法尼基焦磷酸合成酶在血管紧张素Ⅱ诱导心肌肥厚中研究
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摘要
第一部分抑制法尼基焦磷酸合成酶改善血管紧张素Ⅱ诱导心肌细胞肥厚
研究背景：
心肌肥厚作为一种常见疾病,可被许多体液因子如血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)诱导。各种细胞通路参与了AngⅡ诱导的心肌肥厚,其中包括RhoA/ROCK通路。
以往研究发现阿伦磷酸钠(alendronate)重要机制主要是通过抑制甲羟戊酸途径中的一种关键酶-法尼基焦磷酸合成酶(farnesyl pyrophosphate synthase, FPPS),从而抑制类异戊烯化包括了法尼基化和牻牛儿牻牛基儿化进而减少类异戊二烯化产物的产生包括法尼基焦磷酸(farnesylpyrophosphate, FPP)和牻牛儿牻牛儿焦磷酸(geranylgeranylpyrophosphate, GGPP).而GGPP对于RhoA的牻牛儿牻牛儿焦磷酸化和活化非常重要。
目的：
本研究主要探索alendronate抑制FPPS可否影响AngⅡ诱导的心肌肥厚,同时研究该作用是否与RhoA/ROCK通路相关。
方法：
(1)加入3～30 (μM alendronate先孵育30 min然后加入AngⅡ(1μM)共同作用48h,加药前细胞在无血清培养基DMEM中孵育24h。检测肥厚指标包括细胞面积,细胞蛋白合成和基因BNP mRNA水平改变等。
(2)在心肌细胞中加入30μM alendronate及相同浓度的GGOH先孵育30 min,然后与AngⅡ(1μM)共同孵育48h,加药前细胞在无血清培养基DMEM中孵育24 h。检测肥厚指标包括细胞面积,细胞蛋白合成和基因(?)NP mRNA水平改变。
(3)在心肌细胞中加入alendronate, RhoA/ROCK通路抑制剂包括牻牛儿牻牛儿基转移酶抑制剂GGTI-286, Rho的抑制剂C3 exoenzyme或ROCK的抑制剂Y-27632先孵育30 min,然后与AngⅡ(1μM)共同孵育48 h,加药前细胞在无血清培养基DMEM中孵育24 h。检测肥厚指标包括细胞面积,细胞蛋白合成和基因BNP mRNA水平改变。
(4)在心肌细胞中加入30μM alendronate及相同浓度的GGOH或加入GGTI-286孵育48 h,然后加入AngⅡ(1μM)共同孵育15 min,用商业化pull down试剂盒和western-blot技术检测了RhoA活性和RhoA总量的改变。
结果：
(1) Alendronate作为FPPS抑制剂可剂量依赖性抑制Ang II诱导的乳鼠心肌细胞面积增大,细胞蛋白合成量的增加及基因BNP表达的上调。
(2)同浓度GGOH部分逆转了30μM alendronate的抗心肌肥厚反应。
(3) Rho/ROCK通路抑制剂GGTI-286, C3 exoenzyme以及Y-27632可抑制Ang II诱导的心肌细胞肥厚反应。
(4) 30μM alendronate抑制了AngⅡ诱导增加的RhoA活性,该作用可被相同浓度GGOH部分逆转。牻牛儿牻牛儿基化过程抑制剂GGTI-286也抑制了Ang II对RhoA激活。
结论：
本研究实验结果表明alendronate通过抑制FPPS可以有效逆转AngⅡ诱导心肌细胞肥厚反应,这种作用与抑制RhoA的牻牛儿牻牛儿化过程从而降低RhoA的活性相关。
第二部分RNA干扰技术沉默法尼基焦磷酸合成酶基因改善血管紧张素Ⅱ诱导心肌肥厚
研究背景：
第一部分实验结果表明抑制法尼基焦磷酸合成酶(Farnesylpyrophosphate synthase, FPPS)可以逆转血管紧张素Ⅱ(angiotensinⅡ, Ang II)诱导的心肌细胞肥厚发生,提示FPPS作为AngⅡ介导的心肌肥厚发生的重要治疗靶点。
我们实验组前期高通量RNA阵列技术(RNA array)结果表明FPPS的mRNA水平在18周自发高血压大鼠(spontaneously hypertensive rats, SHR)肥厚心脏组织比同周龄WKY大鼠(Wistar-Kyoto rats, WKY)高。而18周SHR的心肌肥厚与其局部RAS系统激活Ang II含量升高相关。
上述所有证据都表明FPPS在Ang II介导心肌肥厚中可能的重要性。
目的：
本实验探究了FPPS水平是否在Ang II介导的心肌肥厚模型有所改变,同时在此基础上研究通过RNA干扰技术沉默FPPS基因的表达,观察是否影响到心肌肥厚的发生,并进一步研究FPPS下游主要细胞通路的改变。
方法：
(1)使用real-time PCR和western-blot技术检测Ang II介导的心肌肥厚模型中FPPS的转录和翻译水平。
(2)设计并构建针对四个不同靶序列FPPS干扰质粒载体PGC-sh-FPPS和带有绿色荧光蛋白的FPPS融合蛋白质粒(FPPS-GFP).通过外源表达系统在HEK-293 T细胞共表达,以免疫荧光和western-blot险测GFP表达判断最有效靶序列。
