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. 2021 Aug 30;22(17):9408.
doi: 10.3390/ijms22179408.

Pemafibrate Prevents Retinal Dysfunction in a Mouse Model of Unilateral Common Carotid Artery Occlusion

Deokho Lee  1   2 , Yohei Tomita  1   2   3 , Heonuk Jeong  1   2 , Yukihiro Miwa  1   2   4 , Kazuo Tsubota  5 , Kazuno Negishi  2 , Toshihide Kurihara  1   2
Affiliations

Pemafibrate Prevents Retinal Dysfunction in a Mouse Model of Unilateral Common Carotid Artery Occlusion

Deokho Lee et al. Int J Mol Sci. .

Abstract

Cardiovascular diseases lead to retinal ischemia, one of the leading causes of blindness. Retinal ischemia triggers pathological retinal glial responses and functional deficits. Therefore, maintaining retinal neuronal activities and modulating pathological gliosis may prevent loss of vision. Previously, pemafibrate, a selective peroxisome proliferator-activated receptor alpha modulator, was nominated as a promising drug in retinal ischemia. However, a protective role of pemafibrate remains untouched in cardiovascular diseases-mediated retinal ischemia. Therefore, we aimed to unravel systemic and retinal alterations by treating pemafibrate in a new murine model of retinal ischemia caused by cardiovascular diseases. Adult C57BL/6 mice were orally administered pemafibrate (0.5 mg/kg) for 4 days, followed by unilateral common carotid artery occlusion (UCCAO). After UCCAO, pemafibrate was continuously supplied to mice until the end of experiments. Retinal function (a-and b-waves and the oscillatory potentials) was measured using electroretinography on day 5 and 12 after UCCAO. Moreover, the retina, liver, and serum were subjected to qPCR, immunohistochemistry, or ELISA analysis. We found that pemafibrate enhanced liver function, elevated serum levels of fibroblast growth factor 21 (FGF21), one of the neuroprotective molecules in the eye, and protected against UCCAO-induced retinal dysfunction, observed with modulation of retinal gliosis and preservation of oscillatory potentials. Our current data suggest a promising pemafibrate therapy for the suppression of retinal dysfunction in cardiovascular diseases.

Keywords: common carotid artery occlusion; electroretinography; fibroblast growth factor 21; pemafibrate; peroxisome proliferator-activated receptor alpha; retinal ischemia.

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Conflict of interest statement

Yukihiro Miwa is employed by Tokyo Animal Eye Clinic and Kazuo Tsubota is CEO in Tsubota Laboratory, Inc. The remaining authors declare no conflict of interest.

