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CXCR3 antagonism of SDF-1(5-67) restores trabecular function and prevents retinal neurodegeneration in a rat model of ocular hypertension.

Denoyer A, Godefroy D, Célérier I, Frugier J, Degardin J, Harrison JK, Brignole-Baudouin F, Picaud S, Baleux F, Sahel JA, Rostène W, Baudouin C - PLoS ONE (2012)

Bottom Line: Current antiglaucoma therapy does not target the causal trabecular pathology, which may explain why treatment failure is often observed.The protective effect of CXCR3 antagonism is related to restoration of the trabecular function.These data demonstrate that proteolytic cleavage of CXCL12 is involved in trabecular pathophysiology, and that local administration of a selective CXCR3 antagonist may be a beneficial therapeutic strategy for treating ocular hypertension and subsequent retinal degeneration.

View Article: PubMed Central - PubMed

Affiliation: UPMC University Paris 6, Institut de la Vision, UMRS968, Paris, France. alexandre.denoyer@gmail.com

ABSTRACT
Glaucoma, the most common cause of irreversible blindness, is a neuropathy commonly initiated by pathological ocular hypertension due to unknown mechanisms of trabecular meshwork degeneration. Current antiglaucoma therapy does not target the causal trabecular pathology, which may explain why treatment failure is often observed. Here we show that the chemokine CXCL12, its truncated form SDF-1(5-67), and the receptors CXCR4 and CXCR3 are expressed in human glaucomatous trabecular tissue and a human trabecular cell line. SDF-1(5-67) is produced under the control of matrix metallo-proteinases, TNF-α, and TGF-β2, factors known to be involved in glaucoma. CXCL12 protects in vitro trabecular cells from apoptotic death via CXCR4 whereas SDF-1(5-67) induces apoptosis through CXCR3 and caspase activation. Ocular administration of SDF-1(5-67) in the rat increases intraocular pressure. In contrast, administration of a selective CXCR3 antagonist in a rat model of ocular hypertension decreases intraocular pressure, prevents retinal neurodegeneration, and preserves visual function. The protective effect of CXCR3 antagonism is related to restoration of the trabecular function. These data demonstrate that proteolytic cleavage of CXCL12 is involved in trabecular pathophysiology, and that local administration of a selective CXCR3 antagonist may be a beneficial therapeutic strategy for treating ocular hypertension and subsequent retinal degeneration.

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CXCL12, CXCR3, and CXCR4 expression by human glaucomatous trabecular tissue and a trabecular cell line.(A–C) The chemokine CXCL12 (A) and receptors CXCR3 (B) and CXCR4 (C) are detected in unstimulated human glaucomatous trabecular cells HTM3 by indirect immunofluorescence (secondary antibody in green, propidium iodide in red, scale bar: 50 µm, magnification ×200). (D) Chemokine receptor CXCR4 appears as distinct spots located at the cell membrane surface (scale bar: 5 µm, mag. ×800). Representative images of three independent experiments are depicted. (D) Cell expression of CXCL12 and receptors is also detected and quantified by immunoflowcytometry. Representative results obtained over 6 independent experiments, mean ± SEM of positive cells. (E) Chemokine and receptor mRNAs are detected in human glaucomatous trabecular tissues (n = 15) and in the HTM3 trabecular cell line. Data in the bar graph are presented as means ± SEM.
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pone-0037873-g001: CXCL12, CXCR3, and CXCR4 expression by human glaucomatous trabecular tissue and a trabecular cell line.(A–C) The chemokine CXCL12 (A) and receptors CXCR3 (B) and CXCR4 (C) are detected in unstimulated human glaucomatous trabecular cells HTM3 by indirect immunofluorescence (secondary antibody in green, propidium iodide in red, scale bar: 50 µm, magnification ×200). (D) Chemokine receptor CXCR4 appears as distinct spots located at the cell membrane surface (scale bar: 5 µm, mag. ×800). Representative images of three independent experiments are depicted. (D) Cell expression of CXCL12 and receptors is also detected and quantified by immunoflowcytometry. Representative results obtained over 6 independent experiments, mean ± SEM of positive cells. (E) Chemokine and receptor mRNAs are detected in human glaucomatous trabecular tissues (n = 15) and in the HTM3 trabecular cell line. Data in the bar graph are presented as means ± SEM.

Mentions: We first checked whether human glaucomatous trabecular tissue and cells were able to express the chemokine and chemokine receptors. CXCL12, CXCR3, and CXCR4 were detected by immunofluorescence and flow cytometry in unstimulated cells (Fig. 1A–E). mRNAs for CXCL12, CXCR3 and CXCR4 were also detected in both human tissue and trabecular cell line (Fig. 1F). Finally, TCs were able to release CXCL12 as determined by ELISA of cell supernatants (22.8±5.28 pg/ml [2.9±0.7 pM] from 100,000 cells/mL grown during 48 h). Western blot analysis of unstimulated HTM3 cells, using a polyclonal anti-SDF-1(5-67) neoepitope-specific antibody, identified SDF-1(5-67) (Fig. 2A,B). Incubation (24-h) with MMP inhibitors batimastat (100 nM) or TIMP-1 (0.5 nM) significantly reduced the amount of SDF-1(5-67), confirming that it originated from MMP activity produced by TCs. 24-h incubation of TCs with either TNF-α (50 ng/mL [2.9 nM]) or TGF-β2 (10 ng/mL [0.8 nM]), both known to be involved in glaucoma, increased the production of SDF-1(5-67) (Fig. 2A,B).


