Differential effects of angiotensin II type I receptor blockers on reducing intraocular pressure and TGFβ signaling in the mouse retina

Abstract

Purpose: Angiotensin II type 1 receptor blockers (ARBs) have been investigated for their neuroprotective and intraocular pressure (IOP) lowering effects in treating glaucoma, but the reports have been inconsistent possibly because different compounds and models have been used. Here we selected three ARBs for head-to-head comparisons of their effects on IOP and transforming growth factor β (TGFβ) signaling, which is believed to play an important role in glaucoma pathogenesis.

Methods: Three ARBs (losartan, irbesartan or telmisartan) or vehicle controls were administered via chow to C57BL/6J mice for up to 7 days. Drug concentrations in the eye, brain, and plasma were evaluated by liquid chromatography mass spectrometry. Cohorts of mice were randomly assigned to different treatments. IOP and blood pressure were measured before and after ARB treatment. Effects of ARBs on TGFβ signaling in the retina were evaluated by phosphorylated Smad2 (pSmad2) immunohistochemistry.

Results: Physiologically relevant concentrations of losartan, irbesartan and telmisartan were detected in eye, brain and plasma after drug administration (n = 11 mice/treatment). Blood pressure was significantly reduced by all ARBs compared to vehicle-fed controls (all p-values < 0.001, n = 8-15 mice/treatment). Compared to vehicle control, IOP was significantly reduced by irbesartan (p = 0.030) and telmisartan (p = 0.019), but not by losartan (n = 14-17 mice/treatment). Constitutive pSmad2 fluorescence observed in retinal ganglion cells was significantly reduced by telmisartan (p = 0.034), but not by losartan or irbesartan (n = 3-4 mice/treatment).

Conclusions: Administration via chow is an effective delivery method for ARBs, as evidenced by lowered blood pressure. ARBs vary in their abilities to lower IOP or reduce TGFβ signaling. Considering the significant roles of IOP and TGFβ in glaucoma pathogenesis, specific ARBs with dual effects, such as telmisartan, may be more effective than other ARBs for treating glaucoma.

Fig 1. Tissue distribution of losartan after delivery via drinking water.

Concentrations of losartan (left) and its active metabolite, EXP 3174 (right) in the plasma (red symbols) and eye (blue symbols) were determined by LC/MS after 3 days of treatment with 1.2 g/L losartan in water available ad libidum. At different time points, mice (n = 3) were sacrificed and eyes and plasma were collected for LC/MS. Approximate peak concentrations were determined by gathering samples at the end of the active nocturnal phase. Approximate trough concentrations were determined by gathering samples at the end of the inactive daytime phase. Symbols represent mean/SD.

Fig 2. Tissue distribution of ARBs after delivery via chow.

Mice (n = 11) were fed chow containing losartan, irbesartan or telmisartan available ad libidum. After 3 days, mice were sacrificed and eyes, brain, and plasma were collected for LC/MS analysis of drug concentrations. Irbesartan, telmisartan, losartan and EXP 3174 were detected in eyes, brain, and plasma samples. Solid symbols represent data from individual mice, with mean indicated as horizontal line for each group. Data are visualized on logarithmic scale.

Fig 3. Systolic BP before and after treatment with ARBs for 3 days.

Systolic BP was measured before (day 0) and after (day 3) feeding mice chow containing losartan (n = 15, green), irbesartan (n = 15, blue) or telmisartan (n = 15, violet) at 2 g drug/kg chow, or normal chow (n = 8, orange) available ad libidum. Compared to mice fed normal chow, mice fed losartan, irbesartan or telmisartan chow had significantly lower BP (p < 10⁻⁴). Box plots show the median (thick line), first and third quatriles (lower and upper box sides), with vertical lines representing 5^th and 95^th percentiles. Data outside the 5^th to 95^th percentiles are shown as individual data points (black symbols).

Fig 4. Change in IOP after treatment with ARBs for 3 and 7 days.

Significant reductions of IOP were found for mice treated with irbesartan (p = 0.016 and 0.013) and telmisartan (p = 0.012 and 0.008) at day 3 and 7, respectively, compared to mice fed normal chow, but not for losartan treated mice. IOP decreased significantly faster for mice treated with irbesartan (p = 0.030) and telmisartan (p = 0.019), compared to mice fed normal chow, while losartan had no significant effect. Symbols and error bars represent mean and 95% confidence intervals; orange: normal chow, green: losartan, blue: irbesartan, purple: telmisartan. Data are from 14–17 mice/treatment for days 0 and 3 and 10 mice/treatment for day 7. Median IOP values for day 0 were 17.2, 16.9, 17.0 and 16.7 for normal, losartan, irbesartan and telmisartan-treated mice, respectively.

Fig 5. Reduced TGFβ signal transduction in the RGC layer of ARB-treated mice.

(A) Representative immunostaining for pSmad2 (red, left column) and DAPI-staining of cell nuclei (blue, middle column) shows nuclear pSmad2 in overlay images (pink, right column). Pattern indicates constitutive TGFβ signal transduction in the inner nuclear and RGC layers of mice fed normal chow (upper row) that is reduced in mice fed ARB-containing chows (lower three rows), most strongly by telmisartan (lower row). IgG negative control (B) shows lack of non-specific staining. Quantification of pSmad2 fluorescence (red) in the RGC layer (C) shows statistically significant reduction in telmisartan-treated mice, with a 70% reduction compared to normal fed mice (p = 0.034). Results are from duplicate experiments from one eye of each individual mouse; n = 4 for normal, irbesartan and telmisartan; n = 3 for losartan. Symbols represent the average pSmad2 red fluorescence for each mouse with mean/SD for each treatment shown in (C). Retinal layers are indicated (upper right panel, A): RGC = RGC layer; IPL = inner plexiform layer; INL = inner nuclear layer. Scale bars = 50 μm.