Endothelial PRMT5 plays a crucial role in angiogenesis after acute ischemic injury
Qing Ye, Jian Zhang, Chen Zhang, Bing Yi, Kyosuke Kazama, Wennan Liu, Xiaobo Sun, Yan Liu, Jianxin Sun
Qing Ye, Jian Zhang, Chen Zhang, Bing Yi, Kyosuke Kazama, Wennan Liu, Xiaobo Sun, Yan Liu, Jianxin Sun
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Research Article Angiogenesis Vascular biology

Endothelial PRMT5 plays a crucial role in angiogenesis after acute ischemic injury

Abstract

Arginine methylation mediated by protein arginine methyltransferases (PRMTs) has been shown to be an important posttranslational mechanism involved in various biological processes. Herein, we sought to investigate whether PRMT5, a major type II enzyme, is involved in pathological angiogenesis and, if so, to elucidate the molecular mechanism involved. Our results show that PRMT5 expression is significantly upregulated in ischemic tissues and hypoxic endothelial cells (ECs). Endothelial-specific Prmt5-KO mice were generated to define the role of PRMT5 in hindlimb ischemia–induced angiogenesis. We found that these mice exhibited impaired recovery of blood perfusion and motor function of the lower limbs, an impairment that was accompanied by decreased vascular density and increased necrosis as compared with their WT littermates. Furthermore, both pharmacological and genetic inhibition of PRMT5 significantly attenuated EC proliferation, migration, tube formation, and aortic ring sprouting. Mechanistically, we showed that inhibition of PRMT5 markedly attenuated hypoxia-induced factor 1-α (HIF-1α) protein stability and vascular endothelial growth factor–induced (VEGF-induced) signaling pathways in ECs. Our results provide compelling evidence demonstrating a crucial role of PRMT5 in hypoxia-induced angiogenesis and suggest that inhibition of PRMT5 may provide novel therapeutic strategies for the treatment of abnormal angiogenesis-related diseases, such as cancer and diabetic retinopathy.

Authors

Qing Ye, Jian Zhang, Chen Zhang, Bing Yi, Kyosuke Kazama, Wennan Liu, Xiaobo Sun, Yan Liu, Jianxin Sun

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Figure 5

Inhibition of PRMT5 attenuates endothelial cell proliferation and migration.

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Inhibition of PRMT5 attenuates endothelial cell proliferation and migrat...
(A) MLECs isolated from Prmt5fl/fl and EC-Prmt5Δ/Δ mice were cultured in complete ECM and were counted at days 0 and 14. ***P < 0.001, using 2-tailed Student’s t test. n = 4. (B) HUVECs were incubated with 0.1% DMSO or 10.0 μM EPZ015666 for indicated days, and the cell numbers were calculated by hemocytometer. **P < 0.01 and ***P < 0.001 versus 0.1% DMSO at same time point, using 2-tailed Student’s t test. n = 3. (C) HUVECs were seeded into a 96-well plate at 5%–10% density and incubated with either 0.1% DMSO or EPZ015666 at indicated doses for 0, 1, 2, 3, 4, and 6 days. Cell proliferation was determined by CCK-8 assay. ***P < 0.001 versus 0.1% DMSO, using 2-way ANOVA coupled with Tukey’s multiple-comparison post hoc test. n = 3. (D) CCK-8 assay was performed in HUVECs incubated with increasing concentrations of PRMT5 inhibitor EPZ015666 for 6 days. n =3, IC50 = 1294 nM. (E and F) Wound healing assay was performed in HUVECs incubated with 0.1% DMSO or 10.0 μM EPZ015666 for 4 days, in the presence or absence of VEGF stimulation (50 ng/mL). The representative images and quantification are shown. Scale bars: 100 μm. ***P < 0.001, using 2-tailed Student’s t test. n = 3. (G and H) HUVECs were infected with lentivirus expressing control shRNA (sh-Ctrl) or PRMT5 shRNA (sh-PRMT5). Three days after infection, wound healing assays were performed to determine cell migration. The representative images and quantification are shown. Scale bar: 100 μm. **P < 0.01, using 2-tailed Student’s t test. n = 3. (I) HUVECs were treated with EPZ015666 at indicated doses for 3 days, followed by 12-hour starvation. NO concentration in the culture media was determined after stimulation of VEGF for 6 hours. *P < 0.05 versus 0 μM, using 1-way ANOVA coupled with Tukey’s post hoc test. n = 3. All data were exhibited as mean ± SD.