Both Jagged1and Accumulation Of P53Participated In The Impairment Of Micro Vasculatures In Pathological Cardiac Hypertrophy

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 vitroObjective: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-PCRResults: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.