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Complement anaphylatoxin C3a is a potent inducer of embryonic chick retina regeneration.

Haynes T, Luz-Madrigal A, Reis ES, Echeverri Ruiz NP, Grajales-Esquivel E, Tzekou A, Tsonis PA, Lambris JD, Del Rio-Tsonis K - Nat Commun (2013)

Bottom Line: Identifying the initiation signals for tissue regeneration in vertebrates is one of the major challenges in regenerative biology.Much of the research thus far has indicated that certain growth factors have key roles.This activation sets forth regulation of Wnt2b, Six3 and Sox2, genes associated with retina stem and progenitor cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Miami University, Oxford, Ohio 45056, USA.

ABSTRACT
Identifying the initiation signals for tissue regeneration in vertebrates is one of the major challenges in regenerative biology. Much of the research thus far has indicated that certain growth factors have key roles. Here we show that complement fragment C3a is sufficient to induce complete regeneration of the embryonic chick retina from stem/progenitor cells present in the eye, independent of fibroblast growth factor receptor signaling. Instead, C3a induces retina regeneration via STAT3 activation, which in turn activates the injury- and inflammation-responsive factors, IL-6, IL-8 and TNF-α. This activation sets forth regulation of Wnt2b, Six3 and Sox2, genes associated with retina stem and progenitor cells. Thus, our results establish a mechanism for retina regeneration based on injury and inflammation signals. Furthermore, our results indicate a unique function for complement anaphylatoxins that implicate these molecules in the induction and complete regeneration of the retina, opening new avenues of experimentation in the field.

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C3a-p induction of chick retina regeneration is independent of FGF2.(a) RT–qPCR shows the level of FGF2 expression stimulated by treatment with C3a-p in the CM at 2 h PR and 24 h PR. The level of FGF2 expression was normalized to the level expressed in the CM of eyes undergoing retinectomy without treatment. Error bars represent s.e.m. The mean ratio of FGF2 expression to GAPDH expression, together with the s.e.m. and range, is provided in Supplementary Table S5. Significance was determined by comparing the expression of FGF2 in eyes treated with C3a-p to that in eyes treated with scrambled peptide (Scr). P-value=0.0003 using the Student’s t-test.; n=3 biological samples done in triplicate. ***P<0.001. (b–g) Histological analysis at 3 days (d) PR after treatment with C3a-p+PD173074 (b), C3a-p+DMSO (c), FGF2+PD173074 (d). FGF2+an antibody that blocks the C3a receptor (C3aR-Ab) (e), C3a-p+C3aR-Ab (f), or C3a-p+pre-immune serum for C3aR (IgG) (g). The scale bar in (g) represents 100 μm and also applies to (b–f). (h) Quantification of regeneration shows there is a significant difference in the amount of regeneration between eyes treated with C3a-p+IgG (n=7; 5 with regeneration) and C3a-p+C3aR-Ab (n=9; 1 with regeneration) (P-value=0.0376 using a two-tailed permutation test). There is also significant difference in the amount of regeneration between eyes treated with FGF2 and those treated with FGF2+PD173074 (n=5; 0 with regeneration) (P=0.0011 using a two-tailed permutation test). *P<0.05. Error bars represent s.e.m. n=9; 7 with regeneration for C3a-p+DMSO-treated eyes, n=8; 6 with regeneration for C3a-p+PD173074-treated eyes and n=14; 12 with regeneration for FGF2+C3aR-Ab-treated eyes. The mean amount of regeneration, s.e.m. and range are provided in Supplementary Table S7. NS, not significant; cr, regeneration from the ciliary margin; L, lens.
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f4: C3a-p induction of chick retina regeneration is independent of FGF2.(a) RT–qPCR shows the level of FGF2 expression stimulated by treatment with C3a-p in the CM at 2 h PR and 24 h PR. The level of FGF2 expression was normalized to the level expressed in the CM of eyes undergoing retinectomy without treatment. Error bars represent s.e.m. The mean ratio of FGF2 expression to GAPDH expression, together with the s.e.m. and range, is provided in Supplementary Table S5. Significance was determined by comparing the expression of FGF2 in eyes treated with C3a-p to that in eyes treated with scrambled peptide (Scr). P-value=0.0003 using the Student’s t-test.; n=3 biological samples done in triplicate. ***P<0.001. (b–g) Histological analysis at 3 days (d) PR after treatment with C3a-p+PD173074 (b), C3a-p+DMSO (c), FGF2+PD173074 (d). FGF2+an antibody that blocks the C3a receptor (C3aR-Ab) (e), C3a-p+C3aR-Ab (f), or C3a-p+pre-immune serum for C3aR (IgG) (g). The scale bar in (g) represents 100 μm and also applies to (b–f). (h) Quantification of regeneration shows there is a significant difference in the amount of regeneration between eyes treated with C3a-p+IgG (n=7; 5 with regeneration) and C3a-p+C3aR-Ab (n=9; 1 with regeneration) (P-value=0.0376 using a two-tailed permutation test). There is also significant difference in the amount of regeneration between eyes treated with FGF2 and those treated with FGF2+PD173074 (n=5; 0 with regeneration) (P=0.0011 using a two-tailed permutation test). *P<0.05. Error bars represent s.e.m. n=9; 7 with regeneration for C3a-p+DMSO-treated eyes, n=8; 6 with regeneration for C3a-p+PD173074-treated eyes and n=14; 12 with regeneration for FGF2+C3aR-Ab-treated eyes. The mean amount of regeneration, s.e.m. and range are provided in Supplementary Table S7. NS, not significant; cr, regeneration from the ciliary margin; L, lens.

