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Netrin-1-Induced Stem Cell Bioactivity Contributes to the Regeneration of Injured Tissues via the Lipid Raft-Dependent Integrin α 6 β 4 Signaling Pathway

View Article: PubMed Central - PubMed

ABSTRACT

Netrin-1 (Ntn-1) is a multifunctional neuronal signaling molecule; however, its physiological significance, which improves the tissue-regeneration capacity of stem cells, has not been characterized. In the present study, we investigate the mechanism by which Ntn-1 promotes the proliferation of hUCB-MSCs with regard to the regeneration of injured tissues. We found that Ntn-1 induces the proliferation of hUCB-MSCs mainly via Inα6β4 coupled with c-Src. Ntn-1 induced the recruitment of NADPH oxidases and Rac1 into membrane lipid rafts to facilitate ROS production. The Inα6β4 signaling of Ntn-1 through ROS production is uniquely mediated by the activation of SP1 for cell cycle progression and the transcriptional occupancy of SP1 on the VEGF promoter. Moreover, Ntn-1 has the ability to induce the F-actin reorganization of hUCB-MSCs via the Inα6β4 signaling pathway. In an in vivo model, transplantation of hUCB-MSCs pre-treated with Ntn-1 enhanced the skin wound healing process, where relatively more angiogenesis was detected. The potential effect of Ntn-1 on angiogenesis is further verified by the mouse hindlimb ischemia model, where the pre-activation of hUCB-MSCs with Ntn-1 significantly improved vascular regeneration. These results demonstrate that Ntn-1 plays an important role in the tissue regeneration process of hUCB-MSC via the lipid raft-mediated Inα6β4 signaling pathway.

No MeSH data available.


Related in: MedlinePlus

Effects of Ntn-1 on mouse skin wound healing.(A) Representative gross images on skin wound healing on day 0, 5, 9, and 12 are shown (left panel). Mouse skin wounds were made by 6-mm-diameter biopsy punch and treated with vehicle, Ntn-1, hUCB-MSC + vehicle, and hUCB-MSC + Ntn-1, respectively. (B) Quantifications of wound sizes relative to original wound size for 15 days are shown. Data represent the mean ± S.E. n = 7. *P < 0.05 vs. vehicle alone. #P < 0.05 vs. hUCB-MSC + vehicle. (C) Representative wound tissues stained with H&E on day 12 are shown (Top panel). n = 7. Scale bars, 100 μm. Abbreviations: Ep, epidermis; W, wound bed; CL, cornified layer. Histological scores in re-epithelialization were quantified according to the Supplementary Table 1 (right panel). (D) Representative images of blood vessels in wounds on day 12. n = 7 (left panel). Vessel densities relative to the group treated with Vehicle alone were quantified by using Image J program (middle panel). ROD, relative optical density. Histological scores in angiogenesis were quantified according to the Supplementary Table 1 (right panel). (E) Upon postoperative day 12, Miles assay was performed to measure blood vessel permeability. Wound tissue samples (100 mg) were incubated with 500 μL formamide to release the extravagated Evans blue for 24 h. Optical density was measured at 610 nm and the measurements converted into ng dye extravagated per mg tissue (n = 7). $P < 0.01 versus Normal. *P < 0.01 versus vehicle. #P < 0.05 versus hUCB-MSCs alone.
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f6: Effects of Ntn-1 on mouse skin wound healing.(A) Representative gross images on skin wound healing on day 0, 5, 9, and 12 are shown (left panel). Mouse skin wounds were made by 6-mm-diameter biopsy punch and treated with vehicle, Ntn-1, hUCB-MSC + vehicle, and hUCB-MSC + Ntn-1, respectively. (B) Quantifications of wound sizes relative to original wound size for 15 days are shown. Data represent the mean ± S.E. n = 7. *P < 0.05 vs. vehicle alone. #P < 0.05 vs. hUCB-MSC + vehicle. (C) Representative wound tissues stained with H&E on day 12 are shown (Top panel). n = 7. Scale bars, 100 μm. Abbreviations: Ep, epidermis; W, wound bed; CL, cornified layer. Histological scores in re-epithelialization were quantified according to the Supplementary Table 1 (right panel). (D) Representative images of blood vessels in wounds on day 12. n = 7 (left panel). Vessel densities relative to the group treated with Vehicle alone were quantified by using Image J program (middle panel). ROD, relative optical density. Histological scores in angiogenesis were quantified according to the Supplementary Table 1 (right panel). (E) Upon postoperative day 12, Miles assay was performed to measure blood vessel permeability. Wound tissue samples (100 mg) were incubated with 500 μL formamide to release the extravagated Evans blue for 24 h. Optical density was measured at 610 nm and the measurements converted into ng dye extravagated per mg tissue (n = 7). $P < 0.01 versus Normal. *P < 0.01 versus vehicle. #P < 0.05 versus hUCB-MSCs alone.

