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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

VASN protein secretion and localization. (A) Western blot analysis of VASN protein in cell extracts and supernatant of HepG2 cells. (B) Electron micrograph of exosomes isolated from supernatants of HepG2 cells. Bar represents 100 nm. (C) VASN expression in exosomes, supernatant, and exosomes-depleted supernatants. The exosomal marker protein CD63 was also detected. (D) Western blot analysis of VASN protein in exosomes derived from HepG2, L02, and HUVECs. CD63 was used as a loading control. (E) Western blot analysis of VASN protein in cell lysates (top panel) and exosomes (bottom panel) from HepG2 cells transfected with NC or VASN siRNA (siVASN). GAPDH and CD63 were used as loading controls for cell lysates and exosomes, respectively.
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Figure 2: VASN protein secretion and localization. (A) Western blot analysis of VASN protein in cell extracts and supernatant of HepG2 cells. (B) Electron micrograph of exosomes isolated from supernatants of HepG2 cells. Bar represents 100 nm. (C) VASN expression in exosomes, supernatant, and exosomes-depleted supernatants. The exosomal marker protein CD63 was also detected. (D) Western blot analysis of VASN protein in exosomes derived from HepG2, L02, and HUVECs. CD63 was used as a loading control. (E) Western blot analysis of VASN protein in cell lysates (top panel) and exosomes (bottom panel) from HepG2 cells transfected with NC or VASN siRNA (siVASN). GAPDH and CD63 were used as loading controls for cell lysates and exosomes, respectively.

Mentions: Cancer cells may communicate with endothelial cells by secreting free vascular endothelial growth factors such as VEGF11, or by releasing membrane vesicles such as “microvesicles” and “exosomes” to transfer functional molecules including “oncoproteins” into recipient cells12, 13. We showed that VASN was detectable in HepG2 supernatant (Fig. 2A). The shift rate of VASN in supernatant is fast than that in whole cell extract because the former is cleaved by TACE and lack of intracellular domain. We then purified exosomes from the supernatant of HepG2 cells and confirmed them by TEM (Fig. 2B). VASN expression in exosomes was confirmed by western blotting. VASN was detected both in isolated exosomes and supernatant, but its expression was low in exosomes-depleted supernatant (Fig. 2C). CD63, an exosomal marker protein, was also detecteded. Consistent with the intracellular expression levels of VASN in these cell lines, VASN expression was found to be higher in HepG2-derived exosomes than in L02-derived or HUVECs-derived exosomes (Fig. 2D). When HepG2 cells were treated with VASN siRNA, the expression levels of VASN in exosomes derived from these cells were decreased (Fig. 2E), indicating exosomal protein levels correlate with intracellular VASN expression levels.


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)

VASN protein secretion and localization. (A) Western blot analysis of VASN protein in cell extracts and supernatant of HepG2 cells. (B) Electron micrograph of exosomes isolated from supernatants of HepG2 cells. Bar represents 100 nm. (C) VASN expression in exosomes, supernatant, and exosomes-depleted supernatants. The exosomal marker protein CD63 was also detected. (D) Western blot analysis of VASN protein in exosomes derived from HepG2, L02, and HUVECs. CD63 was used as a loading control. (E) Western blot analysis of VASN protein in cell lysates (top panel) and exosomes (bottom panel) from HepG2 cells transfected with NC or VASN siRNA (siVASN). GAPDH and CD63 were used as loading controls for cell lysates and exosomes, respectively.
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Related In: Results  -  Collection

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Figure 2: VASN protein secretion and localization. (A) Western blot analysis of VASN protein in cell extracts and supernatant of HepG2 cells. (B) Electron micrograph of exosomes isolated from supernatants of HepG2 cells. Bar represents 100 nm. (C) VASN expression in exosomes, supernatant, and exosomes-depleted supernatants. The exosomal marker protein CD63 was also detected. (D) Western blot analysis of VASN protein in exosomes derived from HepG2, L02, and HUVECs. CD63 was used as a loading control. (E) Western blot analysis of VASN protein in cell lysates (top panel) and exosomes (bottom panel) from HepG2 cells transfected with NC or VASN siRNA (siVASN). GAPDH and CD63 were used as loading controls for cell lysates and exosomes, respectively.
Mentions: Cancer cells may communicate with endothelial cells by secreting free vascular endothelial growth factors such as VEGF11, or by releasing membrane vesicles such as “microvesicles” and “exosomes” to transfer functional molecules including “oncoproteins” into recipient cells12, 13. We showed that VASN was detectable in HepG2 supernatant (Fig. 2A). The shift rate of VASN in supernatant is fast than that in whole cell extract because the former is cleaved by TACE and lack of intracellular domain. We then purified exosomes from the supernatant of HepG2 cells and confirmed them by TEM (Fig. 2B). VASN expression in exosomes was confirmed by western blotting. VASN was detected both in isolated exosomes and supernatant, but its expression was low in exosomes-depleted supernatant (Fig. 2C). CD63, an exosomal marker protein, was also detecteded. Consistent with the intracellular expression levels of VASN in these cell lines, VASN expression was found to be higher in HepG2-derived exosomes than in L02-derived or HUVECs-derived exosomes (Fig. 2D). When HepG2 cells were treated with VASN siRNA, the expression levels of VASN in exosomes derived from these cells were decreased (Fig. 2E), indicating exosomal protein levels correlate with intracellular VASN expression levels.

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