Jagged1和p53共同参与病理性心肌肥厚中心肌微血管内皮细胞损伤的分子机制

发布时间：2018-05-09 03:17

本文选题：P53 + Jaggedl　；参考：《复旦大学》2012年博士论文

【摘要】：众所周知,高血压可以引起心肌肥厚,起初的病理性心肌肥厚是心肌细胞适应压力负荷的一种代偿机制,但长时间的肥厚刺激,加之肥厚自身的有害性最终将导致心室的扩大、左室功能的减退和慢性心力衰竭的发生；目前来讲,慢性心力衰竭在我们当今社会中仍然存在着较高的病死率,如何延缓心肌肥厚到心力衰竭的进展仍然是我们需要解决的话题。因此,对心肌肥厚发生发展机制的阐述具有其一定的必要性及现实性。以往的研究认为,在心肌肥厚发生发展的过程中,存在着心肌细胞和微血管数量之间的不平衡,使得心肌细胞相对缺氧,最终导致心肌肥厚发展到心力衰竭。尽管肾素-血管紧张素系统,尤其是血管紧张素Ⅱ(AngⅡ)在心肌肥厚发生发展中的重要作用早已明确,伴随着血管紧张素转换酶抑制剂(ACEⅠ)及血管紧张素Ⅱ受体阻断剂(ARBs)在临床中的广泛应用,AngⅡ在心肌肥厚治疗中的地位亦被大多数学者所认可,但以往基于AngⅡ对心脏影响的研究主要集中在其对心肌细胞凋亡和心肌重构的探讨上,其对心脏微血管的作用目前尚鲜有研究、作用机理亦尚未明确,因此本研究主要探讨AngⅡ对心脏微血管的作用及其可能的分子机制,以期对心肌肥厚发生发展机理提供新的研究思路。 P53,肿瘤抑制因子,由393个氨基酸组成,是调节细胞周期、细胞衰老及凋亡的重要转录调控因子。生理条件下,p53蛋白在心脏组织中维持在较低的水平上,但在心肌细胞缺氧的条件下,p53蛋白被上调、左心室收缩功能障碍,但其并不直接导致心肌细胞死亡,而是通过抑制心脏血管的新生间接导致了心肌细胞死亡,进而影响了左室的收缩功能。当p53发生磷酸化之后,不但失去野生型p53抑制肿瘤增殖的作用,而且突变本身又使该基因具备癌基因功能。突变的p53蛋白与野生型p53蛋白相结合所形成的寡聚蛋白不能结合DNA,使得一些癌变基因转录失控最终导致肿瘤的发生。例如,p53可以被ATM, ATR和DNA-PK等在Ser15和Ser37位磷酸化,从而抑制p53的泛素化降解,促进p53的激活和积累。Chkl和Chk2可以磷酸化p53的Ser20,促进p53的四聚化,增强其稳定性和活性。P53的Ser46磷酸化和其诱导细胞凋亡密切相关。P53的Ser392可以被CAK磷酸化,该位点磷酸化和p53的抑制生长功能及其与DNA结合并转录激活有关。2007年,日本Komuro团队发表在Nature的研究发现,压力负荷下,p53累积,其下游保护性因子Hif-1a表达减少,使得心肌肥厚发展到心力衰竭,其在研究中提出此机制可能通过抑制心脏血管的新生间接导致心肌细胞死亡的这一假设,但尚未阐述p53对心肌微血管影响的具体机制。本文就此机理进行进一步的阐述。 Jagged1,Ⅰ型膜蛋白,Noth信号通路的一个配体,通过其Delta/Serrate/Lag2与Noth受体的EGF repeat结合参与诸如细胞表型、差异、分化、迁移、凋亡和血管新生等生理过程。尽管Jagged1在胚胎期动静脉形成中所起的作用不可替代,但其在出生后的血管形成中并非必不可少。目前Jagged1在血管新生方面的研究主要集中在肿瘤领域的研究上,本实验室先前的研究也主要集中在Jagged1在动脉粥样硬化中动脉内皮、动脉平滑肌的研究上,Jagged1在成年啮齿类动物心肌微血管内皮中的作用本实验室及以外尚未有此方面的报道、其作用机制尚不是很明确。鉴于此,我们假设Angll作用于心肌微血管内皮细胞(CMVEC)上的AT1R,引起细胞膜上Notch配体-Jagged1的下调,进而增加了p53在细胞浆中的蓄积,联动保护性因子Hif-1a下调,血管新生因子VEGF分泌减少,心脏血管新生障碍。本实验共分为三个部分分别从离体和在体两个层面上对这一设想进行阐述。第一部分P53参与血管紧张素Ⅱ致心肌微血管内皮细胞功能障碍的分子机制(离体实验) 目的：P53在Angll致CMVEC功能障碍中的作用。方法：1.心肌植块法培养成年Wistar雄性大鼠的原代CMVEC,通过免疫荧光染色法鉴定细胞表面分子(vWF、CD31)的表达确定此种方法所培养的细胞为CMVEC,体外迁移实验及体外管腔样结构形成实验明确CMVEC的体外血管新生能力。 2.10-6MAngⅡ干预第二或第三代CMVEC18hrs后,体外管腔样结构形成实验观察其体外血管新生能力,同时采用western blotting方法观察细胞中磷酸化p53(S392)、p53、Hif-1a及Jagged1的表达变化；realtime RT-PCR方法检测AngⅡ干预后细胞中VEGF基因的变化；ELISA方法观察CMVEC分泌到培养基中VEGF蛋白的变化。 3.在50μMpifithrin-a (PFT-a)阻断p53的基础上,AngⅡ干预CMVEC18hrs后,观察CMVEC中上述指标(phospho-p53(S392)、p53、Hif-1a、Jagged1、gene/protein of VEGF)的变化(实验所用方法同方法2)。结果：1.心肌植块法培养出的成年大鼠原代CMVEC具有很高的纯度(约95%),此方法培养的CMVEC具备诸如体外迁移、体外血管新生的能力。 2.10-6M AngⅡ干预CMVEC后,上调了细胞核中p53的磷酸化,并使得胞浆中p53的蓄积增加,同时下调了细胞核中的Hif-1a蛋白,上调了细胞膜上Jagged1的表达、胞浆内VEGF在RNA水平的表达,但减少了分泌到培养基中的VEGF蛋白。 3.50gM的PFT-a可以很好的阻断p53的表达,在PFT-a阻断p53的基础上, AngⅡ干预CMVEC后,与单纯AngⅡ干预组比较,CMVEC体外管腔样结构形成能力增强,胞核中Hif-la表达上调,分泌到培养基中的VEGF增加,胞膜上的Jagged1表达减少。结论：10-6MAngⅡ干预CMVEC,引起CMVEC体外管腔样结构形成障碍,这种损伤作用通过磷酸化细胞核中的p53,实现p53在胞浆中的蓄积,进而引起胞核中具有保护作用的Hif-1a下调,血管新生因子VEGF分泌减少,从而损伤了CMVEC的体外血管新生能力。上调的Jagged1配体可能作为一种代偿机制参与了AngⅡ致CMVEC的血管新生损伤作用。