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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.

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Related in: MedlinePlus

Effects of Ntn-1 on vascular regeneration in mouse hindlimb ischemia model.(A) The ratio of blood perfusion (blood flow in the left ischemic limb/blood flow in the right non-ischemic limb) was measured using Laser Doppler perfusion imaging analysis in the ischemic limbs of nude mice injected with PBS, hUCB-MSC, or hUCB-MSC + Ntn-1 on postoperative days 0, 5, 10, 15, 20, and 25 (left and middle panels). Gross morphologies of mice hindlimb on postoperative day 15 are shown (right panel). Data represent the mean ± SE. n = 5. **P < 0.01 vs. vehicle, ##P < 0.01 vs. hUCB-MSC + Ntn-1. (B) The survival of transplanted hUCB-MSCs was assessed by immunofluorescent staining with Cleaved Caspase-3 (red) and anti-human nuclei antigen (HNA, green) antibodies. DAPI was used as nuclear control (blue). n = 3. Scale bar, 50 μm. Apoptotic cells were quantified as the number of HNA- and Cleaved Caspase-3-positive cells. (C) The level of apoptosis-related proteins in the mice treated with vehicle, hUCB-MSCs, and hUCB-MSCs + Ntn-1 on 3 days was determined by Western blot with Bcl-2, Bax, and Cleaved Caspase-3 antibodies. n = 3. *P < 0.01 versus vehicle. #P < 0.05 versus hUCB-MSCs alone. Data represent the means ± S.E. Upon postoperative day 15, ischemic limb tissue samples were immunostained with an anti-CD31 (D) and anti-α-SMA (E) for assessment of capillary density and arteriole density, respectively. n = 3. Scale bar, 50 μm. The density of capillary and arteriole was quantified as the number of CD31- and α-SMA-positive cells. $P < 0.01 versus Normal. *P < 0.01 versus vehicle. #P < 0.05 versus hUCB-MSCs alone. (F) The level of FGF and VEGF in the mice treated with vehicle, hUCB-MSCs, and hUCB-MSCs + Ntn-1 for 15 days was determined by Western blot. *P < 0.01 versus vehicle. #P < 0.05 versus hUCB-MSCs alone. Data represent the means ± S.E. n = 3. (G) A hypothetical model for Ntn-1-induced signaling pathway in promoting mouse wound healing and vascular regeneration. (C,F) ROD is the abbreviation for relative optical density.
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f7: Effects of Ntn-1 on vascular regeneration in mouse hindlimb ischemia model.(A) The ratio of blood perfusion (blood flow in the left ischemic limb/blood flow in the right non-ischemic limb) was measured using Laser Doppler perfusion imaging analysis in the ischemic limbs of nude mice injected with PBS, hUCB-MSC, or hUCB-MSC + Ntn-1 on postoperative days 0, 5, 10, 15, 20, and 25 (left and middle panels). Gross morphologies of mice hindlimb on postoperative day 15 are shown (right panel). Data represent the mean ± SE. n = 5. **P < 0.01 vs. vehicle, ##P < 0.01 vs. hUCB-MSC + Ntn-1. (B) The survival of transplanted hUCB-MSCs was assessed by immunofluorescent staining with Cleaved Caspase-3 (red) and anti-human nuclei antigen (HNA, green) antibodies. DAPI was used as nuclear control (blue). n = 3. Scale bar, 50 μm. Apoptotic cells were quantified as the number of HNA- and Cleaved Caspase-3-positive cells. (C) The level of apoptosis-related proteins in the mice treated with vehicle, hUCB-MSCs, and hUCB-MSCs + Ntn-1 on 3 days was determined by Western blot with Bcl-2, Bax, and Cleaved Caspase-3 antibodies. n = 3. *P < 0.01 versus vehicle. #P < 0.05 versus hUCB-MSCs alone. Data represent the means ± S.E. Upon postoperative day 15, ischemic limb tissue samples were immunostained with an anti-CD31 (D) and anti-α-SMA (E) for assessment of capillary density and arteriole density, respectively. n = 3. Scale bar, 50 μm. The density of capillary and arteriole was quantified as the number of CD31- and α-SMA-positive cells. $P < 0.01 versus Normal. *P < 0.01 versus vehicle. #P < 0.05 versus hUCB-MSCs alone. (F) The level of FGF and VEGF in the mice treated with vehicle, hUCB-MSCs, and hUCB-MSCs + Ntn-1 for 15 days was determined by Western blot. *P < 0.01 versus vehicle. #P < 0.05 versus hUCB-MSCs alone. Data represent the means ± S.E. n = 3. (G) A hypothetical model for Ntn-1-induced signaling pathway in promoting mouse wound healing and vascular regeneration. (C,F) ROD is the abbreviation for relative optical density.

