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Macrophage-Induced Blood Vessels Guide Schwann Cell-Mediated Regeneration of Peripheral Nerves.

Cattin AL, Burden JJ, Van Emmenis L, Mackenzie FE, Hoving JJ, Garcia Calavia N, Guo Y, McLaughlin M, Rosenberg LH, Quereda V, Jamecna D, Napoli I, Parrinello S, Enver T, Ruhrberg C, Lloyd AC - Cell (2015)

Bottom Line: Here we show that blood vessels direct the migrating cords of Schwann cells.Importantly, disrupting the organization of the newly formed blood vessels in vivo, either by inhibiting the angiogenic signal or by re-orienting them, compromises Schwann cell directionality resulting in defective nerve repair.This study provides important insights into how the choreography of multiple cell-types is required for the regeneration of an adult tissue.

View Article: PubMed Central - PubMed

Affiliation: MRC Laboratory for Molecular Cell Biology, UCL, Gower Street, London WC1E 6BT, UK.

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Inactivation of Vegfa in Macrophages Inhibits Vascularization of the Nerve Bridge after Nerve Transection(A) Representative images of longitudinal sections of injured sciatic nerves from Vegfafl/fl (control), Vegfafl/flLysmCre, and Vegfafl/flTie2-Cre mice, Day 5 after transection, immunostained to detect ECs (CD31+, red) and SCs (p75NTR+, green). Scale bar, 50 μm.(B) Quantification of (A) showing the proportion of CD31-positive area per bridge area and shows that the vascularization of the bridge is significantly reduced in mutants animals (n = 5).(C) Quantification of (A) showing the area of SC influx from the proximal and distal stumps in Vegfafl/fl versus Vegfafl/flTie2-Cre animals (n = 5).(D) Representative images of longitudinal sections of injured sciatic nerves from wild-type that have received bone marrow from Vegfafl/fl (control) or Vegfafl/flTie2-Cre mice immunostained to detect ECs (CD31+, red), SCs (p75NTR+, green), and axons (NF+, blue), Day 5 after transection. Scale bar, 100 μm.(E) Quantification of (D) showing the proportion of CD31-positive area per bridge area (n = 3 for each group).(F) Representative images of longitudinal sections of injured sciatic nerves of Vegfafl/flTie2-Cre mice, Day 5 after transection following injection of PBS or VEGF-A188 into the bridges at Day 4. Scale bar, 100 μm.(G and H) Quantification of (F) showing the blood vessel density (G) or area of infiltrating SCs (H) (n = 4). For reconstruction of longitudinal sections shown in (A), (D) and (F), multiple images from the same sample were acquired using the same microscope settings.Graphs show mean value ± SEM. See also Figure S6.
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fig6: Inactivation of Vegfa in Macrophages Inhibits Vascularization of the Nerve Bridge after Nerve Transection(A) Representative images of longitudinal sections of injured sciatic nerves from Vegfafl/fl (control), Vegfafl/flLysmCre, and Vegfafl/flTie2-Cre mice, Day 5 after transection, immunostained to detect ECs (CD31+, red) and SCs (p75NTR+, green). Scale bar, 50 μm.(B) Quantification of (A) showing the proportion of CD31-positive area per bridge area and shows that the vascularization of the bridge is significantly reduced in mutants animals (n = 5).(C) Quantification of (A) showing the area of SC influx from the proximal and distal stumps in Vegfafl/fl versus Vegfafl/flTie2-Cre animals (n = 5).(D) Representative images of longitudinal sections of injured sciatic nerves from wild-type that have received bone marrow from Vegfafl/fl (control) or Vegfafl/flTie2-Cre mice immunostained to detect ECs (CD31+, red), SCs (p75NTR+, green), and axons (NF+, blue), Day 5 after transection. Scale bar, 100 μm.(E) Quantification of (D) showing the proportion of CD31-positive area per bridge area (n = 3 for each group).(F) Representative images of longitudinal sections of injured sciatic nerves of Vegfafl/flTie2-Cre mice, Day 5 after transection following injection of PBS or VEGF-A188 into the bridges at Day 4. Scale bar, 100 μm.(G and H) Quantification of (F) showing the blood vessel density (G) or area of infiltrating SCs (H) (n = 4). For reconstruction of longitudinal sections shown in (A), (D) and (F), multiple images from the same sample were acquired using the same microscope settings.Graphs show mean value ± SEM. See also Figure S6.

