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Synthetic RGDS peptide attenuates lipopolysaccharide-induced pulmonary inflammation by inhibiting integrin signaled MAP kinase pathways.

Moon C, Han JR, Park HJ, Hah JS, Kang JL - Respir. Res. (2009)

Bottom Line: RGDS inhibited LPS-induced phosphorylation of focal adhesion kinase and MAP kinases, including ERK, JNK, and p38 MAP kinase, in lung tissue.Similarly, a blocking antibody against integrin alphav significantly inhibited LPS-induced inflammatory cell migration into the lung, protein accumulation and proinflammatory mediator production in BAL fluid, at 4 or 24 h post-LPS.These results suggest that RGDS with high specificity for alphavintegrins attenuates inflammatory cascade during LPS-induced development of acute lung injury.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology, Division of Cell Biology, Ewha Medical Research Center, School of Medicine, Ewha Womans University, Seoul, Seoul, Korea. 94cmoon@hanmail.net

ABSTRACT

Background: Synthetic peptides containing the RGD sequence inhibit integrin-related functions in different cell systems. Here, we investigated the effects of synthetic Arg-Gly-Asp-Ser (RGDS) peptide on key inflammatory responses to intratracheal (i.t.) lipopolysaccharide (LPS) treatment and on the integrin signaled mitogen-activated protein (MAP) kinase pathway during the development of acute lung injury.

Methods: Saline or LPS (1.5 mg/kg) was administered i.t. with or without a single dose of RGDS (1, 2.5, or 5 mg/kg, i.p.), anti-alphav or anti-beta3 mAb (5 mg/kg, i.p.). Mice were sacrificed 4 or 24 h post-LPS.

Results: A pretreatment with RGDS inhibited LPS-induced increases in neutrophil and macrophage numbers, total protein levels and TNF-alpha and MIP-2 levels, and matrix metalloproteinase-9 activity in bronchoalveolar lavage (BAL) fluid at 4 or 24 h post-LPS treatment. RGDS inhibited LPS-induced phosphorylation of focal adhesion kinase and MAP kinases, including ERK, JNK, and p38 MAP kinase, in lung tissue. Importantly, the inhibition of the inflammatory responses and the kinase pathways were still evident when this peptide was administered 2 h after LPS treatment. Similarly, a blocking antibody against integrin alphav significantly inhibited LPS-induced inflammatory cell migration into the lung, protein accumulation and proinflammatory mediator production in BAL fluid, at 4 or 24 h post-LPS. Anti-beta3 also inhibited all LPS-induced inflammatory responses, except the accumulation of BAL protein at 24 h post-LPS.

Conclusion: These results suggest that RGDS with high specificity for alphavintegrins attenuates inflammatory cascade during LPS-induced development of acute lung injury.

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Effects of RGDS on FAK phosphorylation in lung tissue (A, B), and fibrinogen binding activity in alveolar macrophages (C, D). Where indicated, mice were administered RGDS or RGES (5 mg/kg, i.p.) once 1 h before LPS treatment and sacrificed 4 h post-LPS. Mice were also administered this peptide (5 mg/kg, i.p.) once 1 h before or 2 h after LPS and sacrificed 24 h post-LPS. (A, B) Western blotting with each anti-specific (phospho) FAK Ab was performed on lung tissue homogenates. Relative values for phosphorylated FAK versus FAK are indicated below the gel. Values represent means ± SEM of 5 mice per group. *Significantly different from saline treated controls, p < 0.05; +significantly different from animals treated with LPS only, p < 0.05. (C, D) Alveolar macrophages were incubated with fibrinogen conjugated to Alexafluor-488 for 20 min. After exposure to fibrinogen for 20 min, cells were fixed and then analyzed by confocal fluorescence microscopy. The results are representative results obtained from 5 mice per group.
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Figure 5: Effects of RGDS on FAK phosphorylation in lung tissue (A, B), and fibrinogen binding activity in alveolar macrophages (C, D). Where indicated, mice were administered RGDS or RGES (5 mg/kg, i.p.) once 1 h before LPS treatment and sacrificed 4 h post-LPS. Mice were also administered this peptide (5 mg/kg, i.p.) once 1 h before or 2 h after LPS and sacrificed 24 h post-LPS. (A, B) Western blotting with each anti-specific (phospho) FAK Ab was performed on lung tissue homogenates. Relative values for phosphorylated FAK versus FAK are indicated below the gel. Values represent means ± SEM of 5 mice per group. *Significantly different from saline treated controls, p < 0.05; +significantly different from animals treated with LPS only, p < 0.05. (C, D) Alveolar macrophages were incubated with fibrinogen conjugated to Alexafluor-488 for 20 min. After exposure to fibrinogen for 20 min, cells were fixed and then analyzed by confocal fluorescence microscopy. The results are representative results obtained from 5 mice per group.