第二部分在体实验验证p53参与血管紧张素Ⅱ致心肌微血管内皮细胞功能损伤的分子机制目的：对p53在AngⅡ致CMVEC功能障碍中的分子机理进行在体验证。方法：采用皮下埋藏微量泵的方法持续给予6-8周龄C57/BL6雄性小鼠200ng/kg/min AngⅡ,给药时间为2周(预实验建立给药时间为1周、2周及4周的实验动物模型)；在建立AngⅡ干预引起心肌肥厚模型的同时,设置p53抑制组,即在AngⅡ干预前一天腹腔注射3.0mg/kg PFT-a一次,随后每间隔三天同剂量腹腔给药一次,直至AngⅡ微量泵埋藏的第14天PFT-a给药结束。AngⅡ给药结束取材前,小动物心脏超声测量小鼠左心室的肥厚程度,并用尾动脉压测量装置无创测量小鼠尾动脉压力；取材时,称量小鼠体重及其心脏重量,并在心脏的中1/2处将心脏横断为两部分,将心尖所在部分立即投入液氮中备用于分子生物学实验(western blotting, realtime RT-PCR)心底部放入10%中性福尔马林溶液中,用做随后的苏木精-伊红染色(HE)、免疫组织化学染色(CD31)。结果：1.200ng/kg/min AngⅡ持续皮下给药1周、2周、4周引起C57/BL6雄性小鼠心室壁的肥厚,以2周肥厚最为显著；200ng/kg/min AngⅡ干预C57/BL6雄性小鼠2周后,CD31免疫组织化学染色显示心脏微血管数量减少,同时有胞核中p53的磷酸化增加,胞浆中p53的蓄积增多,Hif-1a在胞核中的表达下调,Jagged1在胞膜上的表达上调,VEGF在组织中的表达增加。 2.3.0mg/kg PFT-a腹腔给药,初次给药时间为AngⅡ给药前一天,后续每间隔三天给药一次的方法能够抑制p53的表达,并缓解AngⅡ给药14天所致小鼠左心室的肥厚程度,与单纯AngⅡ给药14天组比较,此种处理增加了Hif-1a在心肌组织细胞核中的表达、减少了VEGF及Jagged1在心肌组织中的表达。结论：200ng/kg/min AngⅡ干预C57/BL6雄性小鼠2周后,引起小鼠左心室肥厚,心脏微血管数量减少,且p53参与了此种损伤作用；抑制p53后,缓解了AngⅡ所致心肌的肥厚程度及心脏微血管数量的减少程度,与离体实验p53参与了AngⅡ致CMVEC体外血管新生障碍这一实验结果相符；但体实验中,组织中增加的VEGF蛋白,可能是心肌细胞通过旁分泌对微血管障碍的一种代偿机制。第三部分Jagged1与p53在参与血管紧张素Ⅱ致心肌微血管内皮细胞功能障碍机理中的关系目的：分析Jagged1在AngⅡ所致CMVEC功能障碍中的角色及其与p53的关系。方法：心肌植块法培养成年Wistar雄性大鼠的原代CMVEC, anti-rat Jagged1siRNA预处置第二或第三代CMVEC后,10-6MAngⅡ干预CMVEC18hrs,体外管腔样结构形成实验观察CMVEC的体外血管新生能力,western blotting方法观察CMVEC中磷酸化p53(S392)、p53、Hif-1a的表达变化,realtime RT-PCR方法检测AngⅡ干预后CMVEC中VEGF基因的变化；ELISA方法观察培养基中分泌性蛋白-VEGF的变化。结果：与单纯AngⅡ干预组相比,anti-rat Jagged1siRNA预处置后、AngⅡ干预,CMVEC体外管腔样结构形成能力增强,胞核中p53的磷酸化减少,但p53在胞浆中的累积、Hif-1a在胞核及胞浆中的表达、分泌到培养基中的VEGF均没有统计学意义的变化。结论：Jagged1在AngⅡ致CMVEC功能损伤作用中,既不是此处p53信号通路的上游分子,亦非其下游分子；而是作为另一个独立信号分子参与此种损伤作用的。
[Abstract]:It is well known that hypertension can cause hypertrophy of the myocardium. At first the pathological myocardial hypertrophy is a compensatory mechanism for the stress load of the myocardium, but the prolonged hypertrophy of hypertrophy and the detrimental of hypertrophy will eventually lead to the enlargement of the ventricle, the decline of left ventricular function and the occurrence of chronic heart failure; at present, chronic heart is concerned. There is still a high mortality rate in our society, and how to delay the progress of cardiac hypertrophy to heart failure is still a topic we need to solve. Therefore, it is necessary and realistic to explain the mechanism of the development of cardiac hypertrophy. In the presence of imbalance between the number of cardiac myocytes and microvessels, myocardial cells are relatively anoxic and eventually lead to cardiac hypertrophy to heart failure. Although the role of the renin angiotensin system, especially angiotensin II (Ang II), in the development of cardiac hypertrophy has long been clear, accompanied by angiotensin conversion Enzyme inhibitor (ACE