Mentions: Macrophages have been shown to promote angiogenesis (Fantin et al., 2010; Pollard, 2009) and autocrine VEGF-A signaling helps to maintain the health of ECs (Lee et al., 2007). We therefore analyzed the vasculature of uninjured nerves from all genotypes but found no differences (Figures S6D and S6E). Remarkably however, nerves from both mutant animals showed a reduction in the vascularization of the bridge following injury (Figure 6A). The extent of the inhibition was more dramatic in the Vegfafl/flTie2-Cre mice, consistent with the greater degree of recombination in these animals, with very few blood vessels detectable within the bridge (Figure 6B). However, there was also a significant decrease in the Vegfafl/flLysmCre mice (Figure 6B). Strikingly, SCs remained in the stumps of the Tie2-Cre mutant animals, consistent with a requirement for blood vessels to provide a “track” for the SCs to enter the bridge (Figure 6C). To confirm this was not due to loss of VEGF-A expression in ECs we (1) performed bone marrow transplant experiments from Vegfafl/flTie2-Cre and control Vegfafl/fl litter-mates into WT mice and found similar defective entry of blood vessels into the bridges of the mice receiving the mutant bone marrow, confirming that cells derived from hematopoietic-stem cells were responsible for the defect (Figures 6D, 6E, S6F, and S6G); and (2) performed rescue experiments in the Vegfafl/flTie2-Cre mice. We injected either VEGF-A or PBS into the bridges of Vegfafl/flTie2-Cre mice on day 4 and found that VEGF-A was able to rescue EC migration into the bridge and that SCs and axons migrated along these blood vessels (Figures 6F–6H). These results show that ECs deleted for VEGF-A are able to migrate and survive in the bridge and also provide a substrate for SC migration. Together, these results show that macrophages in the bridge secrete VEGF-A to enable the formation of a polarized endothelial scaffold that can direct SCs out of the nerve stumps and across the bridge.


Macrophage-Induced Blood Vessels Guide Schwann Cell-Mediated Regeneration of Peripheral Nerves.

Cattin AL, Burden JJ, Van Emmenis L, Mackenzie FE, Hoving JJ, Garcia Calavia N, Guo Y, McLaughlin M, Rosenberg LH, Quereda V, Jamecna D, Napoli I, Parrinello S, Enver T, Ruhrberg C, Lloyd AC - Cell (2015)