Mentions: We also attempted to determine the angiogenic capacity of Ntn-1 on hindlimb ischemia in the mouse model using a laser Doppler imager (Fig. 7A). Upon postoperative day 25, progressive recoveries of blood perfusion and the nearly complete restoration of the hindlimb were observed in the hUCB-MSC transplantation groups. However, the group given hUCB-MSC + Ntn-1 showed an enhanced ischemic/normal hindlimb blood perfusion ratio on postoperative days 15 and 20 compared to the ratio with hUCB-MSC alone. There were no significant differences between the blood perfusion ratio of mice treated with a vehicle or with 50 ng/mL of Ntn-1 alone (data not shown). Upon postoperative day 3, importantly, Ntn-1 significantly reduced the number of apoptotic cells corresponding to the transplanted hUCB-MSC (Fig. 7B). Consistently, the group given hUCB-MSC + Ntn-1 showed an increased Bcl-2 amount but decreased levels of Bax as well as cleaved-caspase-3 compared to the results by hUCB-MSC alone, suggesting that Ntn-1 enhances the survival of transplanted hUCB-MSCs in ischemic tissues (Fig. 7C). To evaluate the role of hUCB-MSC pre-treated with Ntn-1 in neovascularization, CD31-positive capillaries and α-SMA-positive arterioles were quantified in ischemic tissues by immunohistochemistry on postoperative day 15. The results of immunofluorescent staining for CD31 (Fig. 7D) and α-SMA (Fig. 7E) revealed that the number of capillaries and arterioles were significantly increased in the group that received hUCB-MSC + Ntn-1 compared to that by hUCB-MSC alone. Moreover, hUCB-MSCs activated by Ntn-1 were shown to enhance the levels of FGF and VEGF respectively, compared to the results by hUCB-MSC alone (Fig. 7F). These results indicate that the pre-activation of hUCB-MSC with Ntn-1 facilitates vascular regeneration in ischemic tissues.


