尿毒症血清活化Notch信号通路调控血管平滑肌细胞表型转化
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摘要
目的研究尿毒症血清对血管平滑肌细胞(vascular smooth muscle cell,VSMC)表型转化影响,探讨动静脉内瘘(arteriovenous fistula,AVF)狭窄及失功的机制。方法 (1)弹性纤维染色(elastic-Van Gieson staining,EVG)比较内膜厚度,免疫组化法观察α-SMA、FSP-1、PCNA的表达;(2)培养人主动脉平滑肌细胞株(human aortic smooth muscle cells,HASMCs),分为无血清组(control,Ctl)、10%正常血清组(normal serum group,NS)、5%～20%尿毒症血清组(uremia serum group,US),检测平滑肌细胞α-SMA、SMMHC、SM22α、FSP-1、PCNA的表达;(3)Western blot检测各组平滑肌细胞N1ICD蛋白的表达,QPCR检测细胞Notch1-3、Hes1、Hes5的mRNA表达,并比较Notch信号抑制剂DAPT预处理24h对细胞SM22α、SMMHC、FSP-1、PCNA、Hes1的mRNA表达的影响;(4)免疫组化染色检测Jagged1的表达,ELISA法检测人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)Jagged1的表达,并用Western blot法检测HASMCs的Jagged1蛋白表达。结果 (1)内瘘内膜显著增厚,增生内膜α-SMA呈阳性表达;与正常血管比较,增生内膜FSP-1、PCNA阳性表达增加,差异均有统计学意义(FSP-1:Ctl-A:P=0.024,Ctl-V:P=0.011;PCNA:Ctl-A:P=0.002;Ctl-V:P=0.005);(2)与10%NS组相比,10%US组细胞α-SMA、SM22α蛋白表达下降,而FSP-1、PCNA蛋白表达增加,差异均有统计学意义(P=0.002;P=0.001;P=0.001;P<0.001),且上述指标与尿毒症血清呈浓度依赖性;10%US组细胞SM22α、SMMHC的mRNA表达下降,而FSP-1、PCNA的mRNA表达增加,差异有统计学意义(P=0.014;P=0.003;P=0.045;P=0.001);(3)与10%NS组比较,10%US组细胞N1/CD蛋白的表达显著增加,差异有统计学意义(P<0.001);10%US组细胞Notch 1,3、Hes 1、Hes 5的mRNA表达增加,差异有统计学意义(P值分别为<0.001,0.025,<0.001,0.035),Notch2的表达无统计学差异(P=0.907);DAPT预处理可上调SM22α、SMMHC的mRNA表达,下调FSP-1、PCNA及Hes1的mRNA表达,差异均有统计学意义(P=0.003;P=0.020;P=0.036;P=0.009;P<0.001);(4)内瘘组织Jagged1的表达增加,主要位于增生内膜;与10%NS组相比,10%US组HASMCs及HUVEC的Jagged1蛋白表达均增加,差异均有统计学意义(P值分别为0.001,0.005)。结论尿毒症血清通过上调配体Jagged1的表达,激活Notch信号通路,促进VSMC表型由收缩型向合成型转变,可能是AVF内膜增生的机制之一。
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 (1) The 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.(2)Human 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.(3) The expression of N1 ICD 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 24 h, and then the expressions of SM22α, SMMHC, FSP-1, PCNA and Hes1 m RNAs were measured by quantitative PCR.(4)The 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(1)In 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).(2)In US groups, the expressions of α-SMA and SM22α proteins decreased significantly(P=0.002 and0.001, 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).(3)In US group, the expression of N1 ICD 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 m RNA had no significant difference(P=0.907), as compared with those in NS group. Pre-treatment of DAPT up-regulated the expressions of SM22αand SMMHC mRNAs(P=0.003 and 0.020, respectively) and down-regulated the expressions of FSP-1, PCNA and Hes1 m RNAs(P=0.036, 0.009 and <0.001, respectively).(4)In 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.
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 (1) The 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.(2)Human 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.(3) The expression of N1 ICD 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 24 h, and then the expressions of SM22α, SMMHC, FSP-1, PCNA and Hes1 m RNAs were measured by quantitative PCR.(4)The 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(1)In 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).(2)In US groups, the expressions of α-SMA and SM22α proteins decreased significantly(P=0.002 and0.001, 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).(3)In US group, the expression of N1 ICD 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 m RNA had no significant difference(P=0.907), as compared with those in NS group. Pre-treatment of DAPT up-regulated the expressions of SM22αand SMMHC mRNAs(P=0.003 and 0.020, respectively) and down-regulated the expressions of FSP-1, PCNA and Hes1 m RNAs(P=0.036, 0.009 and <0.001, respectively).(4)In 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.
引文
[1]National Kidney Foundation.KDOQI Clinical Practice Guidelines and Clinical Practice Recommendations for2006 Updates:Haemodialysis Adequacy,Peritoneal dialysis Adequacy and Vascular Access[J].Am J Kidney Dis,2010,56:566-577.
