Critical role of SDF-1α-induced progenitor cell recruitment and macrophage VEGF production in the experimental corneal neovascularization

Abstract

Purpose: To address the roles of the stromal derived factor-1 (SDF-1) α in the course of experimental corneal neovascularization (CNV).

Methods: CNV was induced by alkali injury and compared in SDF-1α- or vehicle-treated mice two weeks after injury. Angiogenic factor expression in the early phase after injury was quantified by reverse transcription polymerase chain reaction (RT-PCR). Progenitor cell, macrophage, and monocyte intracorneal accumulation in the early phase after injury was evaluated by flow cytometric analysis.

Results: The mRNA expression of SDF-1α was augmented, together with infiltration of c-kit-positive progenitor cells in the corneas after the alkali injury. Compared with vehicle-treated mice, SDF-1α-treated mice exhibited enhanced CNV two weeks after injury, as evidenced by enlarged cluster of differentiation 31 (CD31)-positive areas. Concomitantly, the intracorneal infiltration of c-kit-positive progenitor cells but not F4/80+ macrophages or Ly-6G+ monocytes was significantly enhanced in SDF-1α-treated mice compared to vehicle-treated mice. SDF-1α enhanced vascular endothelial growth factor (VEGF) expression by murine peritoneal macrophages. Enhancement in intraocular VEGF expression was greater in SDF-1α-treated mice than in control mice after injury. Moreover, local administration of C-X-C chemokine receptor type 4 (CXCR4) antagonist after alkali injury reduced alkali-induced CNV.

Conclusions: SDF-1α-treated mice exhibited enhanced alkali-induced CNV through enhanced intracorneal progenitor cell infiltration and increased VEGF expression by macrophages.

Figure 1

SDF-1α mRNA expression in corneas after alkali injury. A: Semi-quantitative RT–PCR to evaluate mRNA expression of SDF-1α. Corneas were harvested at the indicated time points, and five corneas at each time point were pooled to extract total RNAs. RT–PCR was performed using the obtained total RNAs. B: The ratios of SDF-1α to Actb mRNA were determined. All values represent mean±SEM of three to five independent measurements.

Figure 2

The effects of SDF-1α on Alkali injury-induced CNV. A: Macroscopic appearance of vehicle- or SDF-1α-treated BABL/c mouse eyes 2 weeks after alkali injury. B: Corneal tissues were obtained 2 weeks after injury from vehicle- or SDF-1α-treated BABL/c mice. Tissues were immunostained with anti-CD31 antibody, and representative results from five to eight animals are shown here. Original magnifications, 200×. Scale bar, 50 μm. C: CNV numbers per square millimeter in whole section. D: CNV numbers per square millimeter in hot spots. E: Proportions of CNV areas in hot spots were determined from the corneas obtained from vehicle- or SDF-1α-treated BABL/c mice 2 weeks after injury. Each value represents mean±SEM (n=5–8 animals). *, p<0.05 compared with vehicle-treated mice.

Figure 3

c-kit-, F4/80- or Ly-6G-positive cell numbers in cornea after alkali injury. Corneal tissues were obtained 4 days after injury from vehicle- or SDF-1α-treated BABL/c mice, and the tissues from 7 to 8 mice were combined and were subjected to analysis using a flow cytometer after being immunostained with anti-c-kit, anti-F4/80, or anti-Ly-6G antibody. Isotype IgG derived from the same species of the test antibody was used as negative control. Representative results from three to four tests of intracorneal infitlration of c-kit- (A), F4/80- (B), or Ly-6G-positive cells (C) from either vehicle- (left plot) or SDF-1α-treated mice (right plot) are shown.

Figure 4

VEGF expression in the injured corneas of mice. A: Semi-quantitative RT–PCR to evaluate mRNA expression of VEGF. Corneas were harvested at the indicated time points, and five corneas at each time point were pooled to extract total RNAs. RT–PCR was performed using the obtained total RNAs. Representative results from three independent are shown here. B: The ratios of VEGF to Actb mRNA were determined on vehicle- (open bars) and SDF-1α-treated BABL/c mice (black bars). All values represent mean±SEM of three to five independent measurements. C: Protein extracts were obtained and subjected to western blotting analysis. Representative results from three independent experiments are shown here. D: Ratios of VEGF to β-actin protein bands of vehicle- (open bars) and SDF-1α-treated mice (black bars) were determined as described in Methods. All values represent mean±SEM (n=5–8 animals). *, p<0.05 and ^#, p<0.01 compared with vehicle-treated mice.

Figure 5

SDF-1α-induced VEGF production by peritoneal macrophages. A: CXCR4 expression on F4/80-positive murine peritoneal macrophages was determined by a flow cytometric analysis. Purified mononuclear cells were stained with rat anti-mouse F4/80 mAb and rabbit anti-CXCR4 Abs (filled histogram) or rat anti-mouse F4/80 mAb and non-immunized rabbit IgG (open heavy-lined histogram) as a control followed by staining with FITC-conjugated goat anti-rabbit IgG and PE-conjugated swine anti-rat IgG. A representative result from three independent experiments is shown. B: Peritoneal macrophages from WT mice were incubated with the indicated concentrations of SDF-1α for 12 h. Quantitative RT–PCR was performed on total RNAs extracted from the macrophages as described in Methods. Representative results from 3 independent experiments are shown here. C: VEGF mRNA levels were determined and normalized to Actb mRNA levels. Each value represents the mean and SEM (n=3). ^#, p<0.01 compared with untreated. D: VEGF protein expression was detected by immunocytochemical analysis using anti-VEGF Abs as described in Methods. Representative results from three independent experiments are shown. Original magnification, 400×. Scale bar, 50 μm.

Figure 6

The effects of CXCR4 antagonist on alkali-induced CNV. CNV numbers per square millimeter in whole section (A), CNV numbers per square millimeter in hot spots (B), and percentage CNV areas in hot spots (C) were determined from the corneas obtained from vehicle- or CXCR4 antagonist-treated BABL/c mice 2 weeks after injury. Each value represents mean±SEM (n=5–8 animals). p<0.05 compared with vehicle-treated mice.