Mentions: FGF2 is considered a critical molecule for the induction of retina regeneration in the chick. However, data from RT–qPCR at 2 h PR indicated that C3a-p does not stimulate the expression of FGF2 in the CM above the level induced by retinectomy alone, which is not sufficient to induce regeneration (Fig. 4a). Likewise, FGF2 does not increase the expression of C3 or C3aR at 2 h PR further demonstrating that C3a-p and FGF2 have independent modes to trigger regeneration (Supplementary Fig. S5). However, FGF2 expression was significantly stimulated by C3a-p 24 h later, suggesting that the FGF pathway most likely has a role in events post induction including proliferation, differentiation and cell survival25 (Fig. 4a).


Complement anaphylatoxin C3a is a potent inducer of embryonic chick retina regeneration.

Haynes T, Luz-Madrigal A, Reis ES, Echeverri Ruiz NP, Grajales-Esquivel E, Tzekou A, Tsonis PA, Lambris JD, Del Rio-Tsonis K - Nat Commun (2013)

C3a-p induction of chick retina regeneration is independent of FGF2.(a) RT–qPCR shows the level of FGF2 expression stimulated by treatment with C3a-p in the CM at 2 h PR and 24 h PR. The level of FGF2 expression was normalized to the level expressed in the CM of eyes undergoing retinectomy without treatment. Error bars represent s.e.m. The mean ratio of FGF2 expression to GAPDH expression, together with the s.e.m. and range, is provided in Supplementary Table S5. Significance was determined by comparing the expression of FGF2 in eyes treated with C3a-p to that in eyes treated with scrambled peptide (Scr). P-value=0.0003 using the Student’s t-test.; n=3 biological samples done in triplicate. ***P<0.001. (b–g) Histological analysis at 3 days (d) PR after treatment with C3a-p+PD173074 (b), C3a-p+DMSO (c), FGF2+PD173074 (d). FGF2+an antibody that blocks the C3a receptor (C3aR-Ab) (e), C3a-p+C3aR-Ab (f), or C3a-p+pre-immune serum for C3aR (IgG) (g). The scale bar in (g) represents 100 μm and also applies to (b–f). (h) Quantification of regeneration shows there is a significant difference in the amount of regeneration between eyes treated with C3a-p+IgG (n=7; 5 with regeneration) and C3a-p+C3aR-Ab (n=9; 1 with regeneration) (P-value=0.0376 using a two-tailed permutation test). There is also significant difference in the amount of regeneration between eyes treated with FGF2 and those treated with FGF2+PD173074 (n=5; 0 with regeneration) (P=0.0011 using a two-tailed permutation test). *P<0.05. Error bars represent s.e.m. n=9; 7 with regeneration for C3a-p+DMSO-treated eyes, n=8; 6 with regeneration for C3a-p+PD173074-treated eyes and n=14; 12 with regeneration for FGF2+C3aR-Ab-treated eyes. The mean amount of regeneration, s.e.m. and range are provided in Supplementary Table S7. NS, not significant; cr, regeneration from the ciliary margin; L, lens.
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f4: C3a-p induction of chick retina regeneration is independent of FGF2.(a) RT–qPCR shows the level of FGF2 expression stimulated by treatment with C3a-p in the CM at 2 h PR and 24 h PR. The level of FGF2 expression was normalized to the level expressed in the CM of eyes undergoing retinectomy without treatment. Error bars represent s.e.m. The mean ratio of FGF2 expression to GAPDH expression, together with the s.e.m. and range, is provided in Supplementary Table S5. Significance was determined by comparing the expression of FGF2 in eyes treated with C3a-p to that in eyes treated with scrambled peptide (Scr). P-value=0.0003 