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[3]Al-Jaishi AA,Oliver MJ,Thomas SM,et al.Patency rates of the arteriovenous fistula for hemodialysis:a systematic review and meta-analysis[J].Am J Kidney Dis,2014,63(3):464-478.
[4]Kim YO,Song HC,Yoon SA,et al.Preexisting intimal hyperplasia of radial artery is associated with early failure of radiocephalic arteriovenous fistula in hemodialysis patients[J].Am J Kidney Dis,2003,41(2):422-428.
[5]Liang M,Liang A,Wang Y,et al.Smooth muscle cells from the anastomosed artery are the major precursors for neointima formation in both artery and vein grafts[J].Basic Res Cardiol,2014,109(5):431.
[6]Cheng J,Du J.Mechanical stretch simulates proliferation of venous smooth muscle cells through activation of the insulin-like growth factor-1 receptor[J].Arterioscler Thromb Vasc Biol,2007,27(8):1744-1751.
[7]Cheng J,Wang Y,Liang A,et al.FSP-1 silencing in bone marrow cells suppresses neointima formation in vein graft[J].Circ Res,2012,110(2):230-40.
[8]Liang M,Wang Y,Liang A,et al.Migration of smooth muscle cells from the arterial anastomosis of arteriovenous fistulas requires Notch activation to form neointima[J].Kidney Int,2015,88(3):490-502.
[9]Xiao YG,Wang W,Gong D,et al.γ-Secretase inhibitor DAPT attenuates intimal hyperplasia of vein grafts by inhibition of Notch1 signaling[J].Lab Invest,2014,94(6):654-662.
[10]张紫微,杨丽霞,吕晋琳,等.血管紧张素Ⅱ诱发自噬对血管平滑肌细胞表型转换的调控作用[J].中国动脉硬化杂志,2017,25(5):452-456.
[11]Qi YX,Yao QP,Huang K,et al.Nuclear envelope protein modulate proliferation of vascular smooth muscle cells during cyclic stretch application[N].PNAS,2016,113(19):5293-5298.
[12]朱晋坤,毛华,尹扬光,等.血小板源性生长因子和血小板源性内皮细胞生长因子在内皮细胞和血管平滑肌细胞中的作用研究[J].中国全科医学,2015,18(9):1023-1028.
[13]Fischer C,Schneider M,Carmeliet P.Principles and therapeutic implications of angiogenesis,vasculogenesis and atreriogenesis[J].Handb Exp Pharmacol,2006,(176 Pt 2):157-212.
[14]Song SH,Kim K,Jo EK,et al.Fibroblast Growth Factor12 Is a Novel Regulator of Vascular Smooth Muscle Cell Plasticity and Fate[J].Arterioscler Thromb Vasc Biol,2016,36(9):1928-1936.
[15]Shi N,Chen SY.Mechanisms simultaneously regulate smooth muscle proliferation and diferentiation[J].JBiomed Res,2014,28(1):40-46.
[16]王会琴,宋铁峰,袁颖,等.Myocardin在血管平滑肌细胞表型转化中的作用[J].生命科学,2016,28(7):771-776.
[17]Loomes KM,Taichman DB,Glover CL,et al.Characterization of Notch receptor expression in the developing mammalian heart and liver[J].Am J Med Genet,2002,112(2):181-189.
[18]Joutel A,Andreux F,Gaulis S,et al.The ectodomain of the Notch3 receptor accumulates within the cerebrovasculature of CADASIL patients[J].J Clin Invest,2000,105(5):597-605.
[19]Wu J,Bresnick EH.Glucocorticoid and growth factor synergism requirement for Notch4 chromatin domain activation[J].Mol Cell Biol,2007,27(6):2411-2422.
[20]Hormann JJ,Iruela-Arispe ML.Notch signaling in blood vessels:who is talking to whom about waht?[J].Circ Res,2007,100(11):1556-1568.
[21]Monroy MA,Fang J,Li S,et al.Chronic kidney disease alters vascular smooth muscle cell phenotype[J].Front Biosci(Landmark Ed),2015,20:784-795.
[22]Caza?a-Pérez V,Cidad P,Donate-Correa J,et al.Phenotypic Modulation of Cultured Primary Human Aortic Vascular Smooth Muscle Cells by Uremic Serum[J].Front Physiol,2018,9:89.
[23]Boucher JM,Peterson SM,Urs S,et al.The miR-143/145Cluster Is a Novel Transcriptional Target of Jagged-1/Notch Signaling in Vascular Smooth Muscle Cells[J].JBiol Chem,2011,286(32):28312-28321.
[24]Tang Y,Urs S,Liaw L.Hairy-Related Transcription Factors Inhibit Notch-Induced Smooth Muscleα-Actin Expression by Interfering With Notch Intracellular Domain/CBF-1 Complex Interaction With the CBF-1-Binding Site[J].Circ Res,2008,102(6):661-668.
