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. Jan-Dec 2017;13:1744806917727625.
doi: 10.1177/1744806917727625.

Could an Endoneurial Endothelial Crosstalk Between Wnt/β-catenin and Sonic Hedgehog Pathways Underlie the Early Disruption of the Infra-Orbital Blood-Nerve Barrier Following Chronic Constriction Injury?

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Could an Endoneurial Endothelial Crosstalk Between Wnt/β-catenin and Sonic Hedgehog Pathways Underlie the Early Disruption of the Infra-Orbital Blood-Nerve Barrier Following Chronic Constriction Injury?

Nathan Moreau et al. Mol Pain. .
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Abstract

Background: Blood–nerve barrier disruption is pivotal in the development of neuroinflammation, peripheral sensitization, and neuropathic pain after peripheral nerve injury. Activation of toll-like receptor 4 and inactivation of Sonic Hedgehog signaling pathways within the endoneurial endothelial cells are key events, resulting in the infiltration of harmful molecules and immunocytes within the nerve parenchyma. However, we showed in a previous study that preemptive inactivation of toll-like receptor 4 signaling or sustained activation of Sonic Hedgehog signaling did not prevent the local alterations observed following peripheral nerve injury, suggesting the implication of another signaling pathway.

Methods: Using a classical neuropathic pain model, the infraorbital nerve chronic constriction injury (IoN-CCI), we investigated the role of the Wnt/β-catenin pathway in chronic constriction injury-mediated blood–nerve barrier disruption and in its interactions with the toll-like receptor 4 and Sonic Hedgehog pathways. In the IoN-CCI model versus control, mRNA expression levels and/or immunochemical detection of major Wnt/Sonic Hedgehog pathway (Frizzled-7, vascular endothelial-cadherin, Patched-1 and Gli-1) and/or tight junction proteins (Claudin-1, Claudin-5, and Occludin) readouts were assessed. Vascular permeability was assessed by sodium fluorescein extravasation.

Results: IoN-CCI induced early alterations in the vascular endothelial-cadherin/β-catenin/Frizzled-7 complex, shown to participate in local blood–nerve barrier disruption via a β-catenin-dependent tight junction protein downregulation. Wnt pathway also mediated a crosstalk between toll-like receptor 4 and Sonic Hedgehog signaling within endoneurial endothelial cells. Nevertheless, preemptive inhibition of Wnt/β-catenin signaling before IoN-CCI could not prevent the downregulation of key Sonic Hedgehog pathway readouts or the disruption of the infraorbital blood–nerve barrier, suggesting that Sonic Hedgehog pathway inhibition observed following IoN-CCI is an independent event responsible for blood–nerve barrier disruption.

Conclusion: A crosstalk between Wnt/β-catenin- and Sonic Hedgehog-mediated signaling pathways within endoneurial endothelial cells could mediate the chronic disruption of the blood–nerve barrier following IoN-CCI, resulting in increased irreversible endoneurial vascular permeability and neuropathic pain development.