I) and angiotensin II receptor blocker (ARBs) are widely used in clinical practice. The status of Ang II in the treatment of myocardial hypertrophy is also recognized by most scholars. However, the previous studies based on the effect of Ang II on cardiac muscle cell withering and myocardial remodeling are mainly focused on the cardiac microvascular. In this study, the effect of Ang II on cardiac microvasculature and its possible molecular mechanism are discussed, so as to provide new research ideas for the mechanism of the development of cardiac hypertrophy.
P53, a tumor suppressor, consisting of 393 amino acids, is an important transcriptional regulator that regulates cell cycle, cell senescence and apoptosis. Under physiological conditions, the p53 protein is maintained at a lower level in the cardiac tissue, but the p53 protein is up-regulated and left ventricular systolic dysfunction under the condition of anoxia, but it does not directly lead to the dysfunction of the left ventricle. The death of the cardiac myocytes, but by inhibiting the birth of the heart blood vessels indirectly leads to the death of the cardiomyocytes, and then affects the contractile function of the left ventricle. When p53 is phosphorylated, it not only loses the role of the wild type p53 to inhibit the proliferation of the tumor, but also the mutation itself makes the gene have the function of the oncogene. The mutation of p53 protein and the wild type p53 The oligomeric protein formed by protein binding can not bind to DNA, making some cancerous gene transcriptional out of control eventually leading to the occurrence of tumors. For example, p53 can be phosphorylated by ATM, ATR and DNA-PK in Ser15 and Ser37 sites, thus inhibiting the ubiquitination of p53, promoting the activation of p53 and accumulating.Chkl and Chk2 phosphorylation p53. Four polymerization, enhancing its stability and active.P53 Ser46 phosphorylation and inducing cell apoptosis closely related to.P53 Ser392 can be phosphorylated by CAK, this site phosphorylation and p53 inhibition of growth function and DNA binding and transcriptional activation related.2007 years, Japanese Komuro team published in Nature research found under pressure load, p53 accumulation, The decrease of the protective factor Hif-1a in the lower reaches causes the development of myocardial hypertrophy to heart failure. In the study, it is suggested that this mechanism may inhibit the death of cardiac myocytes indirectly through the inhibition of cardiac angiogenesis, but the specific mechanism of the effect of p53 on myocardial microvessels has not been elaborated. This mechanism is further elaborated in this paper.