using the Student’s t-test.; n=3 biological samples done in triplicate. ***P<0.001. (b–g) Histological analysis at 3 days (d) PR after treatment with C3a-p+PD173074 (b), C3a-p+DMSO (c), FGF2+PD173074 (d). FGF2+an antibody that blocks the C3a receptor (C3aR-Ab) (e), C3a-p+C3aR-Ab (f), or C3a-p+pre-immune serum for C3aR (IgG) (g). The scale bar in (g) represents 100 μm and also applies to (b–f). (h) Quantification of regeneration shows there is a significant difference in the amount of regeneration between eyes treated with C3a-p+IgG (n=7; 5 with regeneration) and C3a-p+C3aR-Ab (n=9; 1 with regeneration) (P-value=0.0376 using a two-tailed permutation test). There is also significant difference in the amount of regeneration between eyes treated with FGF2 and those treated with FGF2+PD173074 (n=5; 0 with regeneration) (P=0.0011 using a two-tailed permutation test). *P<0.05. Error bars represent s.e.m. n=9; 7 with regeneration for C3a-p+DMSO-treated eyes, n=8; 6 with regeneration for C3a-p+PD173074-treated eyes and n=14; 12 with regeneration for FGF2+C3aR-Ab-treated eyes. The mean amount of regeneration, s.e.m. and range are provided in Supplementary Table S7. NS, not significant; cr, regeneration from the ciliary margin; L, lens.
Mentions: FGF2 is considered a critical molecule for the induction of retina regeneration in the chick. However, data from RT–qPCR at 2 h PR indicated that C3a-p does not stimulate the expression of FGF2 in the CM above the level induced by retinectomy alone, which is not sufficient to induce regeneration (Fig. 4a). Likewise, FGF2 does not increase the expression of C3 or C3aR at 2 h PR further demonstrating that C3a-p and FGF2 have independent modes to trigger regeneration (Supplementary Fig. S5). However, FGF2 expression was significantly stimulated by C3a-p 24 h later, suggesting that the FGF pathway most likely has a role in events post induction including proliferation, differentiation and cell survival25 (Fig. 4a).

Bottom Line: Identifying the initiation signals for tissue regeneration in vertebrates is one of the major challenges in regenerative biology.Much of the research thus far has indicated that certain growth factors have key roles.This activation sets forth regulation of Wnt2b, Six3 and Sox2, genes associated with retina stem and progenitor cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Miami University, Oxford, Ohio 45056, USA.

ABSTRACT
Identifying the initiation signals for tissue regeneration in vertebrates is one of the major challenges in regenerative biology. Much of the research thus far has indicated that certain growth factors have key roles. Here we show that complement fragment C3a is sufficient to induce complete regeneration of the embryonic chick retina from stem/progenitor cells present in the eye, independent of fibroblast growth factor receptor signaling. Instead, C3a induces retina regeneration via STAT3 activation, which in turn activates the injury- and inflammation-responsive factors, IL-6, IL-8 and TNF-α. This activation sets forth regulation of Wnt2b, Six3 and Sox2, genes associated with retina stem and progenitor cells. Thus, our results establish a mechanism for retina regeneration based on injury and inflammation signals. Furthermore, our results indicate a unique function for complement anaphylatoxins that implicate these molecules in the induction and complete regeneration of the retina, opening new avenues of experimentation in the field.

Show MeSH
Related in: MedlinePlus