Figures

Figure 1.
Figure 1.
Chronic constriction injury of the infraorbital nerve (IoN-CCI) induces early and transient alterations in the Fzd-7/β-catenin/VE-cadherin AJ complex within endoneurial endothelial cells in association with a transient increased production of VEGF-A protein in the infraorbital nerve. (a) IoN-CCI induces early and transient significant downregulation of Fzd-7 and VE-cadherin mRNAs and early upregulation followed by transient downregulation of β-catenin mRNA expression levels. Changes over time of Fzd-7, β-catenin, and VE-cadherin mRNA levels were assessed in the IoN of sham- or CCI-injured animals using semi-quantitative RT-PCR analyses. Data are presented as relative quantification (R.Q.) in arbitrary units (A.U.) corresponding to the ratio of specific mRNA over RPS18 mRNA. Each bar corresponds to the mean ± SEM of n = 5–8 animals for each time point (post-injury); *p < 0.05, **p < 0.01, ***p < 0.001. A one-way ANOVA followed by Bonferroni post hoc test was used. (b, c) IoN-CCI induces a decrease in Fzd-7 immunoreactivity in endoneurial blood vessels compared to control condition (sham-injured animals) at 24 h post-CCI in longitudinal slices of the injured IoN (b) and in axial slices centered on an endoneurial blood vessel (Scale bar = 100 µm) (c). No Fzd-7 immunoreactivity was observed outside of the endoneurial endothelial cells. Paraformaldehyde-fixed slices of IoN from sham- and CCI-injured rats were incubated with TO-PRO (nuclear stain, blue), anti-Reca 1 antibodies (endothelial marker, red), and anti-Fzd-7 antibodies (green) (Scale bar = 10 µm). (d) IoN-CCI induces a decrease in VE-cadherin immunoreactivity in endoneurial blood vessels compared to control condition (sham-injured animals) at 24 h post-CCI in axial slices of the injured IoN. Paraformaldehyde-fixed slices of IoN from sham- and CCI-injured rats were incubated with TO-PRO (nuclear stain, blue), anti-Reca 1 antibodies (endothelial marker, red), and anti-VE cadherin antibodies (green) (Scale bar = 10 µm). (e) IoN-CCI induces a transient increase in VEGF-A mRNA expression levels at 24 h followed by a significant downregulation between 48 h and seven days (left figure) associated with a significant increase in VEGF-A protein production between 24 h and 48 h post-CCI (right figure). Changes over time of VEGF-A mRNA levels were assessed in the IoN of sham- or CCI-injured animals using semi-quantitative RT-PCR analyses. Data are presented as R.Q. in A.U. corresponding to the ratio of specific mRNA over RPS18 mRNA. Each bar corresponds to the mean ± SEM of n = 5–8 animals for each time point (post-injury); *p < 0.05, **p < 0.01, ***p < 0.001; one-way ANOVA followed by Bonferroni post hoc test was used. Changes over time in VEGF-A protein production in CCI-injured animals (compared to sham-injured animals) were assessed using an enzyme-linked immunosorbent assay (ELISA) technique. Data are presented as the ratio of VEGF-A protein (in pg) over total proteins (in µg). Each bar corresponds to n = 3 rats for each time point (post-injury); *p < 0.05; one-way ANOVA followed by Bonferroni post hoc test was used. VE: vascular endothelial; AJ: adherens junction; VEGF-A: vascular endothelial growth factor-A; RT-PCR: reverse transcription polymerase chain reaction; SEM: standard error of the mean; Fzd-7: Frizzled-7; ANOVA: analysis of variance.
Figure 1.
Figure 1.
Chronic constriction injury of the infraorbital nerve (IoN-CCI) induces early and transient alterations in the Fzd-7/β-catenin/VE-cadherin AJ complex within endoneurial endothelial cells in association with a transient increased production of VEGF-A protein in the infraorbital nerve. (a) IoN-CCI induces early and transient significant downregulation of Fzd-7 and VE-cadherin mRNAs and early upregulation followed by transient downregulation of β-catenin mRNA expression levels. Changes over time of Fzd-7, β-catenin, and VE-cadherin mRNA levels were assessed in the IoN of sham- or CCI-injured animals using semi-quantitative RT-PCR analyses. Data are presented as relative quantification (R.Q.) in arbitrary units (A.U.) corresponding to the ratio of specific mRNA over RPS18 mRNA. Each bar corresponds to the mean ± SEM of n = 5–8 animals for each time point (post-injury); *p < 0.05, **p < 0.01, ***p < 0.001. A one-way ANOVA followed by Bonferroni post hoc test was used. (b, c) IoN-CCI