Form-deprivation myopia induces decreased expression of bone morphogenetic protein-2, 5 in guinea pig sclera

Abstract

Aim: To identify the presence of various bone morphogenetic proteins (BMPs) and their receptors in normal sclera of human, rat and guinea pigs, and to determine whether their expression changed with form-deprivation myopia (FDM) in guinea pig sclera.

Methods: The expression of BMPs and BMP receptors were detected using reverse transcription polymerase chain reaction (RT-PCR) and immunofluorescence. Two-week-old guinea pigs were monocularly form-deprived with a translucent lens. After fourteen days induction of FDM, total RNA was isolated and subjected to RT-PCR to examine the changes of BMPs and BMP receptors in tissues from the posterior sclera. Western blotting analysis was used to investigate their changes in protein levels.

Results: Human sclera expressed mRNAs for BMP-2, -4, -5, -7, -RIA, -RIB and BMP-RII. Conversely, rat sclera only expressed mRNA for BMP-7 and BMP-RIB, while the expression of BMPs and BMP receptors in guinea pigs were similar to that of humans. Human sclera also expresses BMP-2, -4, -5,-7 in protein level. Fourteen days after the induction of myopia, significant decreased expressions for BMP-2 and BMP-5 in the posterior sclera of FDM-affected eyes (P<0.05 vs internal control eyes).

Conclusion: Various BMPs were expressed in human and guinea pig sclera. In the posterior sclera, expressions of BMP-2 and BMP-5 significantly decreased in FDM eyes. This finding indicates that various BMPs as components of the scleral cytokines regulating tissue homeostasis and provide evidence that alterations in the expression of BMP-2 and BMP-5 are associated with sclera remodeling during myopia induction.

Keywords: bone morphogenetic protein; form-deprivation myopia; guinea pig sclera; human sclera.

Figure 1. Semi-quantitative RT-PCR analysis of total RNA from 3 normal human sclera, 3 normal guinea pig sclera and 3 normal rat sclera using specific primers for BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMPR I A, BMPR I B, BMPR II normalized to β-actin at 35 cycles (M: Molecular size marker).

Figure 2. Distribution of BMPs in guinea pig sclera by indirect immunofluorescence

FITC marked the secondary antibody.

Figure 3. Differences in refractive error and axial length after induction of myopia

Differences in refractive error (A) and axial length (B) among FDM (group2), internal control (group1) and normal (group 3) eyes of guinea pigs is illustrated. At 14d, monocularly deprivation produced relative myopia, and axial lengths of FDM eyes were significantly greater than the others. The asterisk ‘a’ denotes ^bP <0.001.

Figure 4. Effect of myopia induction on the mRNA expression for BMP-2 to BMP-7 and BMPRs in guinea pigs sclera

2wk after visual deprivation, Semi-quantitative RT-PCR showed a significant decrease in mRNA expression in BMP-2 (-26.48%, P =0.017) and BMP-5 (-18.67%, P =0.028) but not for BMP-4 compared with the internal control eyes(C) in the same animals. Bar graph shows changes in mRNA expression of BMP subtypes 2 to 7 and BMPRs in the posterior sclera during FDM (F) in guinea pigs. Values (mean±SD) were normalized for β-actin and expressed as ratios of optical density. ^aP<0.05.

Figure 5. Effect of myopia induction on the protein expression of BMP-2,-4,-5. Western blotting showed a significant decrease in protein expression for BMP-2 and BMP-5 but not BMP-4, compared with the internal control (C) eyes

Values (mean±SD) were normalized for β-actin and expressed as ratios of optical density. ^aP<0.05.