Limits...
Development of a novel antimicrobial peptide, AG-30, with angiogenic properties.

Nishikawa T, Nakagami H, Maeda A, Morishita R, Miyazaki N, Ogawa T, Tabata Y, Kikuchi Y, Hayashi H, Tatsu Y, Yumoto N, Tamai K, Tomono K, Kaneda Y - J. Cell. Mol. Med. (2008)

Bottom Line: As a result, AG-30 up-regulated expression of angiogenesis-related cytokines and growth factors for up to 72 hrs in human aortic endothelial cells.To further evaluate the angiogenic effect of AG-30 in vivo, we developed a slow-release AG-30 system utilizing biodegradable gelatin microspheres.In the ischaemic mouse hind limb, slow-release AG-30 treatment results in an increase in angiogenic score, an increase in blood flow (as demonstrated by laser Doppler imaging) and an increase in capillary density (as demonstrated by immunostaining with anti-CD31 antibody).

View Article: PubMed Central - PubMed

Affiliation: Division of Gene Therapy Science, Graduate School of Medicine, Osaka University, Osaka, Japan.

ABSTRACT
The utility of various synthetic peptides has been investigated in clinical trials of the treatment of cancers, infectious diseases and endocrine diseases. In the process of functional gene screening with in silico analysis for molecules with angiogenic properties, we generated a small peptide, angiogenic peptide (AG)-30, that possesses both antimicrobial and pro-inflammatory activities. AG-30 has an alpha-helix structure with a number of hydrophobic or net positively charged amino acids and a propensity to fold into amphipathic structures. Indeed, AG-30 exhibited antimicrobial activity against various bacteria, induced vascular endothelial cell growth and tube formation in a dose-dependent manner and increased neovascularization in a Matrigel plug assay. As a result, AG-30 up-regulated expression of angiogenesis-related cytokines and growth factors for up to 72 hrs in human aortic endothelial cells. To further evaluate the angiogenic effect of AG-30 in vivo, we developed a slow-release AG-30 system utilizing biodegradable gelatin microspheres. In the ischaemic mouse hind limb, slow-release AG-30 treatment results in an increase in angiogenic score, an increase in blood flow (as demonstrated by laser Doppler imaging) and an increase in capillary density (as demonstrated by immunostaining with anti-CD31 antibody). These data suggest that the novel peptide, AG-30, may have therapeutic potential for ischaemic diseases.

Show MeSH

Related in: MedlinePlus

Effect of AG-30 peptide on human aortic endothelial cells (HAEC) and human aortic smooth muscle cells (HASMC). (A) MTS assay with HAEC on days 1, 2 and 4. (B) MTS assay with HASMC on days 1, 3, and 5. (C) Chemokinetic migration assay with HAEC. Lower panel shows representative pictures of each group. (D) Chemotactic migration assay by the addition of AG-30 in the lower chambers (0.1, 1.0 and 10 μg/ml). *P < 0.01 versus NC, ‡P < 0.05 versus NC. n= 8 per group. (E) Tube formation, quantified as area and length. Lower panel shows representative pictures of each group. ‘Ctrl’ indicates treatment with control peptide. ‘AG-30’ indicates treatment with AG-30 peptide (10 μg/ml). ‘LL-37’ indicates treatment with LL-37 peptide (10 μg/ml). Results are expressed as fold-increase relative to the effect of NC (no treatment) for MTS assay and percentage increase to the effect of NC for migration and tube formation, respectively. *P < 0.05 versus NC, †P < 0.05 versus Ctrl, ‡P < 0.05 versus LL-37. n= 8 per group.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3822513&req=5