induces a decrease in Fzd-7 immunoreactivity in endoneurial blood vessels compared to control condition (sham-injured animals) at 24 h post-CCI in longitudinal slices of the injured IoN (b) and in axial slices centered on an endoneurial blood vessel (Scale bar = 100 µm) (c). No Fzd-7 immunoreactivity was observed outside of the endoneurial endothelial cells. Paraformaldehyde-fixed slices of IoN from sham- and CCI-injured rats were incubated with TO-PRO (nuclear stain, blue), anti-Reca 1 antibodies (endothelial marker, red), and anti-Fzd-7 antibodies (green) (Scale bar = 10 µm). (d) IoN-CCI induces a decrease in VE-cadherin immunoreactivity in endoneurial blood vessels compared to control condition (sham-injured animals) at 24 h post-CCI in axial slices of the injured IoN. Paraformaldehyde-fixed slices of IoN from sham- and CCI-injured rats were incubated with TO-PRO (nuclear stain, blue), anti-Reca 1 antibodies (endothelial marker, red), and anti-VE cadherin antibodies (green) (Scale bar = 10 µm). (e) IoN-CCI induces a transient increase in VEGF-A mRNA expression levels at 24 h followed by a significant downregulation between 48 h and seven days (left figure) associated with a significant increase in VEGF-A protein production between 24 h and 48 h post-CCI (right figure). Changes over time of VEGF-A mRNA levels were assessed in the IoN of sham- or CCI-injured animals using semi-quantitative RT-PCR analyses. Data are presented as R.Q. in A.U. corresponding to the ratio of specific mRNA over RPS18 mRNA. Each bar corresponds to the mean ± SEM of n = 5–8 animals for each time point (post-injury); *p < 0.05, **p < 0.01, ***p < 0.001; one-way ANOVA followed by Bonferroni post hoc test was used. Changes over time in VEGF-A protein production in CCI-injured animals (compared to sham-injured animals) were assessed using an enzyme-linked immunosorbent assay (ELISA) technique. Data are presented as the ratio of VEGF-A protein (in pg) over total proteins (in µg). Each bar corresponds to n = 3 rats for each time point (post-injury); *p < 0.05; one-way ANOVA followed by Bonferroni post hoc test was used. VE: vascular endothelial; AJ: adherens junction; VEGF-A: vascular endothelial growth factor-A; RT-PCR: reverse transcription polymerase chain reaction; SEM: standard error of the mean; Fzd-7: Frizzled-7; ANOVA: analysis of variance.
Figure 2.
Figure 2.
In vitro, in the human cerebral microvascular endothelial cell line (hCMEC/D3), modulation of Wnt/β-catenin pathway induces significant expression changes in the VE-cadherin/Fzd-7/β-catenin complex mRNAs levels. (a) Presence of functional Frizzled-7 within the hCMEC/D3 endothelial cells was assessed using immunofluorescent labeling of Frizzled-7, β-catenin, and TO-PRO observed with confocal microscopy imaging. Paraformaldehyde-fixed cells were incubated with anti-Frizzled-7 antibodies (green), anti-β-catenin (cell membrane surrogate marker, red), and TO-PRO (nuclear stain, blue) (Scale bar = 20 µm). (b) Changes in Frizzled-7, β-catenin, and VE-cadherin mRNAs expression levels were assessed in vitro following stimulation with either Wnt agonist I (5 μM; Wnt pathway agonist) or PKF 118-310 (2 μM; Wnt pathway antagonist). Data are presented as relative quantification (R.Q.) in arbitrary units (A.U.) corresponding to the ratio of specific mRNA over RPS18 mRNA. Each bar corresponds to the mean ± SEM of n = 4 experiments for each condition; *p < 0.05, **p < 0.01, ***p < 0.001; Kruskal–Wallis test followed by Dunn’s multiple comparisons test was used. VE: vascular endothelial; SEM: standard error of the mean.
Figure 3.
Figure 3.
In vitro, in the hCMEC/D3 cell line, activation of Wnt/β-catenin pathway downregulates the mRNAs of key Hedgehog pathway readouts and mediates the molecular changes observed following TLR4 stimulation. (a) Changes in Gli-1 or Patched-1 mRNAs expression levels were assessed in vitro following stimulation with either Wnt agonist I (5 μM; Wnt pathway agonist) or PKF 118-310 (2 μM; Wnt pathway antagonist). Data are presented as relative quantification (R.Q.) in arbitrary units (A.U.) corresponding to the ratio of specific mRNA over RPS18 mRNA. Each bar corresponds to the mean ± SEM of n = 4 experiments for each condition; *p < 0.05, **p < 0.01, ***p < 0.001; Kruskal–Wallis test followed by Dunn’s multiple comparisons test was used. (b) In