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
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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。
Although pathological cardiac hypertrophy is initially a compensatory mechanism that a cellular response to increased workload by decreasing wall stress, sustained hypertrophy is deleterious and long-term decompensation may lead to a decline in left ventricular function and congestive heart failure, which has high rate of mortality. The necessity and feasibility of researches about cardiac hypertrophy is still needed. Reports have been postulated that a mismatch between the number of capillaries and the size of cardiomyocytes develops during the development of cardiac hypertrophy, leading to myocardial hypoxia. Though, the renin-angiotensin system(RAS), especially angiotensinll, has previously been established to play an important role in the progression of cardiac remodeling, inhibition of a hyperactive RAS provides protection from cardiac remodeling and subsequent heart failure, studies about AngⅡ were mainly about the apoptosis and remodeling of myocardial cells, effects of Angll on cardiac microvascular endothelial cells(CMVEC) in the heart remains unexplored. Therefore, our studies try to investigate the effects of AngⅡ on CMVEC and the possible mechanisms in order to put a new derivation forward about cardiac hypertrophy.
P53, a tumor inhibitor, contains393amino acids, is a factor that regulated apoptosis, cycles and ageing of cells. Under unstressed conditions, p53protein is generally present at a low concentration, is presumably inactive as a transcription factor and is diffusely distributed throughout the cell. But, it was up-regulated when under myocardial hypoxia, and obstacles of systolic functions of left ventricular occurred, the machanism might be that it can induce apoptosis of myocardial cells through impairment of angiogenesis rather than cause apoptosis directly. The effect of p53on inhibition of tumor proliferation was dispeared when p53was phosphorylated, futhennore, the mutant type can prompt tumors by itself. The oligomeric protein that wild type and mutant type combined together can not combine DNA again, and the combination lead to dys-regulation of genes that prompt tumors, tumors occurred subsequently. P53can be phosphorylated by ATM, ATR and DAN-PK. at both Ser15and Ser37, and the phosphorylation at this point can inhibit the degradation of ubiquitinoylation, promt the accumulation and activation. P53can also be phosphorylated by Chkl and Chk2at Ser20, and this phosphorylation can stumulate tetramerization of itself, enhance the security and activation. Phosphorylation of p53at Ser46was related with the induction of apoptosis. P53can be phosphorylated by CAK at Ser392, phosphorylation at this point is connected with inhibition of growth, combination of DNA and activation of transcription. One paper published in nature by Kumuro group during2007considered that accumulation of p53and reduction of Hif-la promt cardiac hypertrophy to heart failure, and they also made a hypothesis that the machanism was through the inhibition of angiogenesis which lead to apoptosis of cardiomyocyte. However, no discussion about effects of p53on CMVEC was involved. Consequently, we investigate the mechanism intensively here.
Jagged1, a Notch ligand, is a type I transmembrane protein and binds to the EGF repeat of Notch receptor through Delta/Serrate/Lag2domain. The Notch signaling pathway is involved in many biological processes, such as cell fate specification, differentiation, proliferation, apoptosis, migration and angiogenesis. Though, Jagged1plays a critical role during angiogenesis of embryos, its not essential in angiogenesis during adult procedure. Research about effects of Jaggedl on angiogenesis is mainly related to tumors, and the effects on endothelial cells and smooth musules of blood vessels in atherosclerosis were reported in our laboratory. Its effects on CMVEC from adult rodent animals have no reference presently. Therefore, we suppose that AngⅡ combined with AT1R on CMVEC, which lead to up-regulation of Jaggedl, can induce the accumulation of p53and down-regulation of Hif-1α. reduction of VEGF and dysfunction of angiogenesis occurred finally.
This paper is composed of three parts and discussed respectively both in vitro and in vivo as follows.
Part I Mechanisms of p53participated in dysfunctions of cardiac microvascular endothelial cells induced by angiotensinll in vitro
Objective:
Make a disccusion about AngⅡ-induced dysfunctions of CMVEC that p53participated in vitro.
Methods:
1. Wistar rats (male,8-9weeks of age) were selected for primary cell culture by method of planting myocardial tissue. Molecular markers, vWF, CD31were observed and identified by immunocytochemistry to determin the purity of CMVEC. Ability of migration and capillary tube-like formation were also observed to investigate its function.
2. The capillary tube-like formation was observed under a microscope after incubation of CMVEC with AngII(10-6M) for18hrs. Protein expresssion of p53(S392), p53, Hif-1α and Jaggedl was observed by western blotting method, RNA expression of VEGF was observed by realtime RT-PCR, and VEGF secreted in cell culture medium was observed by ELISA method. The scanning of all these molecules was under the same treatment of AngII(10-6M,18hrs).
