Changes in growth factor expression in normal aging of the rat retina

Abstract

Although much is known about the growth factor changes in ocular tissues during various diseases, little is known about normal aging of the retina. In order to further understand normal aging in the retina, we characterized age-related changes of growth factor expression in three different ages of rat retina. Real time PCR and protein analysis was conducted to investigate steady state mRNA expression and protein levels of VEGF, VEGFR2, PEDF, Ang-1, Tie-2, EphB4 and ephrinB2 in the retina of 8-, 22-, and 32-month-old Brown Norway X Fischer 344 F1 hybrid rats. An increase of VEGF protein levels was found at 32months compared to 8 and 22months of age. VEGFR2 protein was found to be increased at 22 and 32months compared to 8months. PEDF protein levels were reduced at 22 and 32months. Tie-2 levels were found to be significantly decreased by 32months compared to 8months of age, while ephrinB2 was found to be significantly lower at both 22 and 32months compared to 8months of age. The increases found in VEGF and its receptor VEGFR2, with the simultaneous decrease of PEDF protein levels, may stimulate an environment that is well suited for neovascularization in the normal aging retina. Overall, these results suggest that normal aging produces substantial changes in gene expression and protein levels.

Figure 1

(A) Bar graph of real time PCR steady state mRNA expression of VEGF in the rat retina. One-way ANOVA tests revealed no significant change with age (N=6). (B) ELISA assay analysis comparing VEGF protein levels at 8, 22, and 32 months of age. Protein is significantly upregulated at 32 months compared to both 8 and 22 months of age (N=6, *P<0.05, ^#P<0.01).

Figure 2

(A) Bar graph of real time PCR steady state mRNA expression of VEGFR2 in the retina found no significant change (N=6). (B) Representative western blot analysis and densitometry found protein expression of VEGFR2 to be significantly increased at 22 months and 32 months of age compared to 8 months of age (N=6, *P<0.05).

Figure 3

(A) Bar graph of PEDF real time PCR steady state mRNA expression found no significant changes at different ages (N=6). (B) Representative western blot and densitometry analysis found protein levels of PEDF to be significantly decreased at 22 and 32 months compared to 8 months of age (N=5, *P<0.01).

Figure 4

(A) Bar graph of real time steady state PCR mRNA expression of Ang-1 in the rat retina revealed a significant increase at 32 months compared to both 8 and 22 months of age (N=6). (B) ELISA assay analysis of Ang-1 protein concluded that there is no change with age (N=6, *P<0.01).

Figure 5

(A) Bar graph of Tie-2 real time PCR steady state mRNA expression representing a significant upregulation at 32 months compared to 8 months of age (N=6, *P<0.05). (B) Representative western blot analysis and densitometry found Tie-2 protein levels to be significantly decreased at 32 months compared to 8 months of age (N=6, *P<0.05).

Figure 6

(A) Bar graph of real time PCR steady state mRNA expression of ephrinB2 showed a trend to decrease from 8 months to 32 months of age. However, it did not decrease to a point of significance (N=7). (B) Representative western blot analysis and densitometry showed a significant decrease of ephrinB2 protein levels at 22 months and 32 months compared to 8 months of age (N=6, *P<0.01).

Figure 7

(A) Bar graph of real time PCR steady state mRNA expression of EphB4 found a significant increase at 22 months compared to 8 months. By 32 months of age, steady state mRNA expression had fallen significantly from 22 months to its 8 month expression level (N=4, *P<0.01, ^#P<0.01). (B) Representative western blot analysis and densitometry showed no significant change in protein levels in the retina as the rats age (N=6).