CXCR3 antagonism of SDF-1(5-67) restores trabecular function and prevents retinal neurodegeneration in a rat model of ocular hypertension.

Denoyer A, Godefroy D, Célérier I, Frugier J, Degardin J, Harrison JK, Brignole-Baudouin F, Picaud S, Baleux F, Sahel JA, Rostène W, Baudouin C - PLoS ONE (2012)

CXCL12, CXCR3, and CXCR4 expression by human glaucomatous trabecular tissue and a trabecular cell line.(A–C) The chemokine CXCL12 (A) and receptors CXCR3 (B) and CXCR4 (C) are detected in unstimulated human glaucomatous trabecular cells HTM3 by indirect immunofluorescence (secondary antibody in green, propidium iodide in red, scale bar: 50 µm, magnification ×200). (D) Chemokine receptor CXCR4 appears as distinct spots located at the cell membrane surface (scale bar: 5 µm, mag. ×800). Representative images of three independent experiments are depicted. (D) Cell expression of CXCL12 and receptors is also detected and quantified by immunoflowcytometry. Representative results obtained over 6 independent experiments, mean ± SEM of positive cells. (E) Chemokine and receptor mRNAs are detected in human glaucomatous trabecular tissues (n = 15) and in the HTM3 trabecular cell line. Data in the bar graph are presented as means ± SEM.
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Related In: Results  -  Collection

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pone-0037873-g001: CXCL12, CXCR3, and CXCR4 expression by human glaucomatous trabecular tissue and a trabecular cell line.(A–C) The chemokine CXCL12 (A) and receptors CXCR3 (B) and CXCR4 (C) are detected in unstimulated human glaucomatous trabecular cells HTM3 by indirect immunofluorescence (secondary antibody in green, propidium iodide in red, scale bar: 50 µm, magnification ×200). (D) Chemokine receptor CXCR4 appears as distinct spots located at the cell membrane surface (scale bar: 5 µm, mag. ×800). Representative images of three independent experiments are depicted. (D) Cell expression of CXCL12 and receptors is also detected and quantified by immunoflowcytometry. Representative results obtained over 6 independent experiments, mean ± SEM of positive cells. (E) Chemokine and receptor mRNAs are detected in human glaucomatous trabecular tissues (n = 15) and in the HTM3 trabecular cell line. Data in the bar graph are presented as means ± SEM.
Mentions: We first checked whether human glaucomatous trabecular tissue and cells were able to express the chemokine and chemokine receptors. CXCL12, CXCR3, and CXCR4 were detected by immunofluorescence and flow cytometry in unstimulated cells (Fig. 1A–E). mRNAs for CXCL12, CXCR3 and CXCR4 were also detected in both human tissue and trabecular cell line (Fig. 1F). Finally, TCs were able to release CXCL12 as determined by ELISA of cell supernatants (22.8±5.28 pg/ml [2.9±0.7 pM] from 100,000 cells/mL grown during 48 h). Western blot analysis of unstimulated HTM3 cells, using a polyclonal anti-SDF-1(5-67) neoepitope-specific antibody, identified SDF-1(5-67) (Fig. 2A,B). Incubation (24-h) with MMP inhibitors batimastat (100 nM) or TIMP-1 (0.5 nM) significantly reduced the amount of SDF-1(5-67), confirming that it originated from MMP activity produced by TCs. 24-h incubation of TCs with either TNF-α (50 ng/mL [2.9 nM]) or TGF-β2 (10 ng/mL [0.8 nM]), both known to be involved in glaucoma, increased the production of SDF-1(5-67) (Fig. 2A,B).

Bottom Line: Current antiglaucoma therapy does not target the causal trabecular pathology, which may explain why treatment failure is often observed.The protective effect of CXCR3 antagonism is related to restoration of the trabecular function.These data demonstrate that proteolytic cleavage of CXCL12 is involved in trabecular pathophysiology, and that local administration of a selective CXCR3 antagonist may be a beneficial therapeutic strategy for treating ocular hypertension and subsequent retinal degeneration.

View Article: PubMed Central - PubMed

Affiliation: UPMC University Paris 6, Institut de la Vision, UMRS968, Paris, France. alexandre.denoyer@gmail.com

ABSTRACT
Glaucoma, the most common cause of irreversible blindness, is a neuropathy commonly initiated by pathological ocular hypertension due to unknown mechanisms of trabecular meshwork degeneration. Current antiglaucoma therapy does not target the causal trabecular pathology, which may explain why treatment failure is often observed. Here we show that the chemokine CXCL12, its truncated form SDF-1(5-67), and the receptors CXCR4 and CXCR3 are expressed in human glaucomatous trabecular tissue and a human trabecular cell line. SDF-1(5-67) is produced under the control of matrix metallo-proteinases, TNF-α, and TGF-β2, factors known to be involved in glaucoma. CXCL12 protects in vitro trabecular cells from apoptotic death via CXCR4 whereas SDF-1(5-67) induces apoptosis through CXCR3 and caspase activation. Ocular administration of SDF-1(5-67) in the rat increases intraocular pressure. In contrast, administration of a selective CXCR3 antagonist in a rat model of ocular hypertension decreases intraocular pressure, prevents retinal neurodegeneration, and preserves visual function. The protective effect of CXCR3 antagonism is related to restoration of the trabecular function. These data demonstrate that proteolytic cleavage of CXCL12 is involved in trabecular pathophysiology, and that local administration of a selective CXCR3 antagonist may be a beneficial therapeutic strategy for treating ocular hypertension and subsequent retinal degeneration.

Show MeSH
Related in: MedlinePlus