3.CMVEC were incubated with AngⅡ(10-6M) for18hrs after inhibition of p53by pifithrin-a(PFT-α,50μM), measurements about capillary-like tubes and molecules were the same as step2above.
Results:
1. Results showed that purity of CMVEC characterized by staining with vWF or CD31was about95%, cells performed a power for migration, and a high ability to form the capillary tube-like structures after grown in matrigel for18hrs. CMVEC cultured by this method were able to be used later.
2. The phosphorylation of p53in nucleus was up-regulated when CMVEC were treated by AngⅡ for18hrs, and the phosphorylation made a accumulation of p53in cytoplasm and a reduction of Hif-la in nucleus. Protein of Jagged1in membrain was also up-regulated under this treatment. But, VEGF secreted into culture medium was down-regulated, gene of VEGF was up-regualted.
3. PFT-a(50μM) inhibited p53perfectly in CMVEC. The ability of CMVEC to form capillary-like tubes was enhanced when p53was inhibited before AngⅡ treatment comparing with groups that AngⅡ treated only, both Hif-1α in nucleus and VEGF that secreted into cell culture medium were up-ragulated, and Jaggedl in membrain was down-ragulated mercifully.
Conclusion:
AngII(10-6M,18hrs) made dysfunctions of CMVEC to form capillary-like tubes. The mechanism might be roles of p53phosphorylation, the phosphorylaiton could accumulate p53in cytoplasm and reduce Hif-la in nucleus. Furthermore, VEGF that secreted into cell culture medium was down-regulated and functions of CMVEC were damaged. The up-regulation of Jagged1might be a compensation that participated in this injury.
Objective:
Verification of AngⅡ-induced dysfunctions of CMVEC that p53participated.
Methods:
6-8weeks old male C57BL/6mice were purchased from Shanghai Animal Administration Center (Shanghai, China). AngⅡ (200ng/kg/min, Sigma-Aldrich) was continuously administered by Alzet micro-osmotic pumps (DURECT Corporation) that implanted subcutaneously. PFT-a (Sigma-Aldrich) was dissolved in DMSO, and was injected into mice intraperitoneally with a3.0mg/kg dose one day before the operation, then was injected every3days once a time. Mice were anesthetized by ketamine (100mg/kg) intraperitoneally injected, and performed by the echocardiography and noninvasive blood pressure (NIBP) when AngⅡ was infused for14days. Excised hearts were perfused with PBS and cut apart into two parts, one part that cantained the bottom was fixed in10%formalin for histological analysis(HE or CD31), and the other was putted in liquid nitrogen for western blotting and realtime RT-PCR
Results:
1.Cardiac hypertrophy appeared when treated male C57/BL6mice with AngⅡ (200ng/kg/min) for2weeks, accompanied by a reduction of the ratio of CD31positive cells/cardiomyocytes. For molecules, Jaggedl in membrane and phosphorylation of p53at S392in nucleus were up-regulated during cardiac hypertrophy, presence of the accumulation of p53in cytoplasm, up-regulation of VEGF in tissues and a reduction of Hif-1a in nucleus.
2. PFT-α(3.0mg/kg) could inhibite p53perfectly by this route of administration, and alleviate the degree of hypertrophy that AngⅡ induced. Meanwhile, Hif-1α was up-regulated, VEGF and Jagged1were down-regulated in the heart.
Conclusion:
P53participated in the impairment of CMVEC induced by AngⅡ in vivo which was the same as results that in vitro. But, VEGF in heart tisssue was up-regulated which was down-regulated in vitro. The reason might be a compensation for dysfunctions of capillaries by cardiomyocyte for its paracrine secretion.
Objective:
To analyze the relationship between Jagged1and p53in dysfunctions of CMVEC induced by Angll.
Methods:
Primary cells of CMVEC were got from aldult male Wistar rats by method of planting myocardial tissue. CMVEC were treated by AngII(10-6M) for18hrs after anti-rat Jagged1siRNA incubation, the capillary tube-like formation was observed under a microscope, phosphorylation of p53at S392, p53and Hif-1α were observed by western blotting method, VEGF that excreted into cell culture medium was also observed under the same treatment by AngⅡ.
Results:
The ability of CMVEC to form capillary-like tubes was enhanced when Jaggedl were silent by anti-rat Jagged1siRNA before AngⅡ treatment comparing with groups that AngⅡ treated only; Accumulation of p53, protein expression of Hif-1α both in nucleus and in cytoplasm, VEGF that secreted into cell culture medium had no significant difference.
Conclusion:
Jaggedl participated in the impairment of CMVEC induced by Angll, but its damageable effect might not be through p53accumulation way.