(3)选择最有效靶序列构建和扩增FPPS干扰重组慢病毒载体,转染乳鼠心肌细胞观察FPPS的下调是否影响β-肌球蛋白重链(β-myosin heavy chain,β-MHC)和B-型脑钠肽(brain natriuretic peptide, BNP)基因及细胞表面积的改变。慢病毒颗粒注射7周SHR大鼠心肌,11周后称重并进行心脏超声学检查。心脏称重获得心脏重／体重比(heart weight/body weight, HW/BW)和左室重／体重比(left ventricular weight/body weight, LVW/BW),并检测左室β-MHC和BNP的改变。
(4)在细胞水平进一步研究FPPS基因沉默后下游主要通路的改变。使用pull down试剂盒和western-blot方法检测RhoA的活性和表达量改变,用细胞免疫荧光激光共聚焦技术检测RhoA的定位,用western-blot检测JNK和P38 MAPK (mitogen-activated protein kinases)的活性。
结果：
(1) AngⅡ(1μM)在诱导心肌细胞肥厚同时上调了FPPS的水平,18周龄SHR肥厚心脏中FPPS的转录和翻译水平也上调。
(2)成功构建FPPS干扰重组慢病毒载体沉默FPPS水平的同时可以明显抑制Ang II诱导的心肌细胞表面积及β-MHC和BNP的mRNA水平。活体实验表明FPPS下调的同时,肥厚指标包括HW/BW和LVW/BW的减少,肥厚标记基因β-MHC和BNP水平的下调。心超结果显示SHR干扰组舒张期室间隔壁厚度(interventricular septum wall thickness at diastolic phase, IVSd)部分逆转,同时左室短轴缩短率(fraction shortening, FS)和射血分数(ejection fraction, EF)均有部分改善。
(3) FPPS基因干扰有效抑制了心肌细胞中AngⅡ诱导的RhoA活性和易位,并逆转了Ang II激活的P-38和JNKMAPK通路。
结论：
FPPS水平在Ang II介导的心肌肥厚上调,提示FPPS在Ang II诱导心肌肥厚中的重要性。同时FPPS调控的RhoA/P-38/JNK MAPK在Ang II介导的心肌肥厚中扮演着重要作用。
第三部分阿伦磷酸钠对血管紧张素Ⅱ诱导心肌成纤维细胞的增殖分化和胶原形成的影响
研究背景：
含氮类双磷酸盐(N-BPs)广泛用于骨骼相关性疾病和肿瘤中。第一部分研究发现阿伦磷酸钠(alendronate),一种权威的N-BPs能有效逆转血管紧张素Ⅱ(angiotensinⅡ, AngⅡ)诱导的心肌细胞肥厚反应。以往研究也报道alendronate可以有效抑制自发性高血压大鼠的心肌肥厚及心肌纤维化。
目的：
本实验为进一步观察alendronate是否可以调节AngⅡ诱导的新生鼠心肌成纤维细胞的增殖、分化及胶原生成。
方法：
予以新生wistar鼠心肌成纤维细胞原代培养,传代第二到四代予以使用。细胞增殖用细胞数目试剂盒(原理是water-soluble tetrazolium, WST-1)进行检测。利用免疫印迹(western blot)方法检测成纤维细胞的分化标志物α-肌动蛋白(a-smooth muscle actin,α-SMA)的表达。胶原合成采用实时定量PCR (real-time polymerase chain reaction, qRT-PCR)技术检测Ⅰ型和Ⅲ型胶原前体的mRNA水平,同时使用商业化试剂盒检测胶原蛋白羟脯氨酸的合成。另外转录生长因子-β1(transforming growth factorβ1, TGF-β1)的含量和和P38促细胞分裂剂激活性蛋白激酶激酶(P38MAPK, P38 mitogen-activated protein kinase)的活性分别用酶联免疫吸附技术(enzyme linked immunosorbent assay, ELISA)和免疫印迹(western blot)技术进行检测。
结果：
Alendronate可以抑制AngⅡ诱导的成纤维细胞的增殖分化及随后的胶原的形
成。同时TGF-β1蛋白的表达和分泌及P38 MAPK的活性都得以被alendronate逆转。结论：
本研究提示alendronate对AngⅡ诱导的细胞增殖,细胞分化及胶原形成可有效抑制。这种作用可能与TGF-β1水平下调,P38 MAPK活性的抑制相关。
Part 1 Inhibition of farnesylpyrophosphate synthase prevents angiotensin II-induced hypertrophic responses in rat neonatal cardiomyocytes:involvement of the RhoA/Rho kinase pathway
Background:
Cardiac hypertophy can be induced by humal factors such as angiotensinⅡ(AngⅡ). Various pathways are involved in AngⅡ-induced hypertrophic response of cardiomyocytes including the RhoA/ROCK signaling.
