen X, Fang T, Li J, Shao N - Int. J. Biol. Sci. (2015)

Bottom Line: We confirmed for the first time that HepG2-derived VASN can be transferred to human umbilical vein endothelial cells (HUVECs) via receptor mediated endocytosis of exosomes, at least in part through HSPGs.The HepG2-derived VASN containing exosomes promote migration of recipient HUVECs cells.Our results identify a novel pathway by which a functional protein expressed in tumor cells affects the biological fate of endothelial cells via exosomes.

View Article: PubMed Central - PubMed

Affiliation: Beijing Institute of Basic Medical Sciences, Beijing 100850, China.

ABSTRACT
Vasorin (VASN) is a type I transmembrane protein that plays important roles in tumor development and vasculogenesis. In this paper, we showed that VASN could be a key mediator of communication between tumor cells and endothelial cells. We confirmed for the first time that HepG2-derived VASN can be transferred to human umbilical vein endothelial cells (HUVECs) via receptor mediated endocytosis of exosomes, at least in part through HSPGs. The HepG2-derived VASN containing exosomes promote migration of recipient HUVECs cells. Our results identify a novel pathway by which a functional protein expressed in tumor cells affects the biological fate of endothelial cells via exosomes.

No MeSH data available.


Related in: MedlinePlus

Transfer of VASN protein from HepG2 to HUVECs via exosomes. (A) Western blot shows increased levels of VASN in HUVECs pretreated with HepG2-derived exosomes. GAPDH was used as a loading control. (B) Western blot analysis of VASN protein in HUVECs treated with exosomes derived from HepG2 cells transfected with NC or VASN siRNAs. GAPDH was used as a loading control. (C) HUVECs were incubated with 20 μg/mL HepG2-derived exosomes in the absence (mock) or in the presence of normal mouse IgG, mouse monoclonal anti-VASN antibody for 24hr in, and then analyzed for VASN levels in the whole cell lysates of incubated HUVECs. GAPDH was used as a loading control. (D) Concentration-dependent uptake of HepG2-derived exosomal VASN in HUVECs. GAPDH was used as a loading control. (E) The exogenous myc tagged VASN expressed in exosomes of HepG2 cells was transferred into HUVECs cells as analyzed by immunofluorescence with anti-myc antibody and fluorescence labeled secondary antibody. HUVECs were incubated with exosomes derived from HepG2 cells transfected with plasmids expressing myc-labeled VASN and analyzed by immunofluorescence under confocal microscopy. DAPI stains showed the nuclear areas.
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Figure 4: Transfer of VASN protein from HepG2 to HUVECs via exosomes. (A) Western blot shows increased levels of VASN in HUVECs pretreated with HepG2-derived exosomes. GAPDH was used as a loading control. (B) Western blot analysis of VASN protein in HUVECs treated with exosomes derived from HepG2 cells transfected with NC or VASN siRNAs. GAPDH was used as a loading control. (C) HUVECs were incubated with 20 μg/mL HepG2-derived exosomes in the absence (mock) or in the presence of normal mouse IgG, mouse monoclonal anti-VASN antibody for 24hr in, and then analyzed for VASN levels in the whole cell lysates of incubated HUVECs. GAPDH was used as a loading control. (D) Concentration-dependent uptake of HepG2-derived exosomal VASN in HUVECs. GAPDH was used as a loading control. (E) The exogenous myc tagged VASN expressed in exosomes of HepG2 cells was transferred into HUVECs cells as analyzed by immunofluorescence with anti-myc antibody and fluorescence labeled secondary antibody. HUVECs were incubated with exosomes derived from HepG2 cells transfected with plasmids expressing myc-labeled VASN and analyzed by immunofluorescence under confocal microscopy. DAPI stains showed the nuclear areas.

Mentions: To determine whether VASN could be transferred between two different cell lines via exosomes, we isolated HepG2-derived exosomes and incubated them with HUVECs for 24 h. Result showed that the protein levels of VASN in whole cell extracts of HUVECs were increased (Fig. 4A). Pre-silencing VASN expression in HepG2 with siRNA could block the VASN elevation in HepG2-derived exosomes treated HUVECs cells maybe because of lower VASN in exosomes (Fig. 4B). Similar results were obtained when mouse monoclonal antibody against VASN was added into the co-culture system of HepG2 derived exosomes and HUVECs (Fig. 4C). The transfer of VASN into HUVECs cells by HepG2-derived exosomes showed a dose-dependent manner (Fig. 4D). The exogenous VASN with myc tag was transiently expressed in HepG2 cells, and the internalization of exosomal myc-VASN into HUVECs cells was visualized by immunofluorescence with anti-myc antibody and fluorescence labeled secondary antibody. (Fig. 4E). All the above implies an exosomes specific intercellular transfer of VASN from HepG2 to HUVECs.


Exosomal transfer of vasorin expressed in hepatocellular carcinoma cells promotes migration of human umbilical vein endothelial cells.