vitro, following pretreatment with LPS (a TLR4 agonist), an important increase in β-catenin immunoreactivity could be observed at the cell membrane and in the nuclear and perinuclear regions observed in confocal microscopy imaging, suggestive of Wnt pathway activation. Paraformaldehyde-fixed cells were incubated with anti-β-catenin antibodies (green) and TO-PRO (nuclear stain, blue) (Scale bar = 30 µm). (c) Changes in Claudin-5, Gli-1, and TLR2 mRNAs expression levels were assessed in vitro, following LPS stimulation or PKF 118-310 pretreatment followed by LPS stimulation. PKF 118-310 pretreatment strongly mitigated the changes in mRNA expression levels of all three markers, suggesting that TLR4-mediated molecular changes (LPS stimulation) are mediated by active Wnt signaling. Data are presented as R.Q. in A.U. corresponding to the ratio of specific mRNA over RPS18 mRNA. Each bar corresponds to the mean ± SEM of n = 4 experiments for each condition; *p < 0.05, **p < 0.01, ***p < 0.001; Kruskal–Wallis test followed by Dunn’s multiple comparisons test was used. hCMEC/D3: human cerebral microvascular endothelial cells; TLR4: toll-like receptor 4; SEM: standard error of the mean; LPS: lipopolysaccharide.
Figure 4.
Figure 4.
In vivo, at 3 h post-injury, inhibition of Wnt/β-catenin signaling could not prevent the molecular and vascular alterations following infraorbital nerve chronic constriction injury (IoN-CCI). (a to d) Changes in mRNAs expression levels of TJ proteins Claudin-1, Claudin-5, Occludin (a), inflammatory markers TLR2 and CD11b (b), Hedgehog pathway markers Patched-1 and Gli-1 (c), and Fzd-7/ β-catenin/VE-cadherin AJ complex proteins (d) were assessed using semi-quantitative RT-PCR following either perineural injections (used as a control condition), or infraorbital nerve chronic constriction injury (CCI) following either PKF 118-310 (50 μM; Wnt pathway antagonist) or NaCl 0.9% injections (three injections, spaced 6 h apart, starting 24 h before injury), as compared to noninjured IoN of naïve rats (serving as baseline values for mRNA levels comparisons). Data are presented as relative quantification (R.Q.) in arbitrary units (A.U.) corresponding to the ratio of specific mRNA over RPS18 mRNA. Each bar corresponds to the mean ± SEM of n = 5–8 animals for each time point (post-injury); *p < 0.05, **p < 0.01, ***p < 0.001. Kruskal–Wallis test followed by Dunn’s multiple comparisons test was used. (e) Fluorescence microscopy observation of paraformaldehyde-fixed axial and longitudinal slices of right IoNs harvested from NaFlu intravenously injected rats submitted to perineural injection or CCI following either PKF 118-310 (Wnt pathway antagonist) or NaCl 0.9% injections (three injections, spaced 6 h apart, starting 24 h before injury) showed an increase in NaFlu fluorescence within the IoN parenchyma following IoN-CCI in both PKF 118-310 and NaCl 0.9% pretreated rats (as compared to control conditions), suggesting that Wnt/β-catenin inhibition could not mitigate the vascular alterations following IoN-CCI (Scale bar = 50 µm). TJ: tight junction; TLR2: toll-like receptor 2; VE: vascular endothelial; Fzd-7: Frizzled-7; AJ: adherens junction; RT-PCR: reverse transcription polymerase chain reaction; NaFlu: sodium fluorescein.
Figure 4.
Figure 4.
In vivo, at 3 h post-injury, inhibition of Wnt/β-catenin signaling could not prevent the molecular and vascular alterations following infraorbital nerve chronic constriction injury (IoN-CCI). (a to d) Changes in mRNAs expression levels of TJ proteins Claudin-1, Claudin-5, Occludin (a), inflammatory markers TLR2 and CD11b (b), Hedgehog pathway markers Patched-1 and Gli-1 (c), and Fzd-7/ β-catenin/VE-cadherin AJ complex proteins (d) were assessed using semi-quantitative RT-PCR following either perineural injections (used as a control condition), or infraorbital nerve chronic constriction injury (CCI) following either PKF 118-310 (50 μM; Wnt pathway antagonist) or NaCl 0.9% injections (three injections, spaced 6 h apart, starting 24 h before injury), as compared to noninjured IoN of naïve rats (serving as baseline values for mRNA levels comparisons). Data are presented as relative quantification (R.Q.) in arbitrary units (A.U.) corresponding to the ratio of specific mRNA over RPS18 mRNA. Each bar corresponds to the mean ± SEM of n = 5–8 animals for each time point (post-injury); *p < 0.05, **p < 0.01, ***p < 0.001. Kruskal–Wallis test followed by Dunn’s multiple comparisons test was used. (e) Fluorescence microscopy observation of paraformaldehyde-fixed axial and longitudinal slices of right IoNs harvested from NaFlu intravenously injected rats submitted to perineural injection or CCI following either PKF 118-310 (Wnt pathway antagonist) or NaCl 0.9% injections (three injections, spaced 6 h apart, starting 24 h before injury) showed an increase in NaFlu fluorescence within the IoN parenchyma following IoN-CCI in both PKF 118-310 and NaCl 0.9% pretreated rats (as compared to control conditions), suggesting that Wnt/β-catenin inhibition could not mitigate the vascular alterations following IoN-CCI (Scale bar = 50 µm). TJ: tight junction; TLR2: toll-like receptor 2; VE: vascular endothelial; Fzd-7: Frizzled-7; AJ: adherens junction; RT-PCR: reverse transcription polymerase chain reaction; NaFlu: sodium fluorescein.
Figure 5.
Figure 5.
In vivo, at 3 h post-injury, CCI of the infraorbital nerve elicited a significant increase in hypoxia marker HIF-1α mRNA expression levels as compared to sham-injured animals. Changes in HIF-1α mRNA levels were assessed in the IoN of CCI-injured animals compared to sham-injured controls, using semi-quantitative RT-PCR analyses. Data are presented as relative quantification (R.Q.) in arbitrary units (A.U.) corresponding to the ratio of specific mRNA over RPS18 mRNA. Each bar corresponds to the mean ± SEM of n = 5–6 animals for each condition; *p < 0.05; Mann–Whitney test was used. CCI: chronic constriction injury; HIF-1α: hypoxia-inducible factor-1α; IoN: infraorbital nerve; RT-PCR: reverse transcription polymerase chain reaction; SEM: standard error of the mean.
Figure 6.
Figure 6.
In vivo, at 3 h post-injury, CCI of the infraorbital nerve elicited a significant increase in chemokine marker CCL2 mRNA expression levels as compared to sham-injured animals. Changes in CCL2 mRNA levels were assessed in the IoN of CCI-injured animals compared to sham-injured controls, using semi-quantitative RT-PCR analyses. Data are presented as relative quantification (R.Q.) in arbitrary units (A.U.) corresponding to the ratio of specific mRNA over RPS18 mRNA. Each bar corresponds to the mean ± SEM of n = 5–6 animals for each condition; *p < 0.05; Mann–Whitney test was used. CCI: chronic constriction injury; CCL2: chemokine (CC motif) ligand 2; IoN: infraorbital nerve; RT-PCR: reverse transcription polymerase chain reaction; SEM: standard error of the mean.
Figure 7.
Figure 7.
Simplified modelization of the neuro-immuno-vascular interactions within endoneurial endothelial cells of nerves subjected to chronic constriction injury (CCI). In this model, Claudin-5 was chosen to illustrate the key role of TJ proteins in the disruption of the blood–nerve barrier following CCI, as it has been previously described as the main endoneurial endothelial TJ protein within peripheral nerves., Other TJ proteins (such as Claudin-1 or Occludin) although not described in this model are of critical importance in the regulation of BNB permeability. Justifications of the proposed model based on past and current results (in italic) and available (nonexhaustive) scientific literature are described hereafter: a, b: Vos et al., Myers et al., Bennett and Xie. c: Myers et al., Bennett and Xie, Walsh et al. d: Myers et al., Nukada et al., Lim et al. e: Figure 5; Lim et al., f: Figure 5; Van Steenwinckel et al., Moreau et al., Sapienza et al. g: Figure 3(b) and (c). h: Figure 5; Coon et al., Walsh et al. i: Lim et al. j: Van Steenwinckel et al., Sapienza et al. k: Van Steenwinckel et al., Sapienza et al. l: Lim et al. m: Figure 1(d) and data not shown (see Results section for details); Gavard, Gavard and Gutkind. n: Disorganization of AJ/TJ proteins induces loss of cell polarity resulting in loss of primary cilium and subsequent inhibition of Hedgehog pathway. o: Gavard and Gutkind, Taddei et al. p: Figures 3(a) and 4(c) . q: Moreau et al., r: Figures 3(c) and 4(a) ; Taddei et al. s: Figure 4(a) and ( e ); Moreau et al.,, Gavard and Gutkind. t: Lim et al., Moreau et al., u: Figure 4(b) ; Lim et al., Moreau et al., v: Moreau et al.,, Myers and Shubayev. w: Kiguchi et al., Xie et al., Basbaum et al.. BNB: blood–nerve barrier; TJ: tight junction.

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