Inactivation of Vegfa in Macrophages Inhibits Vascularization of the Nerve Bridge after Nerve Transection(A) Representative images of longitudinal sections of injured sciatic nerves from Vegfafl/fl (control), Vegfafl/flLysmCre, and Vegfafl/flTie2-Cre mice, Day 5 after transection, immunostained to detect ECs (CD31+, red) and SCs (p75NTR+, green). Scale bar, 50 μm.(B) Quantification of (A) showing the proportion of CD31-positive area per bridge area and shows that the vascularization of the bridge is significantly reduced in mutants animals (n = 5).(C) Quantification of (A) showing the area of SC influx from the proximal and distal stumps in Vegfafl/fl versus Vegfafl/flTie2-Cre animals (n = 5).(D) Representative images of longitudinal sections of injured sciatic nerves from wild-type that have received bone marrow from Vegfafl/fl (control) or Vegfafl/flTie2-Cre mice immunostained to detect ECs (CD31+, red), SCs (p75NTR+, green), and axons (NF+, blue), Day 5 after transection. Scale bar, 100 μm.(E) Quantification of (D) showing the proportion of CD31-positive area per bridge area (n = 3 for each group).(F) Representative images of longitudinal sections of injured sciatic nerves of Vegfafl/flTie2-Cre mice, Day 5 after transection following injection of PBS or VEGF-A188 into the bridges at Day 4. Scale bar, 100 μm.(G and H) Quantification of (F) showing the blood vessel density (G) or area of infiltrating SCs (H) (n = 4). For reconstruction of longitudinal sections shown in (A), (D) and (F), multiple images from the same sample were acquired using the same microscope settings.Graphs show mean value ± SEM. See also Figure S6.
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fig6: Inactivation of Vegfa in Macrophages Inhibits Vascularization of the Nerve Bridge after Nerve Transection(A) Representative images of longitudinal sections of injured sciatic nerves from Vegfafl/fl (control), Vegfafl/flLysmCre, and Vegfafl/flTie2-Cre mice, Day 5 after transection, immunostained to detect ECs (CD31+, red) and SCs (p75NTR+, green). Scale bar, 50 μm.(B) Quantification of (A) showing the proportion of CD31-positive area per bridge area and shows that the vascularization of the bridge is significantly reduced in mutants animals (n = 5).(C) Quantification of (A) showing the area of SC influx from the proximal and distal stumps in Vegfafl/fl versus Vegfafl/flTie2-Cre animals (n = 5).(D) Representative images of longitudinal sections of injured sciatic nerves from wild-type that have received bone marrow from Vegfafl/fl (control) or Vegfafl/flTie2-Cre mice immunostained to detect ECs (CD31+, red), SCs (p75NTR+, green), and axons (NF+, blue), Day 5 after transection. Scale bar, 100 μm.(E) Quantification of (D) showing the proportion of CD31-positive area per bridge area (n = 3 for each group).(F) Representative images of longitudinal sections of injured sciatic nerves of Vegfafl/flTie2-Cre mice, Day 5 after transection following injection of PBS or VEGF-A188 into the bridges at Day 4. Scale bar, 100 μm.(G and H) Quantification of (F) showing the blood vessel density (G) or area of infiltrating SCs (H) (n = 4). For reconstruction of longitudinal sections shown in (A), (D) and (F), multiple images from the same sample were acquired using the same microscope settings.Graphs show mean value ± SEM. See also Figure S6.
Mentions: Macrophages have been shown to promote angiogenesis (Fantin et al., 2010; Pollard, 2009) and autocrine VEGF-A signaling helps to maintain the health of ECs (Lee et al., 2007). We therefore analyzed the vasculature of uninjured nerves from all genotypes but found no differences (Figures S6D and S6E). Remarkably however, nerves from both mutant animals showed a reduction in the vascularization of the bridge following injury (Figure 6A). The extent of the inhibition was more dramatic in the Vegfafl/flTie2-Cre mice, consistent with the greater degree of recombination in these animals, with very few blood vessels detectable within the bridge (Figure 6B). However, there was also a significant decrease in the Vegfafl/flLysmCre mice (Figure 6B). Strikingly, SCs remained in the stumps of the Tie2-Cre mutant animals, consistent with a requirement for blood vessels to provide a “track” for the SCs to enter the bridge (Figure 6C). To confirm this was not due to loss of VEGF-A expression in ECs we (1) performed bone marrow transplant experiments from Vegfafl/flTie2-Cre and control Vegfafl/fl litter-mates into WT mice and found similar defective entry of blood vessels into the bridges of the mice receiving the mutant bone marrow, confirming that cells derived from hematopoietic-stem cells were responsible for the defect (Figures 6D, 6E, S6F, and S6G); and (2) performed rescue experiments in the Vegfafl/flTie2-Cre mice. We injected either VEGF-A or PBS into the bridges of Vegfafl/flTie2-Cre mice on day 4 and found that VEGF-A was able to rescue EC migration into the bridge and that SCs and axons migrated along these blood vessels (Figures 6F–6H). These results show that ECs deleted for VEGF-A are able to migrate and survive in the bridge and also provide a substrate for SC migration. Together, these results show that macrophages in the bridge secrete VEGF-A to enable the formation of a polarized endothelial scaffold that can direct SCs out of the nerve stumps and across the bridge.

Bottom Line: Here we show that blood vessels direct the migrating cords of Schwann cells.Importantly, disrupting the organization of the newly formed blood vessels in vivo, either by inhibiting the angiogenic signal or by re-orienting them, compromises Schwann cell directionality resulting in defective nerve repair.This study provides important insights into how the choreography of multiple cell-types is required for the regeneration of an adult tissue.

View Article: PubMed Central - PubMed

Affiliation: MRC Laboratory for Molecular Cell Biology, UCL, Gower Street, London WC1E 6BT, UK.

Show MeSH
Related in: MedlinePlus