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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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Hypoxia Drives Angiogenesis by a Macrophage-Generated Gradient of VEGF-A(A) Representative images of sections of a rat sciatic nerve bridge, Day 2 and 3 after transection and 30 min after injection of hypoxyprobe-1, immunolabeled to detect hypoxyprobe-1 (green). Scale bar, 25 μm.(B) As in (A) but immunolabeled to detect macrophages (Iba1+, red) and hypoxic cells (hypoxyprobe-1+, green). Scale bar, 25 μm.(C) Graph showing percentage of hypoxic cells (hypoxyprobe-1+) in macrophage (Iba1+) and non-macrophage (Iba1−) populations from rat sciatic nerve bridges cultured at indicated oxygen conditions (n = 3).(D) HUVECs or SCs were placed in the upper compartment of Boyden chambers and allowed to migrate into the lower chamber containing media with no factors (SATO), VEGF-A165, serum, or conditioned medium from bridge cells cultured at 1.5% O2 (n = 5). For (C) and (D) one-way ANOVA test was used for statistical analysis.(E–H) Confocal images of longitudinal cryosections of injured sciatic nerves from PLP-EGFP mice, Day 5 or Day 7 after transection, following gavage of cabozantinib or control solvent on Day 4 (pre-vascularization), immunostained to detect ECs (CD31+, red) and axons (NF+, blue) Scale bar, 50 μm, quantified in (G) and (H) (n = 3).(I) As for (F) but cabozantinib was administered on Day 5 (post-vascularization) and harvested on Day 6, quantified in (J) (n = 3). For reconstruction of longitudinal sections shown in (E), (F), and (I), multiple images from the same sample were acquired using the same microscope settings.Graphs show mean value ± SEM. See also Figure S5.
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fig5: Hypoxia Drives Angiogenesis by a Macrophage-Generated Gradient of VEGF-A(A) Representative images of sections of a rat sciatic nerve bridge, Day 2 and 3 after transection and 30 min after injection of hypoxyprobe-1, immunolabeled to detect hypoxyprobe-1 (green). Scale bar, 25 μm.(B) As in (A) but immunolabeled to detect macrophages (Iba1+, red) and hypoxic cells (hypoxyprobe-1+, green). Scale bar, 25 μm.(C) Graph showing percentage of hypoxic cells (hypoxyprobe-1+) in macrophage (Iba1+) and non-macrophage (Iba1−) populations from rat sciatic nerve bridges cultured at indicated oxygen conditions (n = 3).(D) HUVECs or SCs were placed in the upper compartment of Boyden chambers and allowed to migrate into the lower chamber containing media with no factors (SATO), VEGF-A165, serum, or conditioned medium from bridge cells cultured at 1.5% O2 (n = 5). For (C) and (D) one-way ANOVA test was used for statistical analysis.(E–H) Confocal images of longitudinal cryosections of injured sciatic nerves from PLP-EGFP mice, Day 5 or Day 7 after transection, following gavage of cabozantinib or control solvent on Day 4 (pre-vascularization), immunostained to detect ECs (CD31+, red) and axons (NF+, blue) Scale bar, 50 μm, quantified in (G) and (H) (n = 3).(I) As for (F) but cabozantinib was administered on Day 5 (post-vascularization) and harvested on Day 6, quantified in (J) (n = 3). For reconstruction of longitudinal sections shown in (E), (F), and (I), multiple images from the same sample were acquired using the same microscope settings.Graphs show mean value ± SEM. See also Figure S5.

Mentions: New blood vessels normally form in response to decreased oxygen levels (hypoxia) within a tissue. Upon hypoxia, the transcription factor HIF-1α is stabilized and initiates a transcriptional response that induces angiogenesis by upregulating pro-angiogenic factors such as VEGF (Krock et al., 2011; Pugh and Ratcliffe, 2003). To test whether the nerve bridge was hypoxic, we injected rats with hypoxyprobe-1 (pimonidazole hydrochloride) that forms immunofluorescent detectable protein adducts in hypoxic conditions (pO2 < 10 mm Hg) (Young and Möller, 2010). Immunostaining of day 2 nerve bridges revealed the presence of large numbers of hypoxic cells prior to its vascularization (Figures 5A and S5A). Hypoxic cells were found only in the bridge and at the tips of both the distal and proximal stumps but not further along the stumps or in the uncut nerve (Figure S5B). The proportion of hypoxic cells decreased substantially by day 3, when the bridge had become vascularized (Figures 5A and S5A), consistent with the new blood vessels resolving the hypoxic environment of this new tissue.