Figures

Figure A1
Figure A1
General monitoring for adult mice after consecutive oral administration of pemafibrate. A schematic illustration shows oral administration of pemafibrate (0.5 mg/kg/day) to mice and a time point of the UCCAO surgery and experiments followed. ELISA; enzyme-linked immunosorbent assay, qPCR; quantitative PCR, BW; body weight, IHC; immunohistochemistry, LW; liver weight, ERG; electroretinography, OCT; optical coherence tomography, UCCAO; unilateral common carotid artery occlusion. A schematic illustration of retinal ischemia induction by UCCAO. Retinal ischemia could be induced by occlusion (a blue bar) of the common carotid artery (CCA) as the ophthalmic artery (OpA) is originated from the internal carotid artery (ICA) of CCA. ECA; external carotid artery. Quantitative analyses (n = 5–10 per group) showed that the body weight of mice became lower after UCCAO. There was no dramatic difference in the body weight between pemafibrate-administered mice and PBS-administered mice. However, mice showed a slight decrease in the body weight after consecutive administration of pemafibrate without any statistical significance. p > 0.05. The data were analyzed using two-way ANOVA followed by a Bonferroni post hoc test. The data were presented as mean ± standard deviation. Pema; pemafibrate. ns; not significant.
Figure A2
Figure A2
General measurements of retinal function for adult mice after consecutive oral administration of pemafibrate right before UCCAO. Quantitative analyses (n = 5 per group) showed that oral administration of pemafibrate had no effect on retinal function (a-wave, b-wave, and OPs) in adult naïve mice. p > 0.05. The data (a-and b-waves) were analyzed using two-way ANOVA followed by a Bonferroni post hoc test and presented as mean ± standard error of the mean. The data (OPs) were analyzed using two-tailed Student’s t-test and presented as mean ± standard deviation. Pema; pemafibrate, OPs; oscillatory potentials. ns; not significant.
Figure A3
Figure A3
A slight increase in retinal synaptophysin (SYP) expression by oral administration of pemafibrate in UCCAO-operated mice. A representative image and quantitative analysis (n = 4 per group) showed that SYP expression slightly increased by oral administration of pemafibrate 10 days after UCCAO. p = 0.06. The data were analyzed using Student’s t-test and presented as mean ± standard deviation. Pema; pemafibrate, UC; unilateral common carotid artery occlusion.
Figure A4
Figure A4
No alteration in retinal thickness by oral administration of pemafibrate in UCCAO-operated mice. Representative OCT images (b-scan) in the PBS-and pemafibrate-administered UCCAO-operated retinas and quantitative analyses (n = 5 per group) showed that there was no change in retinal thickness (total, outer, and inner retinal layers) on day 10 after UCCAO. The values in the horizontal axis of the graph stand for 0.2, 0.4, and 0.6 mm distance from the optic nerve head (0) that was detected by the green line (en-face scan). Representative OCT images were taken at 0.4 mm from the optic nerve head. The data were analyzed using two-way ANOVA followed by a Bonferroni post hoc test and presented as a spider diagram (mean ± standard deviation). p > 0.05. Scale bars are 200 (en-face scan; vertical and horizontal bars) and 200 and 100 (b-scan; vertical and horizontal bars) µm, respectively. Pema; pemafibrate, OCT; optical coherence tomography. ns; not significant.
Figure A5
Figure A5
A relationship between pathological retinal gliosis and retinal dysfunction 7 days after UCCAO. Representative images of morphology scoring for pathological retinal gliosis (acquired from our preliminary experiments) and visualization in a correlation between pathological retinal gliosis and the amplitudes of ΣOPs showed that there was a high correlation between pathological gliosis and loss of the amplitudes of ΣOPs in the UCCAO-operated eye, determined by regression analyses. Dots (n = 8) represent each morphology scoring and the amplitude of ΣOPs. A line represents the linear fit of the data points. Scale bar: 50 µm. Slope: −0.0028; Y-intercept: 3.06; r2: 0.68. p < 0.05. GCL: ganglion cell layer; INL: inner nuclear layer; ONL: outer nuclear layer, UC; unilateral common carotid artery occlusion.
Figure 1
Figure 1
Protective effects of pemafibrate against retinal dysfunction on day 5 after UCCAO. (A,B) Representative waveforms (10 cd·s/m2) of a-and b-waves and quantitative analyses (n = 9–10 per group) showed that oral administration of pemafibrate had a slight suppressing tendency in a reduction in the amplitudes of a-wave and b-wave in the UCCAO-operated eye 5 days after UCCAO. The data were analyzed using two-way ANOVA followed by a Bonferroni post hoc test. The data were presented as mean ± standard error of the mean. (C,D) Representative waveforms (10 cd·s/m2) of oscillatory potentials (OPs) and quantitative analyses showed that pemafibrate significantly suppressed reduction in the amplitudes of OPs (OP1, OP2, OP3, and ΣOPs) in UCCAO-induced retinal ischemic mice (n = 9–10 per group). * p < 0.05. The data were analyzed using two-tailed Student’s t-test. The data were presented as mean ± standard deviation. Pema; pemafibrate. UC; unilateral common carotid artery occlusion. ns; not significant.
Figure 2
Figure 2