Mentions: To confirm the functional roles of Ntn-1 in promoting hUCB-MSC proliferation, we further explored the effect of hUCB-MSC pre-treated with 50 ng/mL of Ntn-1 on skin wound healing in mice. On days 9 and 12, the group given hUCB-MSC + Ntn-1 showed an increased extent of wound closure compared to the other groups (Fig. 6A,B). However, our data revealed there was no statistical difference between Ntn-1 alone and a vehicle, although spontaneous wound healing was observed. A histologic examination on day 12 showed that the wound bed was still not completely covered with epidermis in mice treated with the vehicle or Ntn-1 alone (Fig. 6C). However, the hUCB-MSC transplantation groups showed increased re-epithelialization from a mechanical skin wound, but the mice group that received hUCB-MSC + Ntn-1 showed enhanced levels of the wound closure, granulation, and re-epithelialization at mouse skin wound sites, resulting in nearly complete restoration of the epidermis. There were no additional differences between UCB-MSC + Ntn-1 and UCB-MSC on day 15. Histological scores for re-epithelialization and angiogenesis were quantified according to Supplementary Table 1. We found that the group given either hUCB-MSC + Ntn-1 or hUCB-MSC showed significantly augmented levels of re-epithelialization compared to the group treated with the vehicle or Ntn-1 alone (Fig. 6C, bottom panel). In addition, the transplantation of hUCB-MSC + Ntn-1 showed an enhanced level of epidermal organization compared to that by hUCB-MSC alone. Consistently, our results revealed that the group given hUCB-MSC + Ntn-1 showed significantly increased blood vessel density and angiogenesis, with the number of blood vessels toward the wound surface greater than that by hUCB-MSC (Fig. 6D). There were no statistical differences between the groups treated with Ntn-1 alone and the vehicle in terms of wound healing and angiogenic capacity. We performed an additional experiment to assess the degree of blood vessel permeability using a Miles assay, measuring the amount of the extravasation of Evans blue. As shown in Fig. 6E, our results revealed that the level of Evans blue extravasation in mice given hUCB-MSC + Ntn-1 was significantly lower than that by hUCB-MSC alone, resulting in the nearly complete restoration of blood vessel permeability. There were no significant differences between the blood vessel permeability of mice treated with a vehicle or with Ntn-1 alone.