Experimental study has shown that alendronate inhibits farnesylpyrophosphate (FPP) synthase, a key enzyme in the mevalonate pathway, through inhibition of isoprenylation including farnesylation and geranylgeranylation with consecutively decreases of the formation of isoprenoid lipids such as farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP). The latter one is essential for geranylgeranylation and activation of RhoA.
Aim:
We undertook our research to explore whether inhibition of FPP synthase by alendronate could interfere with the hypertrophic responses induced by AngⅡin cultured neonatal ventricular myocytes, and whether it involves RhoA/ROCK pathway.
Methods:
Cardiomyocyte were in serum-free medium (SFM) for 24 h before incubation with various agents with indicated time. Hypertrophy markers including cell surface area and protein content were assayed while gene expression of BNP was measured by quantitative real-time polymerase chain reaction (qRT-PCR).
(1) Myocytes were pre-incubated with 3-30μM alendronate alone or with AngⅡ(1μM) for 48 h.
(2) Myocytes were pre-incubated with 30μM alendronate alone or in combination with equal GGOH in the addition of AngⅡ(1μM) for 48 h.
(3) Myocytes were pre-incubated with alendronate, GGTI-286, C3 exoenzyme or Y-27632 in the addition of AngⅡ(1μM) for 48 h
(4) Myocytes were pre-incubated with 30μM alendronate alone or in combination with equal GGOH for 48 h before incubation with AngⅡfor 15 min. Pull down assay and western blot analysis for RhoA activity and expression were performed.
Results:
(1) Alendronate at 3 to 30μM prevented hypertrophy responses induced by AngⅡ(1μM).
(2) The anti-hypertrophic effects of inhibition of FPP synthase with alendronate in Ang II-cultured neonatal cardiomyocytes hypertrophy were partially reversed by comparative geranylgeranyol (GGOH).
(3) The anti-hypertrophic effects of inhibition of FPP synthase with alendronate could be mimicked by GGTI-286, a geranylgeranyltransferase-I inhibitor, C3 exoenzyme, an inhibitor of Rho or Y-27632, an inhibitor of ROCK.
(4) Pull-down assay showed alendronate reduced-active RhoA by Ang II was also partially antagonized by GGOH.GGTI-286 also abolished the activated RhoA by AngⅡ.
Conlusions:
This study reveals the inhibition of FPP synthase by alendronte reduces RhoA activation by diminishing geranylgeranylation which prevents AngⅡ-induced hypertrophic response of neonatal cardiomyocytes.
Part 2 Konckdown of farnesylpyrophosphate synthase by RNA interference prevents angiotensin II-induced cardiac hypertrophy
Background:
Our first part experimental research suggests inhibition of farnesylpyrophosphate synthase (FPPS) enzyme could reverse cardiac hypertrophy induced by angiotensinⅡ(AngⅡ), which indicates FPPS gene as the therapeutic target involved in AngⅡ-induced cardiac hypertrophy.
The data of our previous RNA array study reported the up-regulation of FPPS mRNA in hypertrophy myocardium of 18-week spontaneously hypertensive rats (SHR) associated with local activated renin-angiotensin system (RAS) and higher AngⅡcontent than the age-matched Wistar-Kyoto rats (WKY).
Collectively, this evidence strongly suggests the probable essential role of FPPS in the development of cardiac hypertrophy by Ang II.
Aim:
The aim of our study was designed to investigate whether the mRNA and protein expression of FPPS are increased in AngⅡ-mediated cardiac hypertrophy, then whether silencing FPPS modulates these hypertrophic responses, and furthermore studied FPPS-related signaling.
Methods:
(1) FPPS expression was measured in cardiac hypertrophy models by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot.
