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 4

PRMT5 inhibitor attenuates aortic ring sprouting and tube formation on Matrigel.

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PRMT5 inhibitor attenuates aortic ring sprouting and tube formation on M...
(A) PRMT5 inhibitor EPZ015666 attenuates neovascularization in the Matrigel plug assays. The VEGF-containing Matrigel plugs with or without 10.0 μM EPZ015666 (dissolved in DMSO, final concentration 0.1%) were implanted and harvested from WT mice after 14 days. The Matrigel plugs were washed and photographed. Scale bar: 2.5 mm. n = 8. (B and C) Sections of Matrigel plugs were double stained with CD31 (green) and DAPI (blue). The representative image and quantification of total vessel areas in each Matrigel plug are shown. Scale bars: 50 μm. ***P < 0.001, using 2-tailed Student’s t test. n = 8. (D and E) The thoracic aortic ring isolated from WT mice were cultured in VEGF-containing Matrigel with or without 10.0 μM EPZ015666 for 4–5 days. The new vessels sprouting was captured and represented as an outline. The representative images and quantification are shown. Scale bar: 200 μm. ***P < 0.001, using 2-tailed Student’s t test. n = 6. (F and G) HUVECs were incubated with 0.1% DMSO or 0.5, 1.0, 5.0, or 10.0 μM EPZ015666 for 4 days and then seeded on Matrigel combined with different concentrations of EPZ015666. Eight hours after seeding, tube formation was captured using EVOS imaging system. The representative images and quantification were shown. Scale bar: 100 μm. ***P < 0.001 versus DMSO, using 1-way ANOVA coupled with Tukey’s multiple-comparison post hoc test. n = 6. (H and I) HUVECs were infected with lentivirus expressing control shRNA (sh-Ctrl) or PRMT5 shRNA (sh-PRMT5). Seventy-two hours after infection, cells were harvested and then seeded on Matrigel for an additional 8 hours to detect tube formation. The representative images and quantification were shown. Scale bar: 100 μm. *P < 0.05, using 2-tailed Student’s t test. n = 6. All data were shown as mean ± SD.