Huang A, Dong J, Li S, Wang C, Ding H, Li H, Su X, Ge X, Sun L, Bai C, Shen X, Fang T, Li J, Shao N - Int. J. Biol. Sci. (2015)

Transfer of VASN protein from HepG2 to HUVECs via exosomes. (A) Western blot shows increased levels of VASN in HUVECs pretreated with HepG2-derived exosomes. GAPDH was used as a loading control. (B) Western blot analysis of VASN protein in HUVECs treated with exosomes derived from HepG2 cells transfected with NC or VASN siRNAs. GAPDH was used as a loading control. (C) HUVECs were incubated with 20 μg/mL HepG2-derived exosomes in the absence (mock) or in the presence of normal mouse IgG, mouse monoclonal anti-VASN antibody for 24hr in, and then analyzed for VASN levels in the whole cell lysates of incubated HUVECs. GAPDH was used as a loading control. (D) Concentration-dependent uptake of HepG2-derived exosomal VASN in HUVECs. GAPDH was used as a loading control. (E) The exogenous myc tagged VASN expressed in exosomes of HepG2 cells was transferred into HUVECs cells as analyzed by immunofluorescence with anti-myc antibody and fluorescence labeled secondary antibody. HUVECs were incubated with exosomes derived from HepG2 cells transfected with plasmids expressing myc-labeled VASN and analyzed by immunofluorescence under confocal microscopy. DAPI stains showed the nuclear areas.
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Related In: Results  -  Collection

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Figure 4: Transfer of VASN protein from HepG2 to HUVECs via exosomes. (A) Western blot shows increased levels of VASN in HUVECs pretreated with HepG2-derived exosomes. GAPDH was used as a loading control. (B) Western blot analysis of VASN protein in HUVECs treated with exosomes derived from HepG2 cells transfected with NC or VASN siRNAs. GAPDH was used as a loading control. (C) HUVECs were incubated with 20 μg/mL HepG2-derived exosomes in the absence (mock) or in the presence of normal mouse IgG, mouse monoclonal anti-VASN antibody for 24hr in, and then analyzed for VASN levels in the whole cell lysates of incubated HUVECs. GAPDH was used as a loading control. (D) Concentration-dependent uptake of HepG2-derived exosomal VASN in HUVECs. GAPDH was used as a loading control. (E) The exogenous myc tagged VASN expressed in exosomes of HepG2 cells was transferred into HUVECs cells as analyzed by immunofluorescence with anti-myc antibody and fluorescence labeled secondary antibody. HUVECs were incubated with exosomes derived from HepG2 cells transfected with plasmids expressing myc-labeled VASN and analyzed by immunofluorescence under confocal microscopy. DAPI stains showed the nuclear areas.
Mentions: To determine whether VASN could be transferred between two different cell lines via exosomes, we isolated HepG2-derived exosomes and incubated them with HUVECs for 24 h. Result showed that the protein levels of VASN in whole cell extracts of HUVECs were increased (Fig. 4A). Pre-silencing VASN expression in HepG2 with siRNA could block the VASN elevation in HepG2-derived exosomes treated HUVECs cells maybe because of lower VASN in exosomes (Fig. 4B). Similar results were obtained when mouse monoclonal antibody against VASN was added into the co-culture system of HepG2 derived exosomes and HUVECs (Fig. 4C). The transfer of VASN into HUVECs cells by HepG2-derived exosomes showed a dose-dependent manner (Fig. 4D). The exogenous VASN with myc tag was transiently expressed in HepG2 cells, and the internalization of exosomal myc-VASN into HUVECs cells was visualized by immunofluorescence with anti-myc antibody and fluorescence labeled secondary antibody. (Fig. 4E). All the above implies an exosomes specific intercellular transfer of VASN from HepG2 to HUVECs.

Bottom Line: We confirmed for the first time that HepG2-derived VASN can be transferred to human umbilical vein endothelial cells (HUVECs) via receptor mediated endocytosis of exosomes, at least in part through HSPGs.The HepG2-derived VASN containing exosomes promote migration of recipient HUVECs cells.Our results identify a novel pathway by which a functional protein expressed in tumor cells affects the biological fate of endothelial cells via exosomes.

View Article: PubMed Central - PubMed

Affiliation: Beijing Institute of Basic Medical Sciences, Beijing 100850, China.

ABSTRACT
Vasorin (VASN) is a type I transmembrane protein that plays important roles in tumor development and vasculogenesis. In this paper, we showed that VASN could be a key mediator of communication between tumor cells and endothelial cells. We confirmed for the first time that HepG2-derived VASN can be transferred to human umbilical vein endothelial cells (HUVECs) via receptor mediated endocytosis of exosomes, at least in part through HSPGs. The HepG2-derived VASN containing exosomes promote migration of recipient HUVECs cells. Our results identify a novel pathway by which a functional protein expressed in tumor cells affects the biological fate of endothelial cells via exosomes.

No MeSH data available.


Related in: MedlinePlus