Dissertation
Dissertation > Medicine, health > Internal Medicine > Endocrine diseases and metabolic diseases > Islet disease > Diabetes

The Effect and Mechanism of High-dose Ang Ⅱ on the Expression of ACE and ACE2in Vascular Endothelial Cells

Author ChenHuan
Tutor ShenJie
School Southern Medical University,
Course Internal Medicine
Keywords Angiotensin Ⅱ Angiotensin transformation enzyme Angiotensin Ⅱ receptorblockers endothelial cells IrbesartanAngiotensin Ⅱ Irbesartan Angiotensin-converting enzyme2 p38 MAPK JNK
CLC R587.1
Type Master's thesis
Year 2013
Downloads 105
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BACKGROUNDThe morbidity of diabetes mellitus has been increasing on global. Wild S predicted that the prevalence of diabetes mellitus in all age-groups worldwide could reach4.4%in2030, and the total number of people with diabetes could rise to366million in2030. Moreover, the increasing of the morbidity of diabetes mellitus is especially astonishing in China. There is a study that suggests the prevalence of diabetes mellitus had risen to9.7%, pre-diabetes15.5%in adult group in China, a quarter of Chinese adults was in the condition of abnormal blood glucose, and the morbidity of diabetes mellitus was4.4%higher, compared with that from2000to2001. With increasing of the morbidity of diabetes mellitus, the prevalence of chronic vascular complications of diabetes become widespread, which is the main cause of death and disability. the occurrence of macroangiopathy and macroangiopathy is closely related to vascular dysfunction and the key factor to vascular dysfunction is he injury of vascular endothelial cells. The metabolic disturbance of patients with diabetes results in the imbalance of microenvironment around vascular endothelial, endothelial cell disfunction and injury. Moreover, the injury of vascular endothelial cells damage the balance between coagulation and fibrinolytic, which induces thrombosis and initiate forming atherosclerotic plaque. At last, the pathological mechanism cause a series of chronic vascular complications of diabetes.One of the critical factor to the injury of vascular endothelial cell is local RAS being activated. After combining with AT1R, the bioactive peptide of RAS, AngⅡ Plays a critical role in injuring the structure and function of vascular endothelial cells. ACE can indirectly damage vascular endothelial cells because of promoting synthesis of Ang Ⅱ. ACE2can protect vascular endothelial cells owing to break down Ang Ⅱ and promoting synthesis of Ang(1-7) antagonizing Ang Ⅱ.Vascular endothelial cells are a continuous layer of squamous cells Covering the increase surface area. The cell biological function consist of barrier function, endocrine function, information passing, coagulation, hemostatic, fibrinolytic, blood transmission,material exchange and so on. The occurrence of chronic complications of diabetes and cardiovascular disease is involved in endothelial cells.In vascular, both of the vascular endothelial cells and smooth muscle cells could secrete Ang Ⅱ。 Ang Ⅱ has inactive effect on the function of vascular endothelial cells, for example, permeability, vasomotor function, Anti-hemoglutination, the expression of adhesion molecule and vascular remodeling.ACE, One of component of RAS, distributes on the vascular endothelial cells and smooth muscle cells. ACE damage to endothelial cells by increasing Ang II quantity. ACE also inactivates bradykinin, and hydrolysis endogenous bioactive peptide to induce vasoconstriction, which injure endothelial cells.ACE2, distributions on the vascular endothelial cells, could decompose Ang Ⅱ to weaken the function of AngⅡ damaging endothelial cells, and synthesize Ang(1-7) antagonizing AngⅡ, so ACE2could protect endothelial cells. ACE2also downregulates AT1R to weaken the function of Ang Ⅱ damaging endothelial cells.Intracellular signals mediates Ang Ⅱ injuring the function of vascular endothelial cells. JNK and p38MAPK mediates the dysfunction of vascular endothelial cells and the effect of AngⅡ on cells. The signals pathway, p38MAPK and JNK are mitogen-activated protein kinase, and important intracellular signals by which extracellular signals are mediated into intracellular。 