Protective effects of pemafibrate against retinal dysfunction 10 days after UCCAO. (A,B) Representative waveforms (10 cd·s/m2) of a-and b-waves and quantitative analyses (n = 5 per group) showed that oral administration of pemafibrate had a suppressing tendency in the reduction in the amplitudes of a-wave and b-wave in the UCCAO-operated eye 10 days after UCCAO with statistical significance. The data were analyzed using two-way ANOVA followed by a Bonferroni post hoc test (a-wave and b-wave). One datum was further analyzed using two-tailed Student’s t-test (b-wave; p = 0.06). The data were presented as mean ± standard error of the mean. (C,D) Representative waveforms (10 cd·s/m2) of oscillatory potentials (OPs) and quantitative analyses showed that pemafibrate significantly suppressed reduction in the amplitudes of OPs (OP1, OP2, OP3, and ΣOPs) in UCCAO-induced retinal ischemic mice (n = 5 per group). * p < 0.05, ** p < 0.01. The data were analyzed using two-tailed Student’s t-test. The data were presented as mean ± standard deviation. Pema; pemafibrate. UC; unilateral common carotid artery occlusion. ns; not significant.
Figure 3
Figure 3
Modulation of pathological reactive gliosis after oral administration of pemafibrate. (A) Representative images and quantitative analyses (n = 4 per group) showed that slightly increased reactive retinal gliosis stained by GFAP in UCCAO-operated mice was reduced by administration of pemafibrate on day 2 after UCCAO. (B) Representative images and quantitative analyses (n = 4–5 per group) showed that dramatically increased reactive retinal gliosis stained by GFAP in UCCAO-operated mice were reduced by the administration of pemafibrate on day 5 after UCCAO. Scale bar: 50 µm. The data were analyzed using two-tailed Student’s t-test. Graphs were presented as mean with ± standard deviation. * p < 0.05. GCL: ganglion cell layer; INL: inner nuclear layer; ONL: outer nuclear layer. Pema; pemafibrate. UC; unilateral common carotid artery occlusion.
Figure 4
Figure 4
Screening of alterations in retinal hypoxia-ischemia-related gene expressions by oral administration of pemafibrate in UCCAO-operated mice. Primarily, genes reported to be slightly or dramatically altered after UCCAO were selected; Epo, Bnip3, Vegfa, Ccl2, Ccl12, and Glut1. Quantitative analyses (n = 6 per group) showed that oral administration of pemafibrate significantly reduced the expression of Glut1 in the retina 1 day after UCCAO. However, the other genes’ expressions were not changed by oral administration of pemafibrate. *** p < 0.001. The data were analyzed using two-tailed Student’s t-test and presented as mean ± standard deviation. Pema; pemafibrate. ns; not significant.
Figure 5
Figure 5
Induction in PPARα downstream gene expressions in the liver by oral administration of pemafibrate in UCCAO-operated mice. (A) Quantitative analyses (n = 4–6 per group) showed that oral administration of pemafibrate did not dramatically increase PPARα downstream gene expressions in the retina. The data were analyzed using two-tailed Student’s t-test and presented as mean ± standard deviation. (B) Quantitative analyses (n = 4–6 per group) showed that the relative liver weight (the liver weight/the body weight) in pemafibrate-administered mice was significantly higher than that in PBS-administered mice. The data were analyzed using two-tailed Student’s t-test and were presented as mean ± standard error of the mean. (C) Quantitative analyses (n = 4–5 per group) showed that oral administration of pemafibrate significantly increased PPARα downstream gene expressions in the liver. The data were analyzed using two-tailed Student’s t-test and presented as mean ± standard deviation. The value for PBS-administered mice was indicated as a dotted line; 1. * p < 0.05, ** p < 0.01, *** p < 0.001. Pema; pemafibrate, UC; unilateral common carotid artery occlusion. ns; not significant.
Figure 6
Figure 6
Changes in serum levels of FGF21, TG, and TC by oral administration of pemafibrate in UCCAO-operated mice. (A) Quantitative analyses (n = 3–9 per group) showed that oral administration of pemafibrate significantly increased serum levels of FGF21. The data were analyzed using two-tailed Student’s t-test and presented as mean ± standard error of the mean. (B,C) Quantitative analyses (n = 3–8 per group) showed that oral administration of pemafibrate significantly decreased serum levels of TG and increased serum levels of TC. The data were analyzed using two-tailed Student’s t-test and presented as mean ± standard error of the mean. * p < 0.05, ** p < 0.01, *** p < 0.001. Pema; pemafibrate, UC; unilateral common carotid artery occlusion, TG; triglyceride, TC; total cholesterol.
Figure 7
Figure 7
A working hypothesis of the protective mechanism against retinal dysfunction by administering pemafibrate in a murine model of retinal ischemia by UCCAO. The possible mechanisms for suppression of retinal dysfunction induced in cardiovascular diseases are that consecutive administration of systemic selective PPARα modulator (SPPARMα) pemafibrate enhances liver function and upregulates PPARα target genes in the liver, and elevated levels of serum FGF21 (one of the strong neuroprotective agents) modulate pathological gliosis and maintain the amplitudes of OPs. Indirectly, a reduction in levels of TG and an induction in levels of TC may have a risk-decreasing effect on developing retinopathy in humans. TG; triglyceride, TC; total cholesterol.
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