Jagged1, type I membrane protein, a ligand of the Noth signaling pathway, which combines its Delta/Serrate/Lag2 with the EGF repeat of the Noth receptor to participate in physiological processes such as cell phenotype, differentiation, differentiation, migration, apoptosis and angiogenesis. Although the role of Jagged1 in the embryonic stage of arteriovenous formation is irreplaceable, its vascular shape after birth Jagged1 is not essential. Current research on angiogenesis is mainly focused on the research in the field of tumor. Previous research in our laboratory is mainly focused on the role of Jagged1 in atherosclerotic artery endothelium, arterial smooth muscle research, and the role of Jagged1 in the myocardial microvascular endothelium of adult rodents. There has not been any report in this area and its mechanism is not very clear.
In view of this, we hypothesized that Angll acts on AT1R on the myocardial microvascular endothelial cells (CMVEC), causing the downregulation of the Notch ligand -Jagged1 on the cell membrane, thereby increasing the accumulation of p53 in the cytoplasm, the down regulation of the protective factor Hif-1a, the decrease of the secretion of angiogenesis factor VEGF, and the cardiac angiogenic disorder. The experiment was divided into three parts. Do not elaborate on this assumption from two levels: in vitro and in vivo.
Part I P53 involved in the molecular mechanism of angiotensin II induced dysfunction of myocardial microvascular endothelial cells (in vitro)
Objective: To investigate the role of P53 in CMVEC dysfunction induced by Angll.
Methods: the primary CMVEC of adult Wistar male rats was cultured by 1. myocardial explant method. The expression of vWF (CD31) was identified by immunofluorescence staining. The cells cultured in this method were CMVEC. The in vitro migration experiment and the formation of the tube like structure in vitro showed that the angiogenesis ability of CMVEC in vitro was confirmed.
After 2.10-6MAng II intervention for second or third generations of CMVEC18hrs, the angiogenesis in vitro was observed in vitro, and the expression of p53 (S392), p53, Hif-1a and Jagged1 in the cells were observed by Western blotting, and realtime RT-PCR square method was used to detect the changes of the genes in the cells after the intervention of Ang II. ISA method was used to observe the changes of CMVEC protein secreted into VEGF medium.
3. on the basis of 50 Mpifithrin-a (PFT-a) blocking p53 and Ang II intervention in CMVEC18hrs, the changes of the above indexes (phospho-p53 (S392), p53, Hif-1a, Jagged1, gene/protein) were observed by Ang II (the method used in the experiment and method 2).
Results: the primary CMVEC cultured in 1. myocardial explants had a high purity (about 95%). The CMVEC culture of this method had the ability to migrate in vitro and in vitro angiogenesis.
After the intervention of CMVEC, 2.10-6M Ang II increased the phosphorylation of p53 in the nucleus and increased the accumulation of p53 in the cytoplasm, down the Hif-1a protein in the nucleus, up the expression of Jagged1 on the cell membrane, and the expression of VEGF in the cytoplasm at RNA level, but reduced the VEGF protein secreted into the culture medium.
The PFT-a of 3.50gM can block the expression of p53 very well. On the basis of blocking p53 by PFT-a, Ang II interfered with CMVEC, and compared with the simple Ang II intervention group, the formation ability of CMVEC in vitro was enhanced, the expression of Hif-la in the nucleus was up, the VEGF increased in the medium, and the Jagged1 expression on the membrane decreased.
Conclusion: the intervention of 10-6MAng II to CMVEC causes the formation of CMVEC in vitro, which can cause the accumulation of p53 in the cytoplasm through the p53 in the phosphorylated nucleus, thereby causing a protective Hif-1a downregulation in the nucleus and reducing the secretion of the neovascularization factor VEGF, thereby damaging the angiogenesis of CMVEC in vitro. Up regulation of Jagged1 ligand may play a compensatory role in Ang II induced CMVEC angiogenesis.