(2) The four different sequences of short interference RNA duplexes (siRNAs) targeting the rat FPPS gene were constructed into shRNA vectors and the FPPS fusion protein was generated by insertion of rat FPPS cDNA into the pEGFP-C1 vector. HEK293 T cells were co-transfected with the rat FPPS fusion protein plus different shRNAs against FPPS or scrambled shRNA using lipofectamine 2000 and then the most active shRNA against rat FPPS was established by observation of GFP expression by immunofluorescence and western blot.
(3) The most active FPPS siRNA was established, synthesized and then incorporated into a lentiviral vector with scrambled shRNA control for lentivirus production in vitro and in vivo gene transfer experiment. Hypertrophy makers in vitro include cell surface area and the mRNA expression of P-myosin heavy chain (β-MHC) and brain natriuretic peptide (BNP) by qRT-PCR. Hypertrophic responses in vivo indexed by heart weight/body weight (HW/BW), left ventricular weight/body weight (LVW/BW) and echocardiography were measured as well as expression ofβ-MHC and BNP mRNA by qRT-PCR.
(4) The involvement of FPPS in Ang II-activated hypertrophic responses was furthered by investigation of downstream signaling in cultured cardiomyocytes. Pull down assay and western blot analysis for RhoA activity and expression were performed. RhoA immunofluorescence confocal microscopy was analysed for RhoA translocation. Besides, the activity of p-38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinases (MAPK) was performed by western blot analysis.
Results:
(1) FPPS expression was elevated both in cultured neonatal cardiomyocytes (NCMs) following AngⅡ(1μM) treatment and in the hypertrophic myocardium of 18-week-old spontaneously hypertensive rats (SHRs).
(2) FPPS shRNA lentivirus was constructed successfully. FPPS silencing in NCMs completely inhibited the hypertrophy marker genes ofβ-myosin heavy chain (β-MHC) and brain natriuretic peptide (BNP), as well as cell surface area. In vivo gene transfer also attenuated hypertrophic responses as indexed by left ventricular weight/body weight, heart weight/body weight as well as expression ofβ-MHC and BNP mRNA in SHRs. Echocardiography also showed the partial attenuation of interventricular septum wall thickness at diastolic phase (IVSd) and improvement of fraction shortening (FS) and ejection fraction (EF) in SHR silencing group.
(2) FPPS knockdown prevented elevated RhoA activity as well as RhoA translocation to plasma membrane in incubation with AngⅡcompared with non-silenced controls in NCMs. Similarly, increased-phosphorylation of P-38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinases (MAPK) by AngⅡwas attenuated.
Conlusions:
In conclusion, the expression of FPPS was elevated in Ang II-mediated cardiac hypertrophy and FPPS with RhoA-associated P38 and JNK MAPK signaling might play an important role in AngⅡ-induced cardiac hypertrophy.
Part 3 Alendronate modulates angiotensinⅡ-induced neonatal cardiac fibroblast proliferation differentiation and collagen production in vitro
Background:
Nitrogen-containing bisphosphonates (N-BPs), are extensively used in bone-related diseases and cancers. Our first part experimental research suggests alendronate could modulate cardiac hypertrophy induced by angiotensinⅡ(AngⅡ). Previous study demonstrated alendronate has beneficial effects on cardiac fibrosis in spontaneously hypertensive rats.
Aims:
This study was designed to examine whether pretreatment with alendronate could modulate Ang II activated early events of cardiac fibrosis in vitro including the increase in cell proliferation, differentiation of neonatal cardiac fibroblasts, and collagen production.
Methods:
Cardiac fibroblasts were prepared from 1 to 2-day-old Wistar rats. The proliferation of cardiac fibroblasts was determined by water-soluble tetrazolium (WST)-1 assay using Cell Counting Kit (CCK-8) and the differentiation marker of cardiac fibroblasts a-smooth muscle actin (a-SMA) was measured by western blot. Collagen production was assayed by the mRNA expression of collagen type II and type II using the real-time polymerase chain reaction (qRT-PCR) method, as well as by the analysis of hydroxyproline levels. Additionally transforming growth factor (TGF)-β1 protein production by cultured neonatal cardiac fibroblasts as well as P38 mitogen-activated protein kinase (MAPK) activity was evaluated by enzyme linked immunosorbent (ELISA) and western blot respectively.
Results:
Alendronate inhibited the proliferation and differentiation of cardiac fibroblasts as well as prevented collagen production by Ang II in vitro. Furthermore, the TGF-β1 protein expression and secretion by cardiac fibroblasts as well as activated P38 MAPK by AngⅡwere all reversed by alendronate.
Conclusion:
The current study demonstrates the inhibitory role of alendronate on Ang II-induced increase in cell proliferation, differentiation of neonatal cardiac fibroblasts and collagen production. The effect is possibly mediated by lowering the TGF-β1 levels and inactivation of P38 MAPK signaling.
