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Involvement of Heme Oxygenase-1 in Orexin-A-induced Angiogenesis in Vascular Endothelial Cells.

Kim MK, Park HJ, Kim SR, Choi YK, Bae SK, Bae MK - Korean J. Physiol. Pharmacol. (2015)

Bottom Line: Furthermore, we showed that inhibition of HO-1 by tin (Sn) protoporphryin-IX (SnPP) reduced orexin-A-induced angiogenesis in vivo and ex vivo.Orexin-A treatment increased the expression of nuclear factor erythroid-derived 2 related factor 2 (Nrf2), and antioxidant response element (ARE) luciferase activity, leading to induction of HO-1.Collectively, these findings indicate that HO-1 plays a role as an important mediator of orexin-A-induced angiogenesis, and provide new possibilities for therapeutic approaches in pathophysiological conditions associated with angiogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral Physiology, School of Dentistry, Pusan National University, Yangsan 626-870, Korea.

ABSTRACT
The cytoprotective enzyme heme oxygenase-1 (HO-1) influences endothelial cell survival, proliferation, inflammatory response, and angiogenesis in response to various angiogenic stimuli. In this study, we investigate the involvement of HO-1 in the angiogenic activity of orexin-A. We showed that orexin-A stimulates expression and activity of HO-1 in human umbilical vein endothelial cells (HUVECs). Furthermore, we showed that inhibition of HO-1 by tin (Sn) protoporphryin-IX (SnPP) reduced orexin-A-induced angiogenesis in vivo and ex vivo. Orexin-A-stimulated endothelial tube formation and chemotactic activity were also blocked in SnPP-treated vascular endothelial cells. Orexin-A treatment increased the expression of nuclear factor erythroid-derived 2 related factor 2 (Nrf2), and antioxidant response element (ARE) luciferase activity, leading to induction of HO-1. Collectively, these findings indicate that HO-1 plays a role as an important mediator of orexin-A-induced angiogenesis, and provide new possibilities for therapeutic approaches in pathophysiological conditions associated with angiogenesis.

No MeSH data available.


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Influence of the ARE-Nrf2 pathway in orexin-A-mediated HO-1 induction and angiogenesis. (A) HMECs transfected with an ARE-luciferase construct were treated with orexin-A (200 nM) for 3 hr. The cell extracts were prepared and analyzed using a luminometer. Three independent experiments were performed. *p<0.05 vs. control. (B) HUVECs were transiently transfected with control or Nrf2 siRNA (1 µM) by Amaxa nucleofector, followed by treatment with orexin-A for 2 hr. The expression of Nrf2 and HO-1 was determined by Western blot analysis. (C) For tube formation assays, Nrf2 siRNA-transfected cells were seeded on growth factor-reduced Matrigel. Cells were incubated with orexin-A (200 nM) for 4 hr. Newly formed tubes were photographed, and tube areas were quantified from at least three individual experiments. *p<0.05 vs. control; #p<0.05 vs. control siRNA with orexin-A.
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Figure 4: Influence of the ARE-Nrf2 pathway in orexin-A-mediated HO-1 induction and angiogenesis. (A) HMECs transfected with an ARE-luciferase construct were treated with orexin-A (200 nM) for 3 hr. The cell extracts were prepared and analyzed using a luminometer. Three independent experiments were performed. *p<0.05 vs. control. (B) HUVECs were transiently transfected with control or Nrf2 siRNA (1 µM) by Amaxa nucleofector, followed by treatment with orexin-A for 2 hr. The expression of Nrf2 and HO-1 was determined by Western blot analysis. (C) For tube formation assays, Nrf2 siRNA-transfected cells were seeded on growth factor-reduced Matrigel. Cells were incubated with orexin-A (200 nM) for 4 hr. Newly formed tubes were photographed, and tube areas were quantified from at least three individual experiments. *p<0.05 vs. control; #p<0.05 vs. control siRNA with orexin-A.

Mentions: Nrf2 is a transcription factor for antioxidative genes and their proteins, including HO-1. Nrf2 induces HO-1 by binding to a cis-acting enhancer element known as the ARE. In order to test the influence of orexin-A on ARE promoter activity, we performed a luciferase reporter gene assay. Vascular endothelial cells were transiently transfected with the ARE-luciferase reporter gene, and luciferase activity was determined after orexin-A treatment. As shown in Fig. 4A, ARE-luciferase activity was increased in orexin-A treated cells. To further determine whether Nrf2 is involved in orexin-A-induced HO-1 expression, we transiently transfected endothelial cells with Nrf2 siRNA or control siRNA. Silencing with Nrf2 siRNA decreased basal levels of Nrf2 and HO-1 protein expression compared with those in control siRNA-transfected cells (Supplemental Fig. 1). The induction of Nrf2 and HO-1 protein by orexin-A was diminished in Nrf2 siRNA-transfected cells when compared with control siRNA-transfected cells (Fig. 4B). Furthermore, as shown in Fig. 4C, knockdown of Nrf2 also decreased angiogenic activity of orexin-A in a tube formation assay. These data suggest that Nrf2-ARE is closely correlated with orexin-A-mediated angiogenesis through HO-1.