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 vascular regeneration in mouse hindlimb ischemia model.(A) The ratio of blood perfusion (blood flow in the left ischemic limb/blood flow in the right non-ischemic limb) was measured using Laser Doppler perfusion imaging analysis in the ischemic limbs of nude mice injected with PBS, hUCB-MSC, or hUCB-MSC + Ntn-1 on postoperative days 0, 5, 10, 15, 20, and 25 (left and middle panels). Gross morphologies of mice hindlimb on postoperative day 15 are shown (right panel). Data represent the mean ± SE. n = 5. **P < 0.01 vs. vehicle, ##P < 0.01 vs. hUCB-MSC + Ntn-1. (B) The survival of transplanted hUCB-MSCs was assessed by immunofluorescent staining with Cleaved Caspase-3 (red) and anti-human nuclei antigen (HNA, green) antibodies. DAPI was used as nuclear control (blue). n = 3. Scale bar, 50 μm. Apoptotic cells were quantified as the number of HNA- and Cleaved Caspase-3-positive cells. (C) The level of apoptosis-related proteins in the mice treated with vehicle, hUCB-MSCs, and hUCB-MSCs + Ntn-1 on 3 days was determined by Western blot with Bcl-2, Bax, and Cleaved Caspase-3 antibodies. n = 3. *P < 0.01 versus vehicle. #P < 0.05 versus hUCB-MSCs alone. Data represent the means ± S.E. Upon postoperative day 15, ischemic limb tissue samples were immunostained with an anti-CD31 (D) and anti-α-SMA (E) for assessment of capillary density and arteriole density, respectively. n = 3. Scale bar, 50 μm. The density of capillary and arteriole was quantified as the number of CD31- and α-SMA-positive cells. $P < 0.01 versus Normal. *P < 0.01 versus vehicle. #P < 0.05 versus hUCB-MSCs alone. (F) The level of FGF and VEGF in the mice treated with vehicle, hUCB-MSCs, and hUCB-MSCs + Ntn-1 for 15 days was determined by Western blot. *P < 0.01 versus vehicle. #P < 0.05 versus hUCB-MSCs alone. Data represent the means ± S.E. n = 3. (G) A hypothetical model for Ntn-1-induced signaling pathway in promoting mouse wound healing and vascular regeneration. (C,F) ROD is the abbreviation for relative optical density.
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f7: Effects of Ntn-1 on vascular regeneration in mouse hindlimb ischemia model.(A) The ratio of blood perfusion (blood flow in the left ischemic limb/blood flow in the right non-ischemic limb) was measured using Laser Doppler perfusion imaging analysis in the ischemic limbs of nude mice injected with PBS, hUCB-MSC, or hUCB-MSC + Ntn-1 on postoperative days 0, 5, 10, 15, 20, and 25 (left and middle panels). Gross morphologies of mice hindlimb on postoperative day 15 are shown (right panel). Data represent the mean ± SE. n = 5. **P < 0.01 vs. vehicle, ##P < 0.01 vs. hUCB-MSC + Ntn-1. (B) The survival of transplanted hUCB-MSCs was assessed by immunofluorescent staining with Cleaved Caspase-3 (red) and anti-human nuclei antigen (HNA, green) antibodies. DAPI was used as nuclear control (blue). n = 3. Scale bar, 50 μm. Apoptotic cells were quantified as the number of HNA- and Cleaved Caspase-3-positive cells. (C) The level of apoptosis-related proteins in the mice treated with vehicle, hUCB-MSCs, and hUCB-MSCs + Ntn-1 on 3 days was determined by Western blot with Bcl-2, Bax, and Cleaved Caspase-3 antibodies. n = 3. *P < 0.01 versus vehicle. #P < 0.05 versus hUCB-MSCs alone. Data represent the means ± S.E. Upon postoperative day 15, ischemic limb tissue samples were immunostained with an anti-CD31 (D) and anti-α-SMA (E) for assessment of capillary density and arteriole density, respectively. n = 3. Scale bar, 50 μm. The density of capillary and arteriole was quantified as the number of CD31- and α-SMA-positive cells. $P < 0.01 versus Normal. *P < 0.01 versus vehicle. #P < 0.05 versus hUCB-MSCs alone. (F) The level of FGF and VEGF in the mice treated with vehicle, hUCB-MSCs, and hUCB-MSCs + Ntn-1 for 15 days was determined by Western blot. *P < 0.01 versus vehicle. #P < 0.05 versus hUCB-MSCs alone. Data represent the means ± S.E. n = 3. (G) A hypothetical model for Ntn-1-induced signaling pathway in promoting mouse wound healing and vascular regeneration. (C,F) ROD is the abbreviation for relative optical density.
Mentions: We also attempted to determine the angiogenic capacity of Ntn-1 on hindlimb ischemia in the mouse model using a laser Doppler imager (Fig. 7A). Upon postoperative day 25, progressive recoveries of blood perfusion and the nearly complete restoration of the hindlimb were observed in the hUCB-MSC transplantation groups. However, the group given hUCB-MSC + Ntn-1 showed an enhanced ischemic/normal hindlimb blood perfusion ratio on postoperative days 15 and 20 compared to the ratio with hUCB-MSC alone. There were no significant differences between the blood perfusion ratio of mice treated with a vehicle or with 50 ng/mL of Ntn-1 alone (data not shown). Upon postoperative day 3, importantly, Ntn-1 significantly reduced the number of apoptotic cells corresponding to the transplanted hUCB-MSC (Fig. 7B). Consistently, the group given hUCB-MSC + Ntn-1 showed an increased Bcl-2 amount but decreased levels of Bax as well as cleaved-caspase-3 compared to the results by hUCB-MSC alone, suggesting that Ntn-1 enhances the survival of transplanted hUCB-MSCs in ischemic tissues (Fig. 7C). To evaluate the role of hUCB-MSC pre-treated with Ntn-1 in neovascularization, CD31-positive capillaries and α-SMA-positive arterioles were quantified in ischemic tissues by immunohistochemistry on postoperative day 15. The results of immunofluorescent staining for CD31 (Fig. 7D) and α-SMA (Fig. 7E) revealed that the number of capillaries and arterioles were significantly increased in the group that received hUCB-MSC + Ntn-1 compared to that by hUCB-MSC alone. Moreover, hUCB-MSCs activated by Ntn-1 were shown to enhance the levels of FGF and VEGF respectively, compared to the results by hUCB-MSC alone (Fig. 7F). These results indicate that the pre-activation of hUCB-MSC with Ntn-1 facilitates vascular regeneration in ischemic tissues.

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