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LL-37 stimulates the functions of adipose-derived stromal/stem cells via early growth response 1 and the MAPK pathway.

Yang Y, Choi H, Seon M, Cho D, Bang SI - Stem Cell Res Ther (2016)

Bottom Line: To examine the effects of LL-37 on ASC function, cell proliferation and migration were measured by a cell counting kit (CCK-8) and a Transwell migration assay.Activation of mitogen-activated protein kinases (MAPKs) was essential not only for LL-37-enhanced ASC proliferation and migration but also EGR1 expression; treatment with a specific inhibitor of extracellular signal-regulated kinase, p38, or c-Jun N-terminal kinase blocked the stimulatory effect of LL-37.This study correlates LL-37 with MSC functions (specifically those of ASCs), including cell expansion, cell migration, and paracrine actions, which may be useful in terms of implantation for tissue regeneration.

View Article: PubMed Central - PubMed

Affiliation: Department of Plastic Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.

ABSTRACT

Background: LL-37 is a naturally occurring antimicrobial peptide found in the wound bed and assists wound repair. No published study has characterized the role of LL-37 in the function(s) of human mesenchymal stem cells (MSCs). This study investigated the functions of adipose-derived stromal/stem cells (ASCs) activated by LL-37 by performing both in vitro assays with cultured cells and in vivo assays with C57BL/6 mice with hair loss.

Methods: Human ASCs were isolated from healthy donors with written informed consent. To examine the effects of LL-37 on ASC function, cell proliferation and migration were measured by a cell counting kit (CCK-8) and a Transwell migration assay. Early growth response 1 (EGR1) mRNA expression was determined by microarray and real-time PCR analyses. The protein levels of EGR1 and regenerative factors were analyzed by specific enzyme-linked immunosorbent assays and western blotting.

Results: LL-37 treatment enhanced the proliferation and migration of human ASCs expressing formyl peptide receptor like-1. Microarray and real-time PCR data showed that EGR1 expression was rapidly and significantly increased by LL-37 treatment. LL-37 treatment also enhanced the production of EGR1. Moreover, small interfering RNA-mediated knockdown of EGR1 inhibited LL-37-enhanced ASC proliferation and migration. Activation of mitogen-activated protein kinases (MAPKs) was essential not only for LL-37-enhanced ASC proliferation and migration but also EGR1 expression; treatment with a specific inhibitor of extracellular signal-regulated kinase, p38, or c-Jun N-terminal kinase blocked the stimulatory effect of LL-37. EGR1 has a strong paracrine capability and can influence angiogenic factors in ASCs; therefore, we evaluated the secretion levels of vascular endothelial growth factor, thymosin beta-4, monocyte chemoattractant protein-1, and stromal cell-derived factor-1. LL-37 treatment increased the secretion of these regenerative factors. Moreover, treatment with the conditioned medium of ASCs pre-activated with LL-37 strongly promoted hair growth in vivo.

Conclusions: These findings show that LL-37 increases EGR1 expression and MAPK activation, and that preconditioning of ASCs with LL-37 has a strong potential to promote hair growth in vivo. This study correlates LL-37 with MSC functions (specifically those of ASCs), including cell expansion, cell migration, and paracrine actions, which may be useful in terms of implantation for tissue regeneration.

No MeSH data available.