Mentions: In vitro inhibition of integrin signaling by RGD peptide has been reported to decrease LPS-induced MAP kinase activity and TNF-α production in macrophages [9]. To investigate the functional link between integrin signaling and LPS-induced MAP kinase activation during the development of acute lung injury, we evaluated the inhibitory effect of RGDS on LPS-induced FAK phosphorylation in lung tissue. RGDS treatment 1 h before LPS completely inhibited LPS-induced this kinase phosphorylation at 4 h post-LPS (Figure 5A). At 24 h post-LPS, pre- or posttreatment with RGDS also completely inhibited LPS-induced FAK phosphorylation with similar potencies (Figure 5B). We also examined the fibrinogen binding activity of alveolar macrophages, because fibrinogen is a specific and physiologic ligand for activated integrin complexes, including αvβ3 [27]. As was expected, alveolar macrophages from mice treated with LPS were found to have increased fibrinogen binding activity in many confocal microscope fields, and RGDS treatment 1 h before or 2 h after LPS suppressed this activity at 4 or 24 h post-LPS, respectively (Figures 5C and 5D). These findings indicate that RGDS inhibits LPS-induced integrin signaling during the development of lung injury.


Synthetic RGDS peptide attenuates lipopolysaccharide-induced pulmonary inflammation by inhibiting integrin signaled MAP kinase pathways.

Moon C, Han JR, Park HJ, Hah JS, Kang JL - Respir. Res. (2009)

Effects of RGDS on FAK phosphorylation in lung tissue (A, B), and fibrinogen binding activity in alveolar macrophages (C, D). Where indicated, mice were administered RGDS or RGES (5 mg/kg, i.p.) once 1 h before LPS treatment and sacrificed 4 h post-LPS. Mice were also administered this peptide (5 mg/kg, i.p.) once 1 h before or 2 h after LPS and sacrificed 24 h post-LPS. (A, B) Western blotting with each anti-specific (phospho) FAK Ab was performed on lung tissue homogenates. Relative values for phosphorylated FAK versus FAK are indicated below the gel. Values represent means ± SEM of 5 mice per group. *Significantly different from saline treated controls, p < 0.05; +significantly different from animals treated with LPS only, p < 0.05. (C, D) Alveolar macrophages were incubated with fibrinogen conjugated to Alexafluor-488 for 20 min. After exposure to fibrinogen for 20 min, cells were fixed and then analyzed by confocal fluorescence microscopy. The results are representative results obtained from 5 mice per group.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2666640&req=5