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。
Although pathological cardiac hypertrophy is initially a compensatory mechanism that a cellular response to increased workload by decreasing wall stress, sustained hypertrophy is deleterious and long-term decompensation may lead to a decline in left ventricular function and congestive heart failure, which has high rate of mortality. The necessity and feasibility of researches about cardiac hypertrophy is still needed. Reports have been postulated that a mismatch between the number of capillaries and the size of cardiomyocytes develops during the development of cardiac hypertrophy, leading to myocardial hypoxia. Though, the renin-angiotensin system(RAS), especially angiotensinll, has previously been established to play an important role in the progression of cardiac remodeling, inhibition of a hyperactive RAS provides protection from cardiac remodeling and subsequent heart failure, studies about AngⅡ were mainly about the apoptosis and remodeling of myocardial cells, effects of Angll on cardiac microvascular endothelial cells(CMVEC) in the heart remains unexplored. Therefore, our studies try to investigate the effects of AngⅡ on CMVEC and the possible mechanisms in order to put a new derivation forward about cardiac hypertrophy.
P53, a tumor inhibitor, contains393amino acids, is a factor that regulated apoptosis, cycles and ageing of cells. Under unstressed conditions, p53protein is generally present at a low concentration, is presumably inactive as a transcription factor and is diffusely distributed throughout the cell. But, it was up-regulated when under myocardial hypoxia, and obstacles of systolic functions of left ventricular occurred, the machanism might be that it can induce apoptosis of myocardial cells through impairment of angiogenesis rather than cause apoptosis directly. The effect of p53on inhibition of tumor proliferation was dispeared when p53was phosphorylated, futhennore, the mutant type can prompt tumors by itself. The oligomeric protein that wild type and mutant type combined together can not combine DNA again, and the combination lead to dys-regulation of genes that prompt tumors, tumors occurred subsequently. P53can be phosphorylated by ATM, ATR and DAN-PK. at both Ser15and Ser37, and the phosphorylation at this point can inhibit the degradation of ubiquitinoylation, promt the accumulation and activation. P53can also be phosphorylated by Chkl and Chk2at Ser20, and this phosphorylation can stumulate tetramerization of itself, enhance the security and activation. Phosphorylation of p53at Ser46was related with the induction of apoptosis. P53can be phosphorylated by CAK at Ser392, phosphorylation at this point is connected with inhibition of growth, combination of DNA and activation of transcription. One paper published in nature by Kumuro group during2007considered that accumulation of p53and reduction of Hif-la promt cardiac hypertrophy to heart failure, and they also made a hypothesis that the machanism was through the inhibition of angiogenesis which lead to apoptosis of cardiomyocyte. However, no discussion about effects of p53on CMVEC was involved. Consequently, we investigate the mechanism intensively here.
Jagged1, a Notch ligand, is a type I transmembrane protein and binds to the EGF repeat of Notch receptor through Delta/Serrate/Lag2domain. The Notch signaling pathway is involved in many biological processes, such as cell fate specification, differentiation, proliferation, apoptosis, migration and angiogenesis. Though, Jagged1plays a critical role during angiogenesis of embryos, its not essential in angiogenesis during adult procedure. Research about effects of Jaggedl on angiogenesis is mainly related to tumors, and the effects on endothelial cells and smooth musules of blood vessels in atherosclerosis were reported in our laboratory. Its effects on CMVEC from adult rodent animals have no reference presently. Therefore, we suppose that AngⅡ combined with AT1R on CMVEC, which lead to up-regulation of Jaggedl, can induce the accumulation of p53and down-regulation of Hif-1α. reduction of VEGF and dysfunction of angiogenesis occurred finally.
This paper is composed of three parts and discussed respectively both in vitro and in vivo as follows.
Part I Mechanisms of p53participated in dysfunctions of cardiac microvascular endothelial cells induced by angiotensinll in vitro
Objective:
Make a disccusion about AngⅡ-induced dysfunctions of CMVEC that p53participated in vitro.
Methods:
1. Wistar rats (male,8-9weeks of age) were selected for primary cell culture by method of planting myocardial tissue. Molecular markers, vWF, CD31were observed and identified by immunocytochemistry to determin the purity of CMVEC. Ability of migration and capillary tube-like formation were also observed to investigate its function.
2. The capillary tube-like formation was observed under a microscope after incubation of CMVEC with AngII(10-6M) for18hrs. Protein expresssion of p53(S392), p53, Hif-1α and Jaggedl was observed by western blotting method, RNA expression of VEGF was observed by realtime RT-PCR, and VEGF secreted in cell culture medium was observed by ELISA method. The scanning of all these molecules was under the same treatment of AngII(10-6M,18hrs).