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)

Hypoxia Drives Angiogenesis by a Macrophage-Generated Gradient of VEGF-A(A) Representative images of sections of a rat sciatic nerve bridge, Day 2 and 3 after transection and 30 min after injection of hypoxyprobe-1, immunolabeled to detect hypoxyprobe-1 (green). Scale bar, 25 μm.(B) As in (A) but immunolabeled to detect macrophages (Iba1+, red) and hypoxic cells (hypoxyprobe-1+, green). Scale bar, 25 μm.(C) Graph showing percentage of hypoxic cells (hypoxyprobe-1+) in macrophage (Iba1+) and non-macrophage (Iba1−) populations from rat sciatic nerve bridges cultured at indicated oxygen conditions (n = 3).(D) HUVECs or SCs were placed in the upper compartment of Boyden chambers and allowed to migrate into the lower chamber containing media with no factors (SATO), VEGF-A165, serum, or conditioned medium from bridge cells cultured at 1.5% O2 (n = 5). For (C) and (D) one-way ANOVA test was used for statistical analysis.(E–H) Confocal images of longitudinal cryosections of injured sciatic nerves from PLP-EGFP mice, Day 5 or Day 7 after transection, following gavage of cabozantinib or control solvent on Day 4 (pre-vascularization), immunostained to detect ECs (CD31+, red) and axons (NF+, blue) Scale bar, 50 μm, quantified in (G) and (H) (n = 3).(I) As for (F) but cabozantinib was administered on Day 5 (post-vascularization) and harvested on Day 6, quantified in (J) (n = 3). For reconstruction of longitudinal sections shown in (E), (F), and (I), multiple images from the same sample were acquired using the same microscope settings.Graphs show mean value ± SEM. See also Figure S5.
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Related In: Results  -  Collection

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fig5: Hypoxia Drives Angiogenesis by a Macrophage-Generated Gradient of VEGF-A(A) Representative images of sections of a rat sciatic nerve bridge, Day 2 and 3 after transection and 30 min after injection of hypoxyprobe-1, immunolabeled to detect hypoxyprobe-1 (green). Scale bar, 25 μm.(B) As in (A) but immunolabeled to detect macrophages (Iba1+, red) and hypoxic cells (hypoxyprobe-1+, green). Scale bar, 25 μm.(C) Graph showing percentage of hypoxic cells (hypoxyprobe-1+) in macrophage (Iba1+) and non-macrophage (Iba1−) populations from rat sciatic nerve bridges cultured at indicated oxygen conditions (n = 3).(D) HUVECs or SCs were placed in the upper compartment of Boyden chambers and allowed to migrate into the lower chamber containing media with no factors (SATO), VEGF-A165, serum, or conditioned medium from bridge cells cultured at 1.5% O2 (n = 5). For (C) and (D) one-way ANOVA test was used for statistical analysis.(E–H) Confocal images of longitudinal cryosections of injured sciatic nerves from PLP-EGFP mice, Day 5 or Day 7 after transection, following gavage of cabozantinib or control solvent on Day 4 (pre-vascularization), immunostained to detect ECs (CD31+, red) and axons (NF+, blue) Scale bar, 50 μm, quantified in (G) and (H) (n = 3).(I) As for (F) but cabozantinib was administered on Day 5 (post-vascularization) and harvested on Day 6, quantified in (J) (n = 3). For reconstruction of longitudinal sections shown in (E), (F), and (I), multiple images from the same sample were acquired using the same microscope settings.Graphs show mean value ± SEM. See also Figure S5.
Mentions: New blood vessels normally form in response to decreased oxygen levels (hypoxia) within a tissue. Upon hypoxia, the transcription factor HIF-1α is stabilized and initiates a transcriptional response that induces angiogenesis by upregulating pro-angiogenic factors such as VEGF (Krock et al., 2011; Pugh and Ratcliffe, 2003). To test whether the nerve bridge was hypoxic, we injected rats with hypoxyprobe-1 (pimonidazole hydrochloride) that forms immunofluorescent detectable protein adducts in hypoxic conditions (pO2 < 10 mm Hg) (Young and Möller, 2010). Immunostaining of day 2 nerve bridges revealed the presence of large numbers of hypoxic cells prior to its vascularization (Figures 5A and S5A). Hypoxic cells were found only in the bridge and at the tips of both the distal and proximal stumps but not further along the stumps or in the uncut nerve (Figure S5B). The proportion of hypoxic cells decreased substantially by day 3, when the bridge had become vascularized (Figures 5A and S5A), consistent with the new blood vessels resolving the hypoxic environment of this new tissue.

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