Related in: MedlinePlus

Preconditioning with LL-37 stimulates hair regeneration in vivo and production of regenerative factors in vitro. a VEGF, (b) TB4, (c) SDF-1α, and (d) MCP-1 mRNA expression was determined by real-time PCR. e VEGF, (f) TB4, (g) SDF-1α, and (h) MCP-1 proteins were detected using the supernatants of confluent cell cultures. ASCs were treated with 20 μg/mL LL-37 for 48 hr, and then the protein levels of VEGF, TB4, SDF-1α, and MCP-1 were analyzed using specific ELISAs. A representative experiment from three independent experiments is shown. Bars represent the mean ± SD. *, P < 0.05 vs. control. i Hair was removed from the backs of C57BL/6 mice and the hair growth rate was monitored for 3 weeks. CM of human ASCs pretreated with or without LL-37 (20 μg/mL) was topically applied daily for up to 18 days to mice with hair loss. Gross views observed by photographs. j Hair growth was scored as described in the Materials and methods section. *, P < 0.05 for control vs. group treated with ASC CM. §, P < 0.05 for group treated with ASC CM vs. group treated with CM of ASCs pre-activated with LL-37. VEGF vascular endothelial growth factor, TB4 thymosin beta-4, SDF-1α stromal cell-derived factor-1α, MCP-1 monocyte chemoattractant protein-1, ASC adipose-derived stromal/stem cell, ELISA enzyme-linked immunosorbent assay, CM conditioned medium
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Fig4: Preconditioning with LL-37 stimulates hair regeneration in vivo and production of regenerative factors in vitro. a VEGF, (b) TB4, (c) SDF-1α, and (d) MCP-1 mRNA expression was determined by real-time PCR. e VEGF, (f) TB4, (g) SDF-1α, and (h) MCP-1 proteins were detected using the supernatants of confluent cell cultures. ASCs were treated with 20 μg/mL LL-37 for 48 hr, and then the protein levels of VEGF, TB4, SDF-1α, and MCP-1 were analyzed using specific ELISAs. A representative experiment from three independent experiments is shown. Bars represent the mean ± SD. *, P < 0.05 vs. control. i Hair was removed from the backs of C57BL/6 mice and the hair growth rate was monitored for 3 weeks. CM of human ASCs pretreated with or without LL-37 (20 μg/mL) was topically applied daily for up to 18 days to mice with hair loss. Gross views observed by photographs. j Hair growth was scored as described in the Materials and methods section. *, P < 0.05 for control vs. group treated with ASC CM. §, P < 0.05 for group treated with ASC CM vs. group treated with CM of ASCs pre-activated with LL-37. VEGF vascular endothelial growth factor, TB4 thymosin beta-4, SDF-1α stromal cell-derived factor-1α, MCP-1 monocyte chemoattractant protein-1, ASC adipose-derived stromal/stem cell, ELISA enzyme-linked immunosorbent assay, CM conditioned medium

Mentions: It was recently reported that EGR1 has a strong paracrine capability and can influence angiogenic factors in ASCs [8, 29]; therefore, we first examined whether LL-37 regulates the expression of various regenerative growth factors and bioactive molecules in ASCs. LL-37 treatment considerably upregulated the mRNA expression of VEGF, TB4, MCP-1, and SDF-1 (Fig. 4a–d). We next confirmed the direct secretion of these growth factors by ASCs using ELISAs. The secretion levels of VEGF, TB4, MCP-1, and SDF-1 were significantly increased by LL-37 treatment (Fig. 4e–h).Fig. 4


LL-37 stimulates the functions of adipose-derived stromal/stem cells via early growth response 1 and the MAPK pathway.

Yang Y, Choi H, Seon M, Cho D, Bang SI - Stem Cell Res Ther (2016)

Preconditioning with LL-37 stimulates hair regeneration in vivo and production of regenerative factors in vitro. a VEGF, (b) TB4, (c) SDF-1α, and (d) MCP-1 mRNA expression was determined by real-time PCR. e VEGF, (f) TB4, (g) SDF-1α, and (h) MCP-1 proteins were detected using the supernatants of confluent cell cultures. ASCs were treated with 20 μg/mL LL-37 for 48 hr, and then the protein levels of VEGF, TB4, SDF-1α, and MCP-1 were analyzed using specific ELISAs. A representative experiment from three independent experiments is shown. Bars represent the mean ± SD. *, P < 0.05 vs. control. i Hair was removed from the backs of C57BL/6 mice and the hair growth rate was monitored for 3 weeks. CM of human ASCs pretreated with or without LL-37 (20 μg/mL) was topically applied daily for up to 18 days to mice with hair loss. Gross views observed by photographs. j Hair growth was scored as described in the Materials and methods section. *, P < 0.05 for control vs. group treated with ASC CM. §, P < 0.05 for group treated with ASC CM vs. group treated with CM of ASCs pre-activated with LL-37. VEGF vascular endothelial growth factor, TB4 thymosin beta-4, SDF-1α stromal cell-derived factor-1α, MCP-1 monocyte chemoattractant protein-1, ASC adipose-derived stromal/stem cell, ELISA enzyme-linked immunosorbent assay, CM conditioned medium
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Related In: Results  -  Collection