[2]中国医院协会,中国医院协会血液净化中心管理分会血液净化通路学组.2014中国血液透析用血管通路专家共识(第1版)[J].中国血液化,2014,13(8):549-558.
[3]Al-Jaishi AA,Oliver MJ,Thomas SM,et al.Patency rates of the arteriovenous fistula for hemodialysis:a systematic review and meta-analysis[J].Am J Kidney Dis,2014,63(3):464-478.
[4]Kim YO,Song HC,Yoon SA,et al.Preexisting intimal hyperplasia of radial artery is associated with early failure of radiocephalic arteriovenous fistula in hemodialysis patients[J].Am J Kidney Dis,2003,41(2):422-428.
[5]Liang M,Liang A,Wang Y,et al.Smooth muscle cells from the anastomosed artery are the major precursors for neointima formation in both artery and vein grafts[J].Basic Res Cardiol,2014,109(5):431.
[6]Cheng J,Du J.Mechanical stretch simulates proliferation of venous smooth muscle cells through activation of the insulin-like growth factor-1 receptor[J].Arterioscler Thromb Vasc Biol,2007,27(8):1744-1751.
[7]Cheng J,Wang Y,Liang A,et al.FSP-1 silencing in bone marrow cells suppresses neointima formation in vein graft[J].Circ Res,2012,110(2):230-40.
[8]Liang M,Wang Y,Liang A,et al.Migration of smooth muscle cells from the arterial anastomosis of arteriovenous fistulas requires Notch activation to form neointima[J].Kidney Int,2015,88(3):490-502.
[9]Xiao YG,Wang W,Gong D,et al.γ-Secretase inhibitor DAPT attenuates intimal hyperplasia of vein grafts by inhibition of Notch1 signaling[J].Lab Invest,2014,94(6):654-662.
[10]张紫微,杨丽霞,吕晋琳,等.血管紧张素Ⅱ诱发自噬对血管平滑肌细胞表型转换的调控作用[J].中国动脉硬化杂志,2017,25(5):452-456.
[11]Qi YX,Yao QP,Huang K,et al.Nuclear envelope protein modulate proliferation of vascular smooth muscle cells during cyclic stretch application[N].PNAS,2016,113(19):5293-5298.
[12]朱晋坤,毛华,尹扬光,等.血小板源性生长因子和血小板源性内皮细胞生长因子在内皮细胞和血管平滑肌细胞中的作用研究[J].中国全科医学,2015,18(9):1023-1028.
[13]Fischer C,Schneider M,Carmeliet P.Principles and therapeutic implications of angiogenesis,vasculogenesis and atreriogenesis[J].Handb Exp Pharmacol,2006,(176 Pt 2):157-212.
[14]Song SH,Kim K,Jo EK,et al.Fibroblast Growth Factor12 Is a Novel Regulator of Vascular Smooth Muscle Cell Plasticity and Fate[J].Arterioscler Thromb Vasc Biol,2016,36(9):1928-1936.
[15]Shi N,Chen SY.Mechanisms simultaneously regulate smooth muscle proliferation and diferentiation[J].JBiomed Res,2014,28(1):40-46.
[16]王会琴,宋铁峰,袁颖,等.Myocardin在血管平滑肌细胞表型转化中的作用[J].生命科学,2016,28(7):771-776.
[17]Loomes KM,Taichman DB,Glover CL,et al.Characterization of Notch receptor expression in the developing mammalian heart and liver[J].Am J Med Genet,2002,112(2):181-189.
[18]Joutel A,Andreux F,Gaulis S,et al.The ectodomain of the Notch3 receptor accumulates within the cerebrovasculature of CADASIL patients[J].J Clin Invest,2000,105(5):597-605.
[19]Wu J,Bresnick EH.Glucocorticoid and growth factor synergism requirement for Notch4 chromatin domain activation[J].Mol Cell Biol,2007,27(6):2411-2422.
[20]Hormann JJ,Iruela-Arispe ML.Notch signaling in blood vessels:who is talking to whom about waht?[J].Circ Res,2007,100(11):1556-1568.
[21]Monroy MA,Fang J,Li S,et al.Chronic kidney disease alters vascular smooth muscle cell phenotype[J].Front Biosci(Landmark Ed),2015,20:784-795.
[22]Caza?a-Pérez V,Cidad P,Donate-Correa J,et al.Phenotypic Modulation of Cultured Primary Human Aortic Vascular Smooth Muscle Cells by Uremic Serum[J].Front Physiol,2018,9:89.
[23]Boucher JM,Peterson SM,Urs S,et al.The miR-143/145Cluster Is a Novel Transcriptional Target of Jagged-1/Notch Signaling in Vascular Smooth Muscle Cells[J].JBiol Chem,2011,286(32):28312-28321.
[24]Tang Y,Urs S,Liaw L.Hairy-Related Transcription Factors Inhibit Notch-Induced Smooth Muscleα-Actin Expression by Interfering With Notch Intracellular Domain/CBF-1 Complex Interaction With the CBF-1-Binding Site[J].Circ Res,2008,102(6):661-668.