Netrin-1-Induced Stem Cell Bioactivity Contributes to the Regeneration of Injured Tissues via the Lipid Raft-Dependent Integrin α 6 β 4 Signaling Pathway
Effects of Ntn-1 on mouse skin wound healing.(A) Representative gross images on skin wound healing on day 0, 5, 9, and 12 are shown (left panel). Mouse skin wounds were made by 6-mm-diameter biopsy punch and treated with vehicle, Ntn-1, hUCB-MSC + vehicle, and hUCB-MSC + Ntn-1, respectively. (B) Quantifications of wound sizes relative to original wound size for 15 days are shown. Data represent the mean ± S.E. n = 7. *P < 0.05 vs. vehicle alone. #P < 0.05 vs. hUCB-MSC + vehicle. (C) Representative wound tissues stained with H&E on day 12 are shown (Top panel). n = 7. Scale bars, 100 μm. Abbreviations: Ep, epidermis; W, wound bed; CL, cornified layer. Histological scores in re-epithelialization were quantified according to the Supplementary Table 1 (right panel). (D) Representative images of blood vessels in wounds on day 12. n = 7 (left panel). Vessel densities relative to the group treated with Vehicle alone were quantified by using Image J program (middle panel). ROD, relative optical density. Histological scores in angiogenesis were quantified according to the Supplementary Table 1 (right panel). (E) Upon postoperative day 12, Miles assay was performed to measure blood vessel permeability. Wound tissue samples (100 mg) were incubated with 500 μL formamide to release the extravagated Evans blue for 24 h. Optical density was measured at 610 nm and the measurements converted into ng dye extravagated per mg tissue (n = 7). $P < 0.01 versus Normal. *P < 0.01 versus vehicle. #P < 0.05 versus hUCB-MSCs alone.
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f6: Effects of Ntn-1 on mouse skin wound healing.(A) Representative gross images on skin wound healing on day 0, 5, 9, and 12 are shown (left panel). Mouse skin wounds were made by 6-mm-diameter biopsy punch and treated with vehicle, Ntn-1, hUCB-MSC + vehicle, and hUCB-MSC + Ntn-1, respectively. (B) Quantifications of wound sizes relative to original wound size for 15 days are shown. Data represent the mean ± S.E. n = 7. *P < 0.05 vs. vehicle alone. #P < 0.05 vs. hUCB-MSC + vehicle. (C) Representative wound tissues stained with H&E on day 12 are shown (Top panel). n = 7. Scale bars, 100 μm. Abbreviations: Ep, epidermis; W, wound bed; CL, cornified layer. Histological scores in re-epithelialization were quantified according to the Supplementary Table 1 (right panel). (D) Representative images of blood vessels in wounds on day 12. n = 7 (left panel). Vessel densities relative to the group treated with Vehicle alone were quantified by using Image J program (middle panel). ROD, relative optical density. Histological scores in angiogenesis were quantified according to the Supplementary Table 1 (right panel). (E) Upon postoperative day 12, Miles assay was performed to measure blood vessel permeability. Wound tissue samples (100 mg) were incubated with 500 μL formamide to release the extravagated Evans blue for 24 h. Optical density was measured at 610 nm and the measurements converted into ng dye extravagated per mg tissue (n = 7). $P < 0.01 versus Normal. *P < 0.01 versus vehicle. #P < 0.05 versus hUCB-MSCs alone.
Mentions: To confirm the functional roles of Ntn-1 in promoting hUCB-MSC proliferation, we further explored the effect of hUCB-MSC pre-treated with 50 ng/mL of Ntn-1 on skin wound healing in mice. On days 9 and 12, the group given hUCB-MSC + Ntn-1 showed an increased extent of wound closure compared to the other groups (Fig. 6A,B). However, our data revealed there was no statistical difference between Ntn-1 alone and a vehicle, although spontaneous wound healing was observed. A histologic examination on day 12 showed that the wound bed was still not completely covered with epidermis in mice treated with the vehicle or Ntn-1 alone (Fig. 6C). However, the hUCB-MSC transplantation groups showed increased re-epithelialization from a mechanical skin wound, but the mice group that received hUCB-MSC + Ntn-1 showed enhanced levels of the wound closure, granulation, and re-epithelialization at mouse skin wound sites, resulting in nearly complete restoration of the epidermis. There were no additional differences between UCB-MSC + Ntn-1 and UCB-MSC on day 15. Histological scores for re-epithelialization and angiogenesis were quantified according to Supplementary Table 1. We found that the group given either hUCB-MSC + Ntn-1 or hUCB-MSC showed significantly augmented levels of re-epithelialization compared to the group treated with the vehicle or Ntn-1 alone (Fig. 6C, bottom panel). In addition, the transplantation of hUCB-MSC + Ntn-1 showed an enhanced level of epidermal organization compared to that by hUCB-MSC alone. Consistently, our results revealed that the group given hUCB-MSC + Ntn-1 showed significantly increased blood vessel density and angiogenesis, with the number of blood vessels toward the wound surface greater than that by hUCB-MSC (Fig. 6D). There were no statistical differences between the groups treated with Ntn-1 alone and the vehicle in terms of wound healing and angiogenic capacity. We performed an additional experiment to assess the degree of blood vessel permeability using a Miles assay, measuring the amount of the extravasation of Evans blue. As shown in Fig. 6E, our results revealed that the level of Evans blue extravasation in mice given hUCB-MSC + Ntn-1 was significantly lower than that by hUCB-MSC alone, resulting in the nearly complete restoration of blood vessel permeability. There were no significant differences between the blood vessel permeability of mice treated with a vehicle or with Ntn-1 alone.

View Article: PubMed Central - PubMed

ABSTRACT

Netrin-1 (Ntn-1) is a multifunctional neuronal signaling molecule; however, its physiological significance, which improves the tissue-regeneration capacity of stem cells, has not been characterized. In the present study, we investigate the mechanism by which Ntn-1 promotes the proliferation of hUCB-MSCs with regard to the regeneration of injured tissues. We found that Ntn-1 induces the proliferation of hUCB-MSCs mainly via In&alpha;6&beta;4 coupled with c-Src. Ntn-1 induced the recruitment of NADPH oxidases and Rac1 into membrane lipid rafts to facilitate ROS production. The In&alpha;6&beta;4 signaling of Ntn-1 through ROS production is uniquely mediated by the activation of SP1 for cell cycle progression and the transcriptional occupancy of SP1 on the VEGF promoter. Moreover, Ntn-1 has the ability to induce the F-actin reorganization of hUCB-MSCs via the In&alpha;6&beta;4 signaling pathway. In an in vivo model, transplantation of hUCB-MSCs pre-treated with Ntn-1 enhanced the skin wound healing process, where relatively more angiogenesis was detected. The potential effect of Ntn-1 on angiogenesis is further verified by the mouse hindlimb ischemia model, where the pre-activation of hUCB-MSCs with Ntn-1 significantly improved vascular regeneration. These results demonstrate that Ntn-1 plays an important role in the tissue regeneration process of hUCB-MSC via the lipid raft-mediated In&alpha;6&beta;4 signaling pathway.

No MeSH data available.


Related in: MedlinePlus