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Fig4: Preconditioning with LL-37 stimulates hair regeneration in vivo and production of regenerative factors in vitro. a VEGF, (b) TB4, (c) SDF-1α, and (d) MCP-1 mRNA expression was determined by real-time PCR. e VEGF, (f) TB4, (g) SDF-1α, and (h) MCP-1 proteins were detected using the supernatants of confluent cell cultures. ASCs were treated with 20 μg/mL LL-37 for 48 hr, and then the protein levels of VEGF, TB4, SDF-1α, and MCP-1 were analyzed using specific ELISAs. A representative experiment from three independent experiments is shown. Bars represent the mean ± SD. *, P < 0.05 vs. control. i Hair was removed from the backs of C57BL/6 mice and the hair growth rate was monitored for 3 weeks. CM of human ASCs pretreated with or without LL-37 (20 μg/mL) was topically applied daily for up to 18 days to mice with hair loss. Gross views observed by photographs. j Hair growth was scored as described in the Materials and methods section. *, P < 0.05 for control vs. group treated with ASC CM. §, P < 0.05 for group treated with ASC CM vs. group treated with CM of ASCs pre-activated with LL-37. VEGF vascular endothelial growth factor, TB4 thymosin beta-4, SDF-1α stromal cell-derived factor-1α, MCP-1 monocyte chemoattractant protein-1, ASC adipose-derived stromal/stem cell, ELISA enzyme-linked immunosorbent assay, CM conditioned medium
Mentions: It was recently reported that EGR1 has a strong paracrine capability and can influence angiogenic factors in ASCs [8, 29]; therefore, we first examined whether LL-37 regulates the expression of various regenerative growth factors and bioactive molecules in ASCs. LL-37 treatment considerably upregulated the mRNA expression of VEGF, TB4, MCP-1, and SDF-1 (Fig. 4a–d). We next confirmed the direct secretion of these growth factors by ASCs using ELISAs. The secretion levels of VEGF, TB4, MCP-1, and SDF-1 were significantly increased by LL-37 treatment (Fig. 4e–h).Fig. 4

Bottom Line: To examine the effects of LL-37 on ASC function, cell proliferation and migration were measured by a cell counting kit (CCK-8) and a Transwell migration assay.Activation of mitogen-activated protein kinases (MAPKs) was essential not only for LL-37-enhanced ASC proliferation and migration but also EGR1 expression; treatment with a specific inhibitor of extracellular signal-regulated kinase, p38, or c-Jun N-terminal kinase blocked the stimulatory effect of LL-37.This study correlates LL-37 with MSC functions (specifically those of ASCs), including cell expansion, cell migration, and paracrine actions, which may be useful in terms of implantation for tissue regeneration.

View Article: PubMed Central - PubMed

Affiliation: Department of Plastic Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.

ABSTRACT

Background: LL-37 is a naturally occurring antimicrobial peptide found in the wound bed and assists wound repair. No published study has characterized the role of LL-37 in the function(s) of human mesenchymal stem cells (MSCs). This study investigated the functions of adipose-derived stromal/stem cells (ASCs) activated by LL-37 by performing both in vitro assays with cultured cells and in vivo assays with C57BL/6 mice with hair loss.

Methods: Human ASCs were isolated from healthy donors with written informed consent. To examine the effects of LL-37 on ASC function, cell proliferation and migration were measured by a cell counting kit (CCK-8) and a Transwell migration assay. Early growth response 1 (EGR1) mRNA expression was determined by microarray and real-time PCR analyses. The protein levels of EGR1 and regenerative factors were analyzed by specific enzyme-linked immunosorbent assays and western blotting.

Results: LL-37 treatment enhanced the proliferation and migration of human ASCs expressing formyl peptide receptor like-1. Microarray and real-time PCR data showed that EGR1 expression was rapidly and significantly increased by LL-37 treatment. LL-37 treatment also enhanced the production of EGR1. Moreover, small interfering RNA-mediated knockdown of EGR1 inhibited LL-37-enhanced ASC proliferation and migration. Activation of mitogen-activated protein kinases (MAPKs) was essential not only for LL-37-enhanced ASC proliferation and migration but also EGR1 expression; treatment with a specific inhibitor of extracellular signal-regulated kinase, p38, or c-Jun N-terminal kinase blocked the stimulatory effect of LL-37. EGR1 has a strong paracrine capability and can influence angiogenic factors in ASCs; therefore, we evaluated the secretion levels of vascular endothelial growth factor, thymosin beta-4, monocyte chemoattractant protein-1, and stromal cell-derived factor-1. LL-37 treatment increased the secretion of these regenerative factors. Moreover, treatment with the conditioned medium of ASCs pre-activated with LL-37 strongly promoted hair growth in vivo.

Conclusions: These findings show that LL-37 increases EGR1 expression and MAPK activation, and that preconditioning of ASCs with LL-37 has a strong potential to promote hair growth in vivo. This study correlates LL-37 with MSC functions (specifically those of ASCs), including cell expansion, cell migration, and paracrine actions, which may be useful in terms of implantation for tissue regeneration.

No MeSH data available.


Related in: MedlinePlus