Synergistic effect with Phe-Gly-Leu-Met-NH2 of the C-terminal of substance P and insulin-like growth factor-1 on epithelial wound healing of rabbit cornea

Abstract

1. We previously reported that substance P and insulin-like growth factor-1 (IGF-1) synergistically stimulate corneal epithelial wound healing in vitro and in vivo. We wished to identify which portion of the amino acid sequence of substance P might be responsible for this synergism. 2. Corneal epithelial migration was not affected by the addition of any one of the following factors: substance P; Phe-Gly-Leu-Met-NH2 (C-terminal of substance P); Val-Gly-Leu-Met-NH2 (C-terminal of neurokinin A, neurokinin B, and kassinin); Tyr-Gly-Leu-Met-NH2 (C-terminal of physalaemin); Ile-Gly-Leu-Met-NH2 (C-terminal of eledoisin); or Gly-Leu-Met-NH2 (common C-terminal of tachykinins). 3. In the presence of IGF-1, only substance P and Phe-Gly-Leu-Met-NH2 were synergistic in stimulating corneal epithelial migration in a dose-dependent fashion. 4. The combination of Phe-Gly-Leu-Met-NH2 and IGF-1 did not affect the incorporation of [3H]-thymidine into corneal epithelial cells. 5. Treatment with Phe-Gly-Leu-Met-NH2 and IGF-1, but not with Phe-Gly-Leu-Met-NH2 or IGF-1 alone, increased attachment of corneal epithelial cells to a fibronectin matrix. 6. The levels of alpha5 and beta1 integrin were not affected by Phe-Gly-Leu-Met-NH2 or IGF-1 alone, but they were significantly increased by the combination of Phe-Gly-Leu-Met-NH2 and IGF-1. 7. Topical application of the same combination facilitated corneal epithelial wound closure in vivo. 8. These results demonstrated that Phe-Gly-Leu-Met-NH2, a sequence of 4 amino-acids of the C-terminal of substance P, is the minimum sequence necessary to produce the synergistic effects of substance P and IGF-1 on corneal epithelial wound healing.

Figure 1

Characterization of rabbit corneal epithelial cells and fibroblasts. Cultured rabbit corneal epithelial cells (A and C) and fibroblasts (B and D) were immunostained with anti-cytokeratin antibody (AE1+AE3, epithelial cell marker) (A and B) or anti-vementin antibody (V9, fibroblastic cell marker) (C and D).

Figure 2

Synergistic effects with various types of C-terminal of tachykinin peptides with IGF-1 on corneal epithelial migration. Corneal blocks were cultured for 24 h in TC-199 containing various kinds of peptides (2×10⁻⁵ M) in the absence or presence of IGF-1 (10 ng ml⁻¹). Error bars represent the s.e.mean from six determinations. ^*P<0.01 versus the corneal blocks cultured without IGF-1.

Figure 3

Synergistic effects with sequence of 7 amino-acids of N-terminal of substance P (SP), SP(1–7), and sequence of 4 amino-acids of C-terminal of SP, SP(8–11), with IGF-1 on corneal epithelial migration. Corneal blocks were cultured for 24 h in TC-199 containing peptides (2×10⁻⁵ M) in the absence or presence of IGF-1 (10 ng ml⁻¹). Error bars represent the s.e.mean from six determinations. ^*P<0.01 versus the corneal blocks cultured without IGF-1.

Figure 4

Synergistic effects of Phe-Gly-Leu-Met-NH₂ (FGLM) with IGF-1 on corneal epithelial migration. Corneal blocks were cultured for 24 h in TC-199 containing: (A) FGLM (0.5, 1, 2×10⁻⁵ M) in the absence or presence of IGF-1 (10 ng ml⁻¹); (B) IGF-1 (0.1, 1, 10 ng ml⁻¹) in the absence or presence of FGLM (2×10⁻⁵ M). Error bars represent the s.e.mean from six determinations. ^*P<0.01 versus the corneal blocks cultured without FGLM or IGF-1.

Figure 5

Synergistic effects of substance P (SP) or Phe-Gly-Leu-Met-NH₂ (FGLM) with IGF-1 on corneal epithelial cell attachment to the fibronectin matrix. Corneal epithelial cells were cultured for 24 h in TC-199 containing SP or FGLM (2×10⁻⁵ M) in the absence or presence of IGF-1 (10 ng ml⁻¹). The dissociated epithelial cells were then plated on the fibronectin (coated at 10 μg ml⁻¹) plus BSA (1 mg ml-1) (A) or BSA matrix (B), and incubated for 45 min. Error bars represent the s.e.mean from three determinations. ^*P<0.01 versus the corneal epithelial cells cultured without IGF-1.

Figure 6

RT–PCR transcription analysis of integrin α5 and β1 expression in corneal epithelial cells after treatment with substance P (SP) or Phe-Gly-Leu-Met-NH₂ (FGLM) and/or IGF-1. Ethidium bromide-staining gels of PCR amplified products of reverse-transcribed epithelial cell RNA. A: None, B: SP (2×10⁻⁵ M), C: FGLM (2×10⁻⁵ M), D: IGF-1 (10 ng ml⁻¹), E: SP plus IGF-1, F: FGLM plus IGF-1.

Figure 7

Effects of substance P (SP) or Phe-Gly-Leu-Met-NH₂ (FGLM) and/or IGF-1 on the gene expression of integrin α5 (A) and integrin β1 (B) in cultured cornea epithelial cells using an RT–PCR. Corneal epithelial cells were cultured for 2 h in TC-199 containing SP or FGLM (2×10⁻⁵ M) and/or IGF-1 (10 ng ml⁻¹), and then RT–PCR was performed. Error bars represent the s.e.mean from three determinations. ^*P<0.05 versus the corneal epithelial cells cultured without SP, FGLM, or IGF-1.

Figure 8

Effects of substances P (SP), Phe-Gly-Leu-Met-NH₂ (FGLM), and/or IGF-1 on the area of the cornea epithelial defect. There were eight eyes per treatment group. Data are expressed as means±s.e.mean. ^*P<0.05; ^**P<0.01 compared with control group.