Involvement of Heme Oxygenase-1 in Orexin-A-induced Angiogenesis in Vascular Endothelial Cells.

Kim MK, Park HJ, Kim SR, Choi YK, Bae SK, Bae MK - Korean J. Physiol. Pharmacol. (2015)

Influence of the ARE-Nrf2 pathway in orexin-A-mediated HO-1 induction and angiogenesis. (A) HMECs transfected with an ARE-luciferase construct were treated with orexin-A (200 nM) for 3 hr. The cell extracts were prepared and analyzed using a luminometer. Three independent experiments were performed. *p<0.05 vs. control. (B) HUVECs were transiently transfected with control or Nrf2 siRNA (1 µM) by Amaxa nucleofector, followed by treatment with orexin-A for 2 hr. The expression of Nrf2 and HO-1 was determined by Western blot analysis. (C) For tube formation assays, Nrf2 siRNA-transfected cells were seeded on growth factor-reduced Matrigel. Cells were incubated with orexin-A (200 nM) for 4 hr. Newly formed tubes were photographed, and tube areas were quantified from at least three individual experiments. *p<0.05 vs. control; #p<0.05 vs. control siRNA with orexin-A.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4499644&req=5

Figure 4: Influence of the ARE-Nrf2 pathway in orexin-A-mediated HO-1 induction and angiogenesis. (A) HMECs transfected with an ARE-luciferase construct were treated with orexin-A (200 nM) for 3 hr. The cell extracts were prepared and analyzed using a luminometer. Three independent experiments were performed. *p<0.05 vs. control. (B) HUVECs were transiently transfected with control or Nrf2 siRNA (1 µM) by Amaxa nucleofector, followed by treatment with orexin-A for 2 hr. The expression of Nrf2 and HO-1 was determined by Western blot analysis. (C) For tube formation assays, Nrf2 siRNA-transfected cells were seeded on growth factor-reduced Matrigel. Cells were incubated with orexin-A (200 nM) for 4 hr. Newly formed tubes were photographed, and tube areas were quantified from at least three individual experiments. *p<0.05 vs. control; #p<0.05 vs. control siRNA with orexin-A.
Mentions: Nrf2 is a transcription factor for antioxidative genes and their proteins, including HO-1. Nrf2 induces HO-1 by binding to a cis-acting enhancer element known as the ARE. In order to test the influence of orexin-A on ARE promoter activity, we performed a luciferase reporter gene assay. Vascular endothelial cells were transiently transfected with the ARE-luciferase reporter gene, and luciferase activity was determined after orexin-A treatment. As shown in Fig. 4A, ARE-luciferase activity was increased in orexin-A treated cells. To further determine whether Nrf2 is involved in orexin-A-induced HO-1 expression, we transiently transfected endothelial cells with Nrf2 siRNA or control siRNA. Silencing with Nrf2 siRNA decreased basal levels of Nrf2 and HO-1 protein expression compared with those in control siRNA-transfected cells (Supplemental Fig. 1). The induction of Nrf2 and HO-1 protein by orexin-A was diminished in Nrf2 siRNA-transfected cells when compared with control siRNA-transfected cells (Fig. 4B). Furthermore, as shown in Fig. 4C, knockdown of Nrf2 also decreased angiogenic activity of orexin-A in a tube formation assay. These data suggest that Nrf2-ARE is closely correlated with orexin-A-mediated angiogenesis through HO-1.

Bottom Line: Furthermore, we showed that inhibition of HO-1 by tin (Sn) protoporphryin-IX (SnPP) reduced orexin-A-induced angiogenesis in vivo and ex vivo.Orexin-A treatment increased the expression of nuclear factor erythroid-derived 2 related factor 2 (Nrf2), and antioxidant response element (ARE) luciferase activity, leading to induction of HO-1.Collectively, these findings indicate that HO-1 plays a role as an important mediator of orexin-A-induced angiogenesis, and provide new possibilities for therapeutic approaches in pathophysiological conditions associated with angiogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral Physiology, School of Dentistry, Pusan National University, Yangsan 626-870, Korea.

ABSTRACT
The cytoprotective enzyme heme oxygenase-1 (HO-1) influences endothelial cell survival, proliferation, inflammatory response, and angiogenesis in response to various angiogenic stimuli. In this study, we investigate the involvement of HO-1 in the angiogenic activity of orexin-A. We showed that orexin-A stimulates expression and activity of HO-1 in human umbilical vein endothelial cells (HUVECs). Furthermore, we showed that inhibition of HO-1 by tin (Sn) protoporphryin-IX (SnPP) reduced orexin-A-induced angiogenesis in vivo and ex vivo. Orexin-A-stimulated endothelial tube formation and chemotactic activity were also blocked in SnPP-treated vascular endothelial cells. Orexin-A treatment increased the expression of nuclear factor erythroid-derived 2 related factor 2 (Nrf2), and antioxidant response element (ARE) luciferase activity, leading to induction of HO-1. Collectively, these findings indicate that HO-1 plays a role as an important mediator of orexin-A-induced angiogenesis, and provide new possibilities for therapeutic approaches in pathophysiological conditions associated with angiogenesis.

No MeSH data available.


Related in: MedlinePlus