The second part is in vivo experiments to verify the molecular mechanism of p53 involved in angiotensin II induced myocardial damage in microvascular endothelial cells.
Objective: To explore the molecular mechanism of p53 in Ang II - induced CMVEC dysfunction.
Methods: 6-8 weeks old C57/BL6 male mice 200ng/kg/min Ang II was continuously given by subcutaneous embedded micropump. The time of administration was 2 weeks (the experimental animal model was set up for 1 weeks, 2 and 4 weeks). The p53 inhibition group was set up at the same time when the Ang II intervention caused the hypertrophy of the myocardial hypertrophy, that is, the day before the intervention of Ang II. Intraperitoneal injection of 3.0mg/kg PFT-a once, followed by three days of the same dose of the same dose of intraperitoneal administration, until the fourteenth day of the Ang II micropump buried at the end of the PFT-a to end.Ang II, the small animal heart ultrasound measurement of the left ventricular hypertrophy of mice, and the tail artery pressure measurement device to measure the tail artery pressure in mice. Weighing the weight of the mice and the weight of the heart, and breaking the heart into two parts at the middle 1/2 of the heart, the part of the tip of the apex was immediately put into liquid nitrogen for use in the molecular biology experiment (Western blotting, realtime RT-PCR) to be placed in the 10% neutral formalin solution for subsequent hematoxylin eosin staining (HE) and immune tissue. Chemical staining (CD31).
Results: 1.200ng/kg/min Ang II sustained subcutaneous administration for 1 weeks, 2 weeks and 4 weeks to cause the hypertrophy of ventricular wall in C57/BL6 male mice, which was the most significant in 2 weeks. After 2 weeks of 200ng/kg/min Ang II intervention in C57/BL6 male mice, CD31 immuno histochemical staining showed that the number of cardiac microvessels decreased, and the phosphorylation of p53 in the nucleus increased and the cytoplasm was P5. The accumulation of Hif-1a increased, the expression of Jagged1 in the nucleus was down regulated, the expression of VEGF on the cell membrane was up-regulated, and the expression of VEGF in the tissue increased.
2.3.0mg/kg PFT-a was administered intraperitoneally for the first time before the time of Ang II administration. The subsequent administration of three days after each interval could inhibit the expression of p53 and alleviate the degree of left ventricular hypertrophy induced by Ang II Administration for 14 days. Compared with the 14 day group of pure Ang II administration, this treatment increased the table of Hif-1a in the nucleus of myocardial tissue. It reduced the expression of VEGF and Jagged1 in myocardium.
Conclusion: 200ng/kg/min Ang II interfered with C57/BL6 male mice for 2 weeks, causing the left ventricular hypertrophy and the decrease in the number of cardiac microvessels, and p53 participated in the injury. After the inhibition of p53, the degree of myocardial hypertrophy and the decrease in the number of cardiac microvessels caused by Ang II were relieved, and p53 in vitro participated in CMVEC in vitro blood of Ang II induced CMVEC in vitro. The results of this experiment are consistent with the experimental results, but in the body experiment, the increased VEGF protein in the tissue may be a compensatory mechanism for the microvascular obstruction by the paracrine of the cardiac myocytes.
The third part is the relationship between Jagged1 and p53 in the mechanism of dysfunction of myocardial microvascular endothelial cells induced by angiotensin II.
Objective: to analyze the role of Jagged1 in CMVEC dysfunction induced by Ang II and its relationship with p53.
Methods: the primary CMVEC of adult Wistar male rats was cultured by myocardial graft method. After anti-rat Jagged1siRNA was predisposed to second or third generations of CMVEC, 10-6MAng II intervened CMVEC18hrs. The angiogenesis of CMVEC in vitro was observed in vitro. Western blotting formula was used to observe the phosphorylation p53. Realtime RT-PCR method was used to detect the change of VEGF gene in CMVEC after Ang II intervention. ELISA method was used to observe the change of secretory protein -VEGF in the medium.
Results: compared with the simple Ang II intervention group, after the anti-rat Jagged1siRNA pretreatment, the Ang II intervention, the enhancement of the formation ability of CMVEC in vitro, the phosphorylation of p53 in the nucleus decreased, but the accumulation of p53 in the cytoplasm, the expression of Hif-1a in the nucleus and cytoplasm, and the secretion of VEGF in the culture medium were not statistically significant.
Conclusion: Jagged1 is not the upstream molecule of the p53 signaling pathway and the downstream molecules of the p53 signaling pathway, but is an independent signal molecule involved in this damage in the function of CMVEC damage induced by Ang II.

【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R363

【共引文献】