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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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Effect of HO-1 inhibition on orexin-A-stimulated angiogenesis ex vivo and in vitro. (A) Rat aortic rings were embedded in Matrigel and cultured with orexin-A (200 nM) for 4 days in the presence or absence of SnPP (20 µM). The sprouted microvessels from aortic rings were photographed under a microscope (left). The newly formed sprouts of endothelial cells were counted (right). (B) HUVECs were seeded on growth factor-reduced Matrigel in the presence or absence of SnPP (20 µM) and treated with orexin-A (200 nM) for 4 hr. Cells were observed under a phase contrast microscope and the number of tube areas was counted. (C) HUVECs were seeded on gelatin-coated filters of transwell chambers. Cells were incubated with orexin-A (200 nM) and SnPP (20 µM) for 4 hr. The filter containing migrated cells was stained with H&E and photographed. The number of migrated cells was counted. All results shown are representative of at least three independent experiments. *p<0.05 vs. control; #p<0.05 vs. orexin-A alone.
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Figure 3: Effect of HO-1 inhibition on orexin-A-stimulated angiogenesis ex vivo and in vitro. (A) Rat aortic rings were embedded in Matrigel and cultured with orexin-A (200 nM) for 4 days in the presence or absence of SnPP (20 µM). The sprouted microvessels from aortic rings were photographed under a microscope (left). The newly formed sprouts of endothelial cells were counted (right). (B) HUVECs were seeded on growth factor-reduced Matrigel in the presence or absence of SnPP (20 µM) and treated with orexin-A (200 nM) for 4 hr. Cells were observed under a phase contrast microscope and the number of tube areas was counted. (C) HUVECs were seeded on gelatin-coated filters of transwell chambers. Cells were incubated with orexin-A (200 nM) and SnPP (20 µM) for 4 hr. The filter containing migrated cells was stained with H&E and photographed. The number of migrated cells was counted. All results shown are representative of at least three independent experiments. *p<0.05 vs. control; #p<0.05 vs. orexin-A alone.

Mentions: To assess the involvement of HO-1 in orexin-A-induced endothelial angiogenesis ex vivo, the aortic ring angiogenesis assay was used. As shown in Fig. 3A, orexin-A promoted endothelial cell sprouting from aortic segments, whereas the presence of SnPP resulted in a significant decrease in orexin-A-stimulated microvessel sprouting. The role of HO-1 in orexin-A-driven angiogenesis was further assessed by measuring the ability of HUVECs to form capillary-like networks. Orexin-A stimulated tubular formation by vascular endothelial cells on Matrigel. Co-treatment of HUVECs with orexin-A and SnPP resulted in a reduction in orexin-A-induced endothelial tube formation (Fig. 3B, left). Tube-like structures formed by HUVECs were quantified by measuring the number of tube areas (Fig. 3B, right). Next, we investigated the influence of HO-1 on the chemotactic motility of HUVECs using a modified Boyden chamber assay. As shown in Fig. 3C, orexin-A stimulated the migration of HUVECs, and substantial inhibition of orexin-A-mediated migration of HUVECs was observed in the presence of SnPP. Collectively, these results show that HO-1 mediates orexin-A-induced endothelial angiogenesis ex vivo and in vitro.


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)

Effect of HO-1 inhibition on orexin-A-stimulated angiogenesis ex vivo and in vitro. (A) Rat aortic rings were embedded in Matrigel and cultured with orexin-A (200 nM) for 4 days in the presence or absence of SnPP (20 µM). The sprouted microvessels from aortic rings were photographed under a microscope (left). The newly formed sprouts of endothelial cells were counted (right). (B) HUVECs were seeded on growth factor-reduced Matrigel in the presence or absence of SnPP (20 µM) and treated with orexin-A (200 nM) for 4 hr. Cells were observed under a phase contrast microscope and the number of tube areas was counted. (C) HUVECs were seeded on gelatin-coated filters of transwell chambers. Cells were incubated with orexin-A (200 nM) and SnPP (20 µM) for 4 hr. The filter containing migrated cells was stained with H&E and photographed. The number of migrated cells was counted. All results shown are representative of at least three independent experiments. *p<0.05 vs. control; #p<0.05 vs. orexin-A alone.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4499644&req=5

Figure 3: Effect of HO-1 inhibition on orexin-A-stimulated angiogenesis ex vivo and in vitro. (A) Rat aortic rings were embedded in Matrigel and cultured with orexin-A (200 nM) for 4 days in the presence or absence of SnPP (20 µM). The sprouted microvessels from aortic rings were photographed under a microscope (left). The newly formed sprouts of endothelial cells were counted (right). (B) HUVECs were seeded on growth factor-reduced Matrigel in the presence or absence of SnPP (20 µM) and treated with orexin-A (200 nM) for 4 hr. Cells were observed under a phase contrast microscope and the number of tube areas was counted. (C) HUVECs were seeded on gelatin-coated filters of transwell chambers. Cells were incubated with orexin-A (200 nM) and SnPP (20 µM) for 4 hr. The filter containing migrated cells was stained with H&E and photographed. The number of migrated cells was counted. All results shown are representative of at least three independent experiments. *p<0.05 vs. control; #p<0.05 vs. orexin-A alone.
Mentions: To assess the involvement of HO-1 in orexin-A-induced endothelial angiogenesis ex vivo, the aortic ring angiogenesis assay was used. As shown in Fig. 3A, orexin-A promoted endothelial cell sprouting from aortic segments, whereas the presence of SnPP resulted in a significant decrease in orexin-A-stimulated microvessel sprouting. The role of HO-1 in orexin-A-driven angiogenesis was further assessed by measuring the ability of HUVECs to form capillary-like networks. Orexin-A stimulated tubular formation by vascular endothelial cells on Matrigel. Co-treatment of HUVECs with orexin-A and SnPP resulted in a reduction in orexin-A-induced endothelial tube formation (Fig. 3B, left). Tube-like structures formed by HUVECs were quantified by measuring the number of tube areas (Fig. 3B, right). Next, we investigated the influence of HO-1 on the chemotactic motility of HUVECs using a modified Boyden chamber assay. As shown in Fig. 3C, orexin-A stimulated the migration of HUVECs, and substantial inhibition of orexin-A-mediated migration of HUVECs was observed in the presence of SnPP. Collectively, these results show that HO-1 mediates orexin-A-induced endothelial angiogenesis ex vivo and in vitro.

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