研究背景：
心肌肥厚作为一种常见疾病,可被许多体液因子如血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)诱导。各种细胞通路参与了AngⅡ诱导的心肌肥厚,其中包括RhoA/ROCK通路。
以往研究发现阿伦磷酸钠(alendronate)重要机制主要是通过抑制甲羟戊酸途径中的一种关键酶-法尼基焦磷酸合成酶(farnesyl pyrophosphate synthase, FPPS),从而抑制类异戊烯化包括了法尼基化和牻牛儿牻牛基儿化进而减少类异戊二烯化产物的产生包括法尼基焦磷酸(farnesylpyrophosphate, FPP)和牻牛儿牻牛儿焦磷酸(geranylgeranylpyrophosphate, GGPP).而GGPP对于RhoA的牻牛儿牻牛儿焦磷酸化和活化非常重要。
目的：
本研究主要探索alendronate抑制FPPS可否影响AngⅡ诱导的心肌肥厚,同时研究该作用是否与RhoA/ROCK通路相关。
方法：
(1)加入3～30 (μM alendronate先孵育30 min然后加入AngⅡ(1μM)共同作用48h,加药前细胞在无血清培养基DMEM中孵育24h。检测肥厚指标包括细胞面积,细胞蛋白合成和基因BNP mRNA水平改变等。
(2)在心肌细胞中加入30μM alendronate及相同浓度的GGOH先孵育30 min,然后与AngⅡ(1μM)共同孵育48h,加药前细胞在无血清培养基DMEM中孵育24 h。检测肥厚指标包括细胞面积,细胞蛋白合成和基因(?)NP mRNA水平改变。
(3)在心肌细胞中加入alendronate, RhoA/ROCK通路抑制剂包括牻牛儿牻牛儿基转移酶抑制剂GGTI-286, Rho的抑制剂C3 exoenzyme或ROCK的抑制剂Y-27632先孵育30 min,然后与AngⅡ(1μM)共同孵育48 h,加药前细胞在无血清培养基DMEM中孵育24 h。检测肥厚指标包括细胞面积,细胞蛋白合成和基因BNP mRNA水平改变。
(4)在心肌细胞中加入30μM alendronate及相同浓度的GGOH或加入GGTI-286孵育48 h,然后加入AngⅡ(1μM)共同孵育15 min,用商业化pull down试剂盒和western-blot技术检测了RhoA活性和RhoA总量的改变。
结果：
(1) Alendronate作为FPPS抑制剂可剂量依赖性抑制Ang II诱导的乳鼠心肌细胞面积增大,细胞蛋白合成量的增加及基因BNP表达的上调。
(2)同浓度GGOH部分逆转了30μM alendronate的抗心肌肥厚反应。
(3) Rho/ROCK通路抑制剂GGTI-286, C3 exoenzyme以及Y-27632可抑制Ang II诱导的心肌细胞肥厚反应。
(4) 30μM alendronate抑制了AngⅡ诱导增加的RhoA活性,该作用可被相同浓度GGOH部分逆转。牻牛儿牻牛儿基化过程抑制剂GGTI-286也抑制了Ang II对RhoA激活。
结论：
本研究实验结果表明alendronate通过抑制FPPS可以有效逆转AngⅡ诱导心肌细胞肥厚反应,这种作用与抑制RhoA的牻牛儿牻牛儿化过程从而降低RhoA的活性相关。
第二部分RNA干扰技术沉默法尼基焦磷酸合成酶基因改善血管紧张素Ⅱ诱导心肌肥厚
研究背景：
第一部分实验结果表明抑制法尼基焦磷酸合成酶(Farnesylpyrophosphate synthase, FPPS)可以逆转血管紧张素Ⅱ(angiotensinⅡ, Ang II)诱导的心肌细胞肥厚发生,提示FPPS作为AngⅡ介导的心肌肥厚发生的重要治疗靶点。
我们实验组前期高通量RNA阵列技术(RNA array)结果表明FPPS的mRNA水平在18周自发高血压大鼠(spontaneously hypertensive rats, SHR)肥厚心脏组织比同周龄WKY大鼠(Wistar-Kyoto rats, WKY)高。而18周SHR的心肌肥厚与其局部RAS系统激活Ang II含量升高相关。
上述所有证据都表明FPPS在Ang II介导心肌肥厚中可能的重要性。
目的：
本实验探究了FPPS水平是否在Ang II介导的心肌肥厚模型有所改变,同时在此基础上研究通过RNA干扰技术沉默FPPS基因的表达,观察是否影响到心肌肥厚的发生,并进一步研究FPPS下游主要细胞通路的改变。
方法：
(1)使用real-time PCR和western-blot技术检测Ang II介导的心肌肥厚模型中FPPS的转录和翻译水平。
(2)设计并构建针对四个不同靶序列FPPS干扰质粒载体PGC-sh-FPPS和带有绿色荧光蛋白的FPPS融合蛋白质粒(FPPS-GFP).通过外源表达系统在HEK-293 T细胞共表达,以免疫荧光和western-blot险测GFP表达判断最有效靶序列。
