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Uremic serum regulates the phenotype transformation of vascular smooth muscle cells through activating the Notch signaling pathway
2019, 18 (01):
35-41.
doi: 10.3969/j.issn.1671-4091.2019.01.008
【Abstract】Objective To study the effect of uremic serum on phenotype transformation of vascular smooth muscle cells and to correlate the results to the mechanism of arteriovenous fistula (AVF) stenosis and dysfunction. Methods ①The intimal thickness of AVF was compared with normal vessles by elastic Van Gieson staining, and the expressions of α-SMA, FSP-1 and PCNA were evaluated by immunohistochemical staining. ②Human aortic smooth muscle cells (HASMCs) were cultured in vitro and divided into 3 groups: serum-free group (Ctrl group), 10% normal serum group (NS group), and 5～20% uremia serum group (US group). The expressions of α-SMA, SM22α, FSP-1 and PCNA proteins and mRNAs were assessed by Western blot and quantitative PCR. ③The expression of N1ICD protein in different groups was assayed by Western blot; the expressions of Notch1-3, Hes1 and Hes5 mRNAs were detected by quantitative PCR. HASMCs were pre-treated with the Notch signal inhibitor DAPT for 24h, and then the expressions of SM22α, SMMHC, FSP-1, PCNA and Hes1 mRNAs were measured by quantitative PCR. ④The expression of Jagged1, a ligand of Notch, was detected by immunohistochemical staining. Jagged1 protein expressed by human umbilical vein endothelial cells (HUVECs) was assayed by ELISA; Jagged1 expression in HASMCs was measured by Western blot. Results ①In AVF, intimal thickness increased significantly, the expression of α-SMA in neointima was positive, and the positive rates of FSP-1 and PCNA were higher than those in normal vascular tissue (P＜0.05). ②In US groups, the expressions of α-SMA and SM22α proteins decreased significantly (P=0.002 and 0.001, respectively), while the expressions of FSP- 1 and PCNA proteins increased significantly (P=0.001 and ＜0.001, respectively), as compared with those in NS group. These changes showed a tendency of uremia serum concentration dependence. In US group, the expressions of SM22α and SMMHC mRNAs decreased significantly (P=0.014 and 0.003, respectively), while the expressions of FSP-1 and PCNA mRNAs increased significantly (P=0.045 and 0.001, respectively). ③In US group, the expression of N1ICD protein increased significantly (P＜0.001), the expressions of Notch1, Notch3, Hes1 and Hes5 mRNAs increased significantly (P＜0.001, 0.025, ＜0.001 and 0.035, respectively), and Notch2 mRNA had no significant difference (P=0.907), as compared with those in NS group. Pre-treatment of DAPT up-regulated the expressions of SM22α and SMMHC mRNAs (P=0.003 and 0.020, respectively) and down-regulated the expressions of FSP-1, PCNA and Hes1 mRNAs (P=0.036, 0.009 and ＜0.001, respectively). ④In AVF, the expression of Jagged1 increased mainly in neointima. In US group, the expression of Jagged1 protein in HASMCs and HUVECs increased significantly (P=0.001 and 0.005, respectively), as compared with those in NS group. Conclusion Uremic serum promoted the phenotype transformation of vascular smooth muscle cells from deflating type to proliferation type through the up-regulation of Notch ligand Jagged1 and then activation of Notch signaling
pathway, probably being the mechanism of neointimal hyperplasia in AVF in uremic patients.
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