Figure 5: Effects of RGDS on FAK phosphorylation in lung tissue (A, B), and fibrinogen binding activity in alveolar macrophages (C, D). Where indicated, mice were administered RGDS or RGES (5 mg/kg, i.p.) once 1 h before LPS treatment and sacrificed 4 h post-LPS. Mice were also administered this peptide (5 mg/kg, i.p.) once 1 h before or 2 h after LPS and sacrificed 24 h post-LPS. (A, B) Western blotting with each anti-specific (phospho) FAK Ab was performed on lung tissue homogenates. Relative values for phosphorylated FAK versus FAK are indicated below the gel. Values represent means ± SEM of 5 mice per group. *Significantly different from saline treated controls, p < 0.05; +significantly different from animals treated with LPS only, p < 0.05. (C, D) Alveolar macrophages were incubated with fibrinogen conjugated to Alexafluor-488 for 20 min. After exposure to fibrinogen for 20 min, cells were fixed and then analyzed by confocal fluorescence microscopy. The results are representative results obtained from 5 mice per group.
Mentions: In vitro inhibition of integrin signaling by RGD peptide has been reported to decrease LPS-induced MAP kinase activity and TNF-α production in macrophages [9]. To investigate the functional link between integrin signaling and LPS-induced MAP kinase activation during the development of acute lung injury, we evaluated the inhibitory effect of RGDS on LPS-induced FAK phosphorylation in lung tissue. RGDS treatment 1 h before LPS completely inhibited LPS-induced this kinase phosphorylation at 4 h post-LPS (Figure 5A). At 24 h post-LPS, pre- or posttreatment with RGDS also completely inhibited LPS-induced FAK phosphorylation with similar potencies (Figure 5B). We also examined the fibrinogen binding activity of alveolar macrophages, because fibrinogen is a specific and physiologic ligand for activated integrin complexes, including αvβ3 [27]. As was expected, alveolar macrophages from mice treated with LPS were found to have increased fibrinogen binding activity in many confocal microscope fields, and RGDS treatment 1 h before or 2 h after LPS suppressed this activity at 4 or 24 h post-LPS, respectively (Figures 5C and 5D). These findings indicate that RGDS inhibits LPS-induced integrin signaling during the development of lung injury.

Bottom Line: RGDS inhibited LPS-induced phosphorylation of focal adhesion kinase and MAP kinases, including ERK, JNK, and p38 MAP kinase, in lung tissue.Similarly, a blocking antibody against integrin alphav significantly inhibited LPS-induced inflammatory cell migration into the lung, protein accumulation and proinflammatory mediator production in BAL fluid, at 4 or 24 h post-LPS.These results suggest that RGDS with high specificity for alphavintegrins attenuates inflammatory cascade during LPS-induced development of acute lung injury.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology, Division of Cell Biology, Ewha Medical Research Center, School of Medicine, Ewha Womans University, Seoul, Seoul, Korea. 94cmoon@hanmail.net

ABSTRACT

Background: Synthetic peptides containing the RGD sequence inhibit integrin-related functions in different cell systems. Here, we investigated the effects of synthetic Arg-Gly-Asp-Ser (RGDS) peptide on key inflammatory responses to intratracheal (i.t.) lipopolysaccharide (LPS) treatment and on the integrin signaled mitogen-activated protein (MAP) kinase pathway during the development of acute lung injury.

Methods: Saline or LPS (1.5 mg/kg) was administered i.t. with or without a single dose of RGDS (1, 2.5, or 5 mg/kg, i.p.), anti-alphav or anti-beta3 mAb (5 mg/kg, i.p.). Mice were sacrificed 4 or 24 h post-LPS.

Results: A pretreatment with RGDS inhibited LPS-induced increases in neutrophil and macrophage numbers, total protein levels and TNF-alpha and MIP-2 levels, and matrix metalloproteinase-9 activity in bronchoalveolar lavage (BAL) fluid at 4 or 24 h post-LPS treatment. RGDS inhibited LPS-induced phosphorylation of focal adhesion kinase and MAP kinases, including ERK, JNK, and p38 MAP kinase, in lung tissue. Importantly, the inhibition of the inflammatory responses and the kinase pathways were still evident when this peptide was administered 2 h after LPS treatment. Similarly, a blocking antibody against integrin alphav significantly inhibited LPS-induced inflammatory cell migration into the lung, protein accumulation and proinflammatory mediator production in BAL fluid, at 4 or 24 h post-LPS. Anti-beta3 also inhibited all LPS-induced inflammatory responses, except the accumulation of BAL protein at 24 h post-LPS.

Conclusion: These results suggest that RGDS with high specificity for alphavintegrins attenuates inflammatory cascade during LPS-induced development of acute lung injury.

Show MeSH
Related in: MedlinePlus