fig02: Effect of AG-30 peptide on human aortic endothelial cells (HAEC) and human aortic smooth muscle cells (HASMC). (A) MTS assay with HAEC on days 1, 2 and 4. (B) MTS assay with HASMC on days 1, 3, and 5. (C) Chemokinetic migration assay with HAEC. Lower panel shows representative pictures of each group. (D) Chemotactic migration assay by the addition of AG-30 in the lower chambers (0.1, 1.0 and 10 μg/ml). *P < 0.01 versus NC, ‡P < 0.05 versus NC. n= 8 per group. (E) Tube formation, quantified as area and length. Lower panel shows representative pictures of each group. ‘Ctrl’ indicates treatment with control peptide. ‘AG-30’ indicates treatment with AG-30 peptide (10 μg/ml). ‘LL-37’ indicates treatment with LL-37 peptide (10 μg/ml). Results are expressed as fold-increase relative to the effect of NC (no treatment) for MTS assay and percentage increase to the effect of NC for migration and tube formation, respectively. *P < 0.05 versus NC, †P < 0.05 versus Ctrl, ‡P < 0.05 versus LL-37. n= 8 per group.

Mentions: Some antibacterial peptides also possess angiogenic properties. In a human endothelial cell viability assay, AG-30 increased MTS activity in a dose-dependent (from 0.1 to 1.0 μg/ml) manner, and was more potent than LL-37 (Fig. 2A). Treatment with AG-30 also increased vascular smooth muscle cell viability in a dose-dependent manner (from 0.1 to 1.0 μg/ ml), as assessed by MTS assay (Fig. 2B). Human cell lines, such as HEK293, SAS (tongue cancer), HuH7 (hepatoma) and HeLa (cervical cancer) did not show significant response in viability in response to AG-30 stimulation (data not shown). In addition, chemokinetic cell migration significantly increased in response to AG-30 (which was more potent than LL-37) but not in response to control peptide (Fig. 2C). We have also addressed the chamotactic action of AG-30 by the addition of AG-30 in the lower chambers and HAEC in the upper chambers. As a result, the treatment of AG-30 with HAEC showed not only chemokinetic migration but also chemotactic action (Fig. 2D), although the chemokinetic action of AG-30 was stronger than chemotactic action. Further, tube formation was greater in response to AG-30 treatment when compared with LL-37 or control peptide treatment (Fig. 2E). These data demonstrate that AG-30 was superior to LL-37 in terms of inducing endothelial cell growth, migration and tube formation.


Development of a novel antimicrobial peptide, AG-30, with angiogenic properties.

Nishikawa T, Nakagami H, Maeda A, Morishita R, Miyazaki N, Ogawa T, Tabata Y, Kikuchi Y, Hayashi H, Tatsu Y, Yumoto N, Tamai K, Tomono K, Kaneda Y - J. Cell. Mol. Med. (2008)

Effect of AG-30 peptide on human aortic endothelial cells (HAEC) and human aortic smooth muscle cells (HASMC). (A) MTS assay with HAEC on days 1, 2 and 4. (B) MTS assay with HASMC on days 1, 3, and 5. (C) Chemokinetic migration assay with HAEC. Lower panel shows representative pictures of each group. (D) Chemotactic migration assay by the addition of AG-30 in the lower chambers (0.1, 1.0 and 10 μg/ml). *P < 0.01 versus NC, ‡P < 0.05 versus NC. n= 8 per group. (E) Tube formation, quantified as area and length. Lower panel shows representative pictures of each group. ‘Ctrl’ indicates treatment with control peptide. ‘AG-30’ indicates treatment with AG-30 peptide (10 μg/ml). ‘LL-37’ indicates treatment with LL-37 peptide (10 μg/ml). Results are expressed as fold-increase relative to the effect of NC (no treatment) for MTS assay and percentage increase to the effect of NC for migration and tube formation, respectively. *P < 0.05 versus NC, †P < 0.05 versus Ctrl, ‡P < 0.05 versus LL-37. n= 8 per group.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3822513&req=5