(3)选择最有效靶序列构建和扩增FPPS干扰重组慢病毒载体,转染乳鼠心肌细胞观察FPPS的下调是否影响β-肌球蛋白重链(β-myosin heavy chain,β-MHC)和B-型脑钠肽(brain natriuretic peptide, BNP)基因及细胞表面积的改变。慢病毒颗粒注射7周SHR大鼠心肌,11周后称重并进行心脏超声学检查。心脏称重获得心脏重／体重比(heart weight/body weight, HW/BW)和左室重／体重比(left ventricular weight/body weight, LVW/BW),并检测左室β-MHC和BNP的改变。
(4)在细胞水平进一步研究FPPS基因沉默后下游主要通路的改变。使用pull down试剂盒和western-blot方法检测RhoA的活性和表达量改变,用细胞免疫荧光激光共聚焦技术检测RhoA的定位,用western-blot检测JNK和P38 MAPK (mitogen-activated protein kinases)的活性。
结果：
(1) AngⅡ(1μM)在诱导心肌细胞肥厚同时上调了FPPS的水平,18周龄SHR肥厚心脏中FPPS的转录和翻译水平也上调。
(2)成功构建FPPS干扰重组慢病毒载体沉默FPPS水平的同时可以明显抑制Ang II诱导的心肌细胞表面积及β-MHC和BNP的mRNA水平。活体实验表明FPPS下调的同时,肥厚指标包括HW/BW和LVW/BW的减少,肥厚标记基因β-MHC和BNP水平的下调。心超结果显示SHR干扰组舒张期室间隔壁厚度(interventricular septum wall thickness at diastolic phase, IVSd)部分逆转,同时左室短轴缩短率(fraction shortening, FS)和射血分数(ejection fraction, EF)均有部分改善。
(3) FPPS基因干扰有效抑制了心肌细胞中AngⅡ诱导的RhoA活性和易位,并逆转了Ang II激活的P-38和JNKMAPK通路。
结论：
FPPS水平在Ang II介导的心肌肥厚上调,提示FPPS在Ang II诱导心肌肥厚中的重要性。同时FPPS调控的RhoA/P-38/JNK MAPK在Ang II介导的心肌肥厚中扮演着重要作用。
第三部分阿伦磷酸钠对血管紧张素Ⅱ诱导心肌成纤维细胞的增殖分化和胶原形成的影响
研究背景：
含氮类双磷酸盐(N-BPs)广泛用于骨骼相关性疾病和肿瘤中。第一部分研究发现阿伦磷酸钠(alendronate),一种权威的N-BPs能有效逆转血管紧张素Ⅱ(angiotensinⅡ, AngⅡ)诱导的心肌细胞肥厚反应。以往研究也报道alendronate可以有效抑制自发性高血压大鼠的心肌肥厚及心肌纤维化。
目的：
本实验为进一步观察alendronate是否可以调节AngⅡ诱导的新生鼠心肌成纤维细胞的增殖、分化及胶原生成。
方法：
予以新生wistar鼠心肌成纤维细胞原代培养,传代第二到四代予以使用。细胞增殖用细胞数目试剂盒(原理是water-soluble tetrazolium, WST-1)进行检测。利用免疫印迹(western blot)方法检测成纤维细胞的分化标志物α-肌动蛋白(a-smooth muscle actin,α-SMA)的表达。胶原合成采用实时定量PCR (real-time polymerase chain reaction, qRT-PCR)技术检测Ⅰ型和Ⅲ型胶原前体的mRNA水平,同时使用商业化试剂盒检测胶原蛋白羟脯氨酸的合成。另外转录生长因子-β1(transforming growth factorβ1, TGF-β1)的含量和和P38促细胞分裂剂激活性蛋白激酶激酶(P38MAPK, P38 mitogen-activated protein kinase)的活性分别用酶联免疫吸附技术(enzyme linked immunosorbent assay, ELISA)和免疫印迹(western blot)技术进行检测。
结果：
Alendronate可以抑制AngⅡ诱导的成纤维细胞的增殖分化及随后的胶原的形
成。同时TGF-β1蛋白的表达和分泌及P38 MAPK的活性都得以被alendronate逆转。结论：
本研究提示alendronate对AngⅡ诱导的细胞增殖,细胞分化及胶原形成可有效抑制。这种作用可能与TGF-β1水平下调,P38 MAPK活性的抑制相关。
Part 1 Inhibition of farnesylpyrophosphate synthase prevents angiotensin II-induced hypertrophic responses in rat neonatal cardiomyocytes:involvement of the RhoA/Rho kinase pathway
Background:
Cardiac hypertophy can be induced by humal factors such as angiotensinⅡ(AngⅡ). Various pathways are involved in AngⅡ-induced hypertrophic response of cardiomyocytes including the RhoA/ROCK signaling.