3.CMVEC were incubated with AngⅡ(10-6M) for18hrs after inhibition of p53by pifithrin-a(PFT-α,50μM), measurements about capillary-like tubes and molecules were the same as step2above.
Results:
1. Results showed that purity of CMVEC characterized by staining with vWF or CD31was about95%, cells performed a power for migration, and a high ability to form the capillary tube-like structures after grown in matrigel for18hrs. CMVEC cultured by this method were able to be used later.
2. The phosphorylation of p53in nucleus was up-regulated when CMVEC were treated by AngⅡ for18hrs, and the phosphorylation made a accumulation of p53in cytoplasm and a reduction of Hif-la in nucleus. Protein of Jagged1in membrain was also up-regulated under this treatment. But, VEGF secreted into culture medium was down-regulated, gene of VEGF was up-regualted.
3. PFT-a(50μM) inhibited p53perfectly in CMVEC. The ability of CMVEC to form capillary-like tubes was enhanced when p53was inhibited before AngⅡ treatment comparing with groups that AngⅡ treated only, both Hif-1α in nucleus and VEGF that secreted into cell culture medium were up-ragulated, and Jaggedl in membrain was down-ragulated mercifully.
Conclusion:
AngII(10-6M,18hrs) made dysfunctions of CMVEC to form capillary-like tubes. The mechanism might be roles of p53phosphorylation, the phosphorylaiton could accumulate p53in cytoplasm and reduce Hif-la in nucleus. Furthermore, VEGF that secreted into cell culture medium was down-regulated and functions of CMVEC were damaged. The up-regulation of Jagged1might be a compensation that participated in this injury.
Objective:
Verification of AngⅡ-induced dysfunctions of CMVEC that p53participated.
Methods:
6-8weeks old male C57BL/6mice were purchased from Shanghai Animal Administration Center (Shanghai, China). AngⅡ (200ng/kg/min, Sigma-Aldrich) was continuously administered by Alzet micro-osmotic pumps (DURECT Corporation) that implanted subcutaneously. PFT-a (Sigma-Aldrich) was dissolved in DMSO, and was injected into mice intraperitoneally with a3.0mg/kg dose one day before the operation, then was injected every3days once a time. Mice were anesthetized by ketamine (100mg/kg) intraperitoneally injected, and performed by the echocardiography and noninvasive blood pressure (NIBP) when AngⅡ was infused for14days. Excised hearts were perfused with PBS and cut apart into two parts, one part that cantained the bottom was fixed in10%formalin for histological analysis(HE or CD31), and the other was putted in liquid nitrogen for western blotting and realtime RT-PCR
Results:
1.Cardiac hypertrophy appeared when treated male C57/BL6mice with AngⅡ (200ng/kg/min) for2weeks, accompanied by a reduction of the ratio of CD31positive cells/cardiomyocytes. For molecules, Jaggedl in membrane and phosphorylation of p53at S392in nucleus were up-regulated during cardiac hypertrophy, presence of the accumulation of p53in cytoplasm, up-regulation of VEGF in tissues and a reduction of Hif-1a in nucleus.
2. PFT-α(3.0mg/kg) could inhibite p53perfectly by this route of administration, and alleviate the degree of hypertrophy that AngⅡ induced. Meanwhile, Hif-1α was up-regulated, VEGF and Jagged1were down-regulated in the heart.
Conclusion:
P53participated in the impairment of CMVEC induced by AngⅡ in vivo which was the same as results that in vitro. But, VEGF in heart tisssue was up-regulated which was down-regulated in vitro. The reason might be a compensation for dysfunctions of capillaries by cardiomyocyte for its paracrine secretion.
Objective:
To analyze the relationship between Jagged1and p53in dysfunctions of CMVEC induced by Angll.
Methods:
Primary cells of CMVEC were got from aldult male Wistar rats by method of planting myocardial tissue. CMVEC were treated by AngII(10-6M) for18hrs after anti-rat Jagged1siRNA incubation, the capillary tube-like formation was observed under a microscope, phosphorylation of p53at S392, p53and Hif-1α were observed by western blotting method, VEGF that excreted into cell culture medium was also observed under the same treatment by AngⅡ.
Results:
The ability of CMVEC to form capillary-like tubes was enhanced when Jaggedl were silent by anti-rat Jagged1siRNA before AngⅡ treatment comparing with groups that AngⅡ treated only; Accumulation of p53, protein expression of Hif-1α both in nucleus and in cytoplasm, VEGF that secreted into cell culture medium had no significant difference.
Conclusion:
Jaggedl participated in the impairment of CMVEC induced by Angll, but its damageable effect might not be through p53accumulation way.
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