Kisspeptin‐10 Prevents the Development of Cerebral Aneurysms by Reducing the Expression of Egr‐1

Figures

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  FIGURE 1. The Kisspeptin‐10 (KP‐10)/GPR54 signaling is dysregulated in both individuals with cerebral aneurysms (CA) and in mice with experimentally induced CA. (A) The serum levels of KP‐10 were measured in both control subjects and patients with CA using an ELISA method; (B) the serum levels of KP‐10 in both control and CA mice were determined using an ELISA; (C) mRNA levels of GPR54 in the COW region of both control and CA mice; (D) protein levels of GPR54 in the COW region of both control and CA mice as measured by immunohistochemistry (^^p < 0.01 vs. Normal group).

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  FIGURE 2. Administration of KP‐10 prevents CA formation in wild type but not GPR54 KO mice. Mice were divided into five groups: WT, GPR54−/−, CA, CA + KP‐10, and CA + GPR54−/− + KP‐10. (A) Representative images of Verhoeff‐van Gieson staining of the vascular wall; (B) measurement of aneurysm size; (C) systolic blood pressure in different groups of mice at 7‐week after the surgery (^,^^ p < 0.05, 0.01 vs. WT group; #,## p < 0.05, 0.01 vs. CA group; *,**p < 0.05, 0.01 vs. CA + KP10 group).

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  FIGURE 3. Administration of KP‐10 inhibits the expression of MMP‐9 in the COW region of CA mice. Mice were divided into five groups: WT, GPR54−/−, CA, CA + KP‐10, and CA + GPR54−/− + KP‐10. (A) mRNA of MMP‐9; (B) protein levels of MMP‐9 as measured by ELISA; (C) the infiltrated macrophages were staining with CD36 and the number was counted (^^ p < 0.01 vs. WT group; ## p < 0.01 vs. CA group; **p < 0.01 vs. CA + KP10 group).

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  FIGURE 4. Administration of KP‐10 inhibits the expression of VEGF‐A in the COW region. (A) mRNA levels of VEGF‐A; (B) the protein levels of VEGF‐A as measured by ELISA (^^ p < 0.01 vs. WT group; ## p < 0.01 vs. CA group; **p < 0.01 vs. CA + KP10 group).

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  FIGURE 5. Treatment with KP‐10 ameliorates Ang II‐ induced cell proliferation and angiogenic tube formation of human BMVECs (HBMVECs). Cells were stimulated with Ang II (10−7 mol/L) with or without KP‐10 (50, 100 nM). (A) Cell proliferation was measured by CCK‐8; (B) angiogenic tube formation rate; (C) mRNA of VEGF‐A; (D) protein levels of VEGF‐A (^^ p < 0.01 vs. Control group; #,## p < 0.05, 0.01 vs. Ang II group).

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  FIGURE 6. Treatment with KP‐10 inhibits Ang II‐ induced expression of Egr‐1 in HBMVECs. Cells were stimulated with Ang II (10−7 mol/L) with or without KP‐10 (50, 100 nM). (A) mRNA of Egr‐1; (B) protein of Egr‐1 (^^ p < 0.01 vs. Control group; #,## p < 0.05, 0.01 vs. Ang II group).

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  FIGURE 7. Overexpression of Egr‐1 abolishes the beneficial effects of KP‐10 in inhibiting the expression of VEGF‐A, MMP‐9, and tube formation of HBMVECs. Cells were transduced with lentiviral Egr‐1, followed by stimulation with Ang II (10−7 mol/L) with or without KP‐10 (100 nM). (A) Western blot results revealed successful overexpression of Egr‐1; (B) mRNA of VEGF‐A; (C) protein of VEGF‐A; (D) mRNA of MMP‐9; (E) protein levels of MMP‐9; (F) total angiogenic tubes of HBMVECs (^^ p < 0.01 vs. Control group; ## p < 0.01 vs. Ang II group; **p < 0.01 vs. Ang II + KP‐10 group).

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  FIGURE 8. Silencing of Gpr54 counteracts the effects of KP‐10 in inhibiting the expression of Egr‐1 in HBMVECs. Cells were transduced with lentiviral Gpr54 shRNA, followed by stimulation with Ang II (10−7 mol/L) with or without KP‐10 (100 nM). (A) Western blot analysis revealed successful silencing of Gpr54; (B) mRNA of Egr‐1; (C) protein of Egr‐1 (^^ p < 0.01 vs. Control group; ## p < 0.01 vs. Ang II group; **p < 0.01 vs. Ang II + KP‐10 group).