Experimental study has shown that alendronate inhibits farnesylpyrophosphate (FPP) synthase, a key enzyme in the mevalonate pathway, through inhibition of isoprenylation including farnesylation and geranylgeranylation with consecutively decreases of the formation of isoprenoid lipids such as farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP). The latter one is essential for geranylgeranylation and activation of RhoA.
Aim:
We undertook our research to explore whether inhibition of FPP synthase by alendronate could interfere with the hypertrophic responses induced by AngⅡin cultured neonatal ventricular myocytes, and whether it involves RhoA/ROCK pathway.
Methods:
Cardiomyocyte were in serum-free medium (SFM) for 24 h before incubation with various agents with indicated time. Hypertrophy markers including cell surface area and protein content were assayed while gene expression of BNP was measured by quantitative real-time polymerase chain reaction (qRT-PCR).
(1) Myocytes were pre-incubated with 3-30μM alendronate alone or with AngⅡ(1μM) for 48 h.
(2) Myocytes were pre-incubated with 30μM alendronate alone or in combination with equal GGOH in the addition of AngⅡ(1μM) for 48 h.
(3) Myocytes were pre-incubated with alendronate, GGTI-286, C3 exoenzyme or Y-27632 in the addition of AngⅡ(1μM) for 48 h
(4) Myocytes were pre-incubated with 30μM alendronate alone or in combination with equal GGOH for 48 h before incubation with AngⅡfor 15 min. Pull down assay and western blot analysis for RhoA activity and expression were performed.
Results:
(1) Alendronate at 3 to 30μM prevented hypertrophy responses induced by AngⅡ(1μM).
(2) The anti-hypertrophic effects of inhibition of FPP synthase with alendronate in Ang II-cultured neonatal cardiomyocytes hypertrophy were partially reversed by comparative geranylgeranyol (GGOH).
(3) The anti-hypertrophic effects of inhibition of FPP synthase with alendronate could be mimicked by GGTI-286, a geranylgeranyltransferase-I inhibitor, C3 exoenzyme, an inhibitor of Rho or Y-27632, an inhibitor of ROCK.
(4) Pull-down assay showed alendronate reduced-active RhoA by Ang II was also partially antagonized by GGOH.GGTI-286 also abolished the activated RhoA by AngⅡ.
Conlusions:
This study reveals the inhibition of FPP synthase by alendronte reduces RhoA activation by diminishing geranylgeranylation which prevents AngⅡ-induced hypertrophic response of neonatal cardiomyocytes.
Part 2 Konckdown of farnesylpyrophosphate synthase by RNA interference prevents angiotensin II-induced cardiac hypertrophy
Background:
Our first part experimental research suggests inhibition of farnesylpyrophosphate synthase (FPPS) enzyme could reverse cardiac hypertrophy induced by angiotensinⅡ(AngⅡ), which indicates FPPS gene as the therapeutic target involved in AngⅡ-induced cardiac hypertrophy.
The data of our previous RNA array study reported the up-regulation of FPPS mRNA in hypertrophy myocardium of 18-week spontaneously hypertensive rats (SHR) associated with local activated renin-angiotensin system (RAS) and higher AngⅡcontent than the age-matched Wistar-Kyoto rats (WKY).
Collectively, this evidence strongly suggests the probable essential role of FPPS in the development of cardiac hypertrophy by Ang II.
Aim:
The aim of our study was designed to investigate whether the mRNA and protein expression of FPPS are increased in AngⅡ-mediated cardiac hypertrophy, then whether silencing FPPS modulates these hypertrophic responses, and furthermore studied FPPS-related signaling.
Methods:
(1) FPPS expression was measured in cardiac hypertrophy models by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot.
(2) The four different sequences of short interference RNA duplexes (siRNAs) targeting the rat FPPS gene were constructed into shRNA vectors and the FPPS fusion protein was generated by insertion of rat FPPS cDNA into the pEGFP-C1 vector. HEK293 T cells were co-transfected with the rat FPPS fusion protein plus different shRNAs against FPPS or scrambled shRNA using lipofectamine 2000 and then the most active shRNA against rat FPPS was established by observation of GFP expression by immunofluorescence and western blot.
