ID1 and ID3 are Negative Regulators of TGFβ2-Induced Ocular Hypertension and Compromised Aqueous Humor Outflow Facility in Mice

Abstract

Purpose: In POAG, elevated IOP remains the major risk factor in irreversible vision loss. Increased TGFβ2 expression in POAG aqueous humor and in the trabecular meshwork (TM) amplifies extracellular matrix (ECM) deposition and reduces ECM turnover in the TM, leading to a decreased aqueous humor (AH) outflow facility and increased IOP. Inhibitor of DNA binding proteins (ID1 and ID3) inhibit TGFβ2-induced fibronectin and PAI-1 production in TM cells. We examined the effects of ID1 and ID3 gene expression on TGFβ2-induced ocular hypertension and decreased AH outflow facility in living mouse eyes.

Methods: IOP and AH outflow facility changes were determined using a mouse model of Ad5-hTGFβ2C226S/C288S-induced ocular hypertension. The physiological function of ID1 and ID3 genes were evaluated using Ad5 viral vectors to enhance or knockdown ID1/ID3 gene expression in the TM of BALB/cJ mice. IOP was measured in conscious mice using a Tonolab impact tonometer. AH outflow facilities were determined by constant flow infusion in live mice.

Results: Over-expressing ID1 and ID3 significantly blocked TGFβ2-induced ocular hypertension (P < 0.0001). Although AH outflow facility was significantly decreased in TGFβ2-transduced eyes (P < 0.04), normal outflow facility was preserved in eyes injected concurrently with ID1 or ID3 along with TGFβ2. Knockdown of ID1 or ID3 expression exacerbated TGFβ2-induced ocular hypertension.

Conclusions: Increased expression of ID1 and ID3 suppressed both TGFβ2-elevated IOP and decreased AH outflow facility. ID1 and/or ID3 proteins thus may show promise as future candidates as IOP-lowering targets in POAG.

Figure 1.

Intravitreal injection of Ad5-TGFβ2^C226/228S elevates IOP. Groups of mice were injected with Ad5-hTGFβ2^C226/228S (n = 5 animals) or Ad5-Null (n = 8 animals) in their left eye (OS) on day = 0 after baseline IOP measurement. Their right eyes (OD) were used as uninjected controls. Error bars represent ± SEM. Significant difference between Ad5-hTGFβ2^C226/228S –injected versus Ad5-Null–injected (P < 0.0001) and uninjected (P < 0.0001) groups as indicated by two-factor ANOVA followed by Tukey's post-hoc test.

Figure 2.

ID1 Blocks TGFβ2-Induced IOP Elevation. Intravitreal injection of Ad5-null + Ad5-hTGFβ2 (n = 5) (OS) resulted in a significant elevation in IOP commencing at Day 8 post-injection, (P < 0.0001; two-factor ANOVA) compared with Ad5 Null + Ad5 Null, Ad5-hID1 + Ad5 Null, Ad5-hID1 + Ad5-hTGFβ2, or naïve (uninjected) eyes. Overexpression of ID1 completely abolished the IOP response to hTGFβ2. Furthermore, IOP in all groups of injected eyes with the exception of those injected with Ad5 Null + Ad5-hTGFβ2 was not significantly different from uninjected control eyes at any time point measured. Error bars represent ± SEM.

Figure 3.

ID3 blocks TGFβ2-induced IOP elevation. Intravitreal injection of Ad5 Null + Ad5-hTGFβ2 (n = 5) (OS) resulted in a significant elevation in IOP commencing at day 8 after injection, (P < 0.0001; two-factor ANOVA) compared with Ad5 Null + Ad5 Null, Ad5-hID3 + Ad5 Null, or Ad5-hID3 + Ad5-hTGFβ2, or naïve (uninjected) eyes. Overexpression of ID3 completely abolished the IOP response to hTGFβ2. Furthermore, IOP in all groups of injected eyes with the exception of those injected with Ad5 Null + Ad5-hTGFβ2 was not significantly different from uninjected control eyes at any time point measured. Error bars represent ± SEM.

Figure 4.

Knockdown of endogenous ID1 enhances TGFβ2-induced IOP elevation. Intravitreal injection of Ad5-hTGFβ2 (n = 5) (OS) resulted in a significant elevation in IOP on days 5 to 25 after injection (P < 0.0001) through day 28 (P < 0.05) (two-factor ANOVA) Ad5-mID1 shRNA + Ad5-null (n = 5), and Ad5-null + Ad5 null (n = 5) (OS) injected eyes. The IOP in Ad5-Null injected eyes (n = 5) (OS) was not significantly different from uninjected control eyes (OD) at any time point measured. Injection of Ad5-mID1 shRNA + Ad5-Null (n = 5) (OS) led to a more modest but still-significant increase in IOP that manifested from day 5 (P < 0.01) until day 22 (P < 0.0001) as compared with Ad5-null injected (n = 5) eyes (OS). Intravitreal injection of Ad5-mID1-shRNA + Ad5-hTGFβ2 (n = 5) (OS) enhanced the TGFβ2-mediated IOP elevation significantly at day 12 (P < 0.01) to day 22 (P < 0.0001). Error bars represent ± SEM.

Figure 5.

Knockdown of endogenous ID3 enhances TGFβ2-induced IOP elevation. Intravitreal injection of Ad5 Null + Ad5-hTGFβ2 (n = 5) (OS) resulted in a significant elevation in IOP, on days 5 to 23 after injection, (P < 0.0001; 2-factor ANOVA) until day 28 (P < 0.05), compared with Ad5-mID3 shRNA + Ad5-null (n = 5), and Ad5-null + Ad5 null (n = 5) (OS) injected eyes. The IOP in Ad5-Null injected eyes (n = 5) (OS) was not significantly different from uninjected control eyes (OD) at any time point measured. Injection of Ad5-mID3 shRNA + Ad5-Null (n = 5) (OS) led to a more modest but still-significant increase in IOP that manifested from day 5 (P < 0.01) until day 22 (P < 0.0001) as compared with Ad5-null injected (n = 5) eyes (OS). Intravitreal injection of Ad5-mID3-shRNA + Ad5-hTGFβ2 (n = 5) (OS) enhanced the TGFβ2-mediated IOP elevation significantly at day 12 (P < 0.01) to day 22 (P < 0.0001). Error bars represent ± SEM.

Figure 6.

ID1 and ID3 block TGFβ2-induced reduction in outflow facility. At day 21 after injection, animals from groups 2, 4, and 6 (Table 1B) were selected for AH outflow facility studies. Animals injected with Ad5-Null + Ad5-hTGFβ2^C226/228S exhibited a significant decrease in AH outflow facility in injected (OS) eyes as compared to their uninjected contralateral control (OD) eyes (n = 10 animals) (P < 0.05, paired Student's t-test). Animals injected with Ad5-hID1 + Ad5-hTGFβ2^C226/228S (n = 8 animals) (OS) exhibited no significant change in AH outflow facility as compared to their uninjected contralateral control (OD) eyes. Animals injected with Ad5-hID3 + Ad5-hTGFβ2^C226/228S (n = 10 animals) (OS) exhibited no significant change in AH outflow facility as compared to their uninjected contralateral control (OD) eyes. Individual data points are plotted. Horizontal bars within boxes represent mean. Limit of boxes above and below mean represent 95% confidence interval of the mean.

Figure 7.

ID1 and ID3 proteins block TGFβ2-mediated effects on AH outflow facility and IOP. Overexpression of TGFβ2 decreases AH outflow facility and increases IOP mouse eyes. Concurrent overexpression of ID1 and ID3 block these effects.