fig02: Effect of AG-30 peptide on human aortic endothelial cells (HAEC) and human aortic smooth muscle cells (HASMC). (A) MTS assay with HAEC on days 1, 2 and 4. (B) MTS assay with HASMC on days 1, 3, and 5. (C) Chemokinetic migration assay with HAEC. Lower panel shows representative pictures of each group. (D) Chemotactic migration assay by the addition of AG-30 in the lower chambers (0.1, 1.0 and 10 μg/ml). *P < 0.01 versus NC, ‡P < 0.05 versus NC. n= 8 per group. (E) Tube formation, quantified as area and length. Lower panel shows representative pictures of each group. ‘Ctrl’ indicates treatment with control peptide. ‘AG-30’ indicates treatment with AG-30 peptide (10 μg/ml). ‘LL-37’ indicates treatment with LL-37 peptide (10 μg/ml). Results are expressed as fold-increase relative to the effect of NC (no treatment) for MTS assay and percentage increase to the effect of NC for migration and tube formation, respectively. *P < 0.05 versus NC, †P < 0.05 versus Ctrl, ‡P < 0.05 versus LL-37. n= 8 per group.
Mentions: Some antibacterial peptides also possess angiogenic properties. In a human endothelial cell viability assay, AG-30 increased MTS activity in a dose-dependent (from 0.1 to 1.0 μg/ml) manner, and was more potent than LL-37 (Fig. 2A). Treatment with AG-30 also increased vascular smooth muscle cell viability in a dose-dependent manner (from 0.1 to 1.0 μg/ ml), as assessed by MTS assay (Fig. 2B). Human cell lines, such as HEK293, SAS (tongue cancer), HuH7 (hepatoma) and HeLa (cervical cancer) did not show significant response in viability in response to AG-30 stimulation (data not shown). In addition, chemokinetic cell migration significantly increased in response to AG-30 (which was more potent than LL-37) but not in response to control peptide (Fig. 2C). We have also addressed the chamotactic action of AG-30 by the addition of AG-30 in the lower chambers and HAEC in the upper chambers. As a result, the treatment of AG-30 with HAEC showed not only chemokinetic migration but also chemotactic action (Fig. 2D), although the chemokinetic action of AG-30 was stronger than chemotactic action. Further, tube formation was greater in response to AG-30 treatment when compared with LL-37 or control peptide treatment (Fig. 2E). These data demonstrate that AG-30 was superior to LL-37 in terms of inducing endothelial cell growth, migration and tube formation.

Bottom Line: As a result, AG-30 up-regulated expression of angiogenesis-related cytokines and growth factors for up to 72 hrs in human aortic endothelial cells.To further evaluate the angiogenic effect of AG-30 in vivo, we developed a slow-release AG-30 system utilizing biodegradable gelatin microspheres.In the ischaemic mouse hind limb, slow-release AG-30 treatment results in an increase in angiogenic score, an increase in blood flow (as demonstrated by laser Doppler imaging) and an increase in capillary density (as demonstrated by immunostaining with anti-CD31 antibody).

View Article: PubMed Central - PubMed

Affiliation: Division of Gene Therapy Science, Graduate School of Medicine, Osaka University, Osaka, Japan.

ABSTRACT
The utility of various synthetic peptides has been investigated in clinical trials of the treatment of cancers, infectious diseases and endocrine diseases. In the process of functional gene screening with in silico analysis for molecules with angiogenic properties, we generated a small peptide, angiogenic peptide (AG)-30, that possesses both antimicrobial and pro-inflammatory activities. AG-30 has an alpha-helix structure with a number of hydrophobic or net positively charged amino acids and a propensity to fold into amphipathic structures. Indeed, AG-30 exhibited antimicrobial activity against various bacteria, induced vascular endothelial cell growth and tube formation in a dose-dependent manner and increased neovascularization in a Matrigel plug assay. As a result, AG-30 up-regulated expression of angiogenesis-related cytokines and growth factors for up to 72 hrs in human aortic endothelial cells. To further evaluate the angiogenic effect of AG-30 in vivo, we developed a slow-release AG-30 system utilizing biodegradable gelatin microspheres. In the ischaemic mouse hind limb, slow-release AG-30 treatment results in an increase in angiogenic score, an increase in blood flow (as demonstrated by laser Doppler imaging) and an increase in capillary density (as demonstrated by immunostaining with anti-CD31 antibody). These data suggest that the novel peptide, AG-30, may have therapeutic potential for ischaemic diseases.

Show MeSH
Related in: MedlinePlus