(3) The most active FPPS siRNA was established, synthesized and then incorporated into a lentiviral vector with scrambled shRNA control for lentivirus production in vitro and in vivo gene transfer experiment. Hypertrophy makers in vitro include cell surface area and the mRNA expression of P-myosin heavy chain (β-MHC) and brain natriuretic peptide (BNP) by qRT-PCR. Hypertrophic responses in vivo indexed by heart weight/body weight (HW/BW), left ventricular weight/body weight (LVW/BW) and echocardiography were measured as well as expression ofβ-MHC and BNP mRNA by qRT-PCR.
(4) The involvement of FPPS in Ang II-activated hypertrophic responses was furthered by investigation of downstream signaling in cultured cardiomyocytes. Pull down assay and western blot analysis for RhoA activity and expression were performed. RhoA immunofluorescence confocal microscopy was analysed for RhoA translocation. Besides, the activity of p-38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinases (MAPK) was performed by western blot analysis.
Results:
(1) FPPS expression was elevated both in cultured neonatal cardiomyocytes (NCMs) following AngⅡ(1μM) treatment and in the hypertrophic myocardium of 18-week-old spontaneously hypertensive rats (SHRs).
(2) FPPS shRNA lentivirus was constructed successfully. FPPS silencing in NCMs completely inhibited the hypertrophy marker genes ofβ-myosin heavy chain (β-MHC) and brain natriuretic peptide (BNP), as well as cell surface area. In vivo gene transfer also attenuated hypertrophic responses as indexed by left ventricular weight/body weight, heart weight/body weight as well as expression ofβ-MHC and BNP mRNA in SHRs. Echocardiography also showed the partial attenuation of interventricular septum wall thickness at diastolic phase (IVSd) and improvement of fraction shortening (FS) and ejection fraction (EF) in SHR silencing group.
(2) FPPS knockdown prevented elevated RhoA activity as well as RhoA translocation to plasma membrane in incubation with AngⅡcompared with non-silenced controls in NCMs. Similarly, increased-phosphorylation of P-38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinases (MAPK) by AngⅡwas attenuated.
Conlusions:
In conclusion, the expression of FPPS was elevated in Ang II-mediated cardiac hypertrophy and FPPS with RhoA-associated P38 and JNK MAPK signaling might play an important role in AngⅡ-induced cardiac hypertrophy.
Part 3 Alendronate modulates angiotensinⅡ-induced neonatal cardiac fibroblast proliferation differentiation and collagen production in vitro
Background:
Nitrogen-containing bisphosphonates (N-BPs), are extensively used in bone-related diseases and cancers. Our first part experimental research suggests alendronate could modulate cardiac hypertrophy induced by angiotensinⅡ(AngⅡ). Previous study demonstrated alendronate has beneficial effects on cardiac fibrosis in spontaneously hypertensive rats.
Aims:
This study was designed to examine whether pretreatment with alendronate could modulate Ang II activated early events of cardiac fibrosis in vitro including the increase in cell proliferation, differentiation of neonatal cardiac fibroblasts, and collagen production.
Methods:
Cardiac fibroblasts were prepared from 1 to 2-day-old Wistar rats. The proliferation of cardiac fibroblasts was determined by water-soluble tetrazolium (WST)-1 assay using Cell Counting Kit (CCK-8) and the differentiation marker of cardiac fibroblasts a-smooth muscle actin (a-SMA) was measured by western blot. Collagen production was assayed by the mRNA expression of collagen type II and type II using the real-time polymerase chain reaction (qRT-PCR) method, as well as by the analysis of hydroxyproline levels. Additionally transforming growth factor (TGF)-β1 protein production by cultured neonatal cardiac fibroblasts as well as P38 mitogen-activated protein kinase (MAPK) activity was evaluated by enzyme linked immunosorbent (ELISA) and western blot respectively.
Results:
Alendronate inhibited the proliferation and differentiation of cardiac fibroblasts as well as prevented collagen production by Ang II in vitro. Furthermore, the TGF-β1 protein expression and secretion by cardiac fibroblasts as well as activated P38 MAPK by AngⅡwere all reversed by alendronate.
Conclusion:
The current study demonstrates the inhibitory role of alendronate on Ang II-induced increase in cell proliferation, differentiation of neonatal cardiac fibroblasts and collagen production. The effect is possibly mediated by lowering the TGF-β1 levels and inactivation of P38 MAPK signaling.
引文
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