AngⅡ promotes the migration, proliferation of smooth muscle cells by p38MAPK and stimulates endothelial cells upregulating adhesion molecule expression to increase leukocytes aggregation by p38MAPK,which lead to vascular inflammation. In addition,JNK mediates endothelial cells apoptosis. JNK mediates Tumor necrosis factor-a upregulating the expression of intercellular adhesion molecule-1to causing vascular inflammation and promotes the proliferation of smooth muscle cells to causing vascular remodeling. In studies of Wang Min and other experts, tumor necrosis factor stimulates endothelial cells secreting ET-1by JNK. JNK is also activated by Ang Ⅱ to increase C-reactive protein which cause vascular inflammationWhen local RAS is activated, Ang Ⅱ increases and damages endothelial cells by varieties of mechanisms that promotes the prevalence of chronic vascular complications of diabetes and cardiovascular disease. In recent years, studies suggest that Ang Ⅱ upregulates ACE expression,and downregulates ACE2expression in some cells and tissue. In that way, the synthesis of Ang Ⅱ increases, but the decomposition decreases. At last, more Ang Ⅱ accumulates and the situation become negative cycle of RAS. Ang II causes the change above of ACE, and ACE2by AT1R. There are few studies on medium downstream of ATIR. After combining with AT1R, most Ang Ⅱ effect is mediated by p38MAPK and JNK. it is unknown whether p38MAPK and JNK also is the medium to AngⅡdownregulating ACE2expression and Ang Ⅱ upregulating ACE expression.However, in renal tubular epithelial cells, experts find that Ang Ⅱ downregulates ACE2expression by p38MAPK. Moreover, studies indicate that ACE itself upregulates ACE expression by JNK when the Serl270at carboxyl end of ACE is phosphorylated in vascular endothelial cells.However, there are no studies on whether Ang Ⅱ upregulates ACE expression,and downregulates ACE2expression in vascular endothelial cells and what molecular mechanism involve in the procedure.In view of that situation, Ang Ⅱ of different concentration was used for culturing vascular endothelial cells, and the expression of ACE, and ACE2were observed. Irbesartan, the blocker of AT2R, the blocker of p38MAPK and the blocker of JNK were used for intervene vascular endothelial cells cultured with Ang Ⅱ, and the expression of ACE, and ACE2were observed. The objective of the study is to approach the effect of Ang Ⅱ on ACE and ACE2, and what molecular signals are involved in the procedure.Chapter1The effect of Ang Ⅱ on mRNA expression of ACE、ACE2in vascular endothelial cells[Objective]To observe the effect of different concentration of Ang Ⅱ on ACE、ACE2in endothelial cells and to approach the role of Ang Ⅱ receptors[Method]1. Cells culturing and distribution:The Human umbilical vein endothelial cells grow in DMEM/F12with10%FBS in incubator at37℃,5%CO2. culture medium was changed every24hours.When umbilical vein endothelial cells contacted each other and they reached to80%-90%confluency in the cell culture flask, cells were trypsinized and passaged.2. Cell distribution1:Control group (DMEM/F12without FBS), Low concentration of Ang Ⅱ group (DMEM/F12without FBS+0.1μmol/L Ang Ⅱ), Middle concentration of AngⅡgroup (DMEM/F12without FBS+1.0μmol/L Ang Ⅱ), High concentration of Ang Ⅱ group (DMEM/F12without FBS+10.0μmol/L Ang Ⅱ). Cell distribution2:Control group (DMEM/F12without FBS), Ang Ⅱ group (DMEM/F12without FBS+1.0μmol/L AngⅡ), AngⅡ+irbesartan group (DMEM/F12without FBS+1.0μmol/L AngⅡ+1.0μmol/L irbesartan), AngⅡ+AT2R blocked group (DMEM/F12without FBS+1.0μmol/L AngⅡ+1.0μmol/L PD123319(AT2R blocker)).After cells cultured in regular condition being trypsinized, cells were inoculated into the petri dish of35mm in accordance with the density of1×106/ml.2ml DMEM/F12with10%FBS was added to every petri dish. When cells reached to80%-90%confluency in the petri dish, cells medium (DMEM/F12with10%FBS) was placed by DMEM/F12without FBS. cells had cultured in DMEM/F12without FBS for24hours in order to insure cells growth state synchronized. While cells growth state was synchronized,different kind of reagent above was used to intervene cells.24hours after being intervened with different reagent, cells were collected to be detected for the mRNA expression of ACE, and ACE2.2. Detection of ACE mRNA and ACE2mRNA with realtime fluores-cence quantitative PCR(1) Trizol was used to extract cell total RNA, lml Trizol was added to every petri dish to decompose cells→mixture in every petri dish was transferred to the Eppendoff of2ml→Add0.2ml chloroform to the Eppendoff above→4℃,12000g, the Eppendoff was centrifuged for15min→After the content in the Eppendoff being stratified, the supernatant was transferred to another new Eppendoff of2ml.→Add equal volume of isopropanol to the new Eppendoff→4℃,12000g, the Eppendoff was centrifuged for10min→Discard the supernatant→Add1ml75%ethanol without water→4℃,7500g, the Eppendoff was centrifuged for10min→Discard the supernatant, Let the white precipitate in the bottom of the Eppendoff dry out in air→Add30ul1%oDEPC to dissolve mRNA (the white precipitate was mRNA), low temperature storage.(2) Detection of RNA concentration and purity:Use Ultraviolet spectrophotometer to detect absorption values for sample at260nm and280nm(OD260, OD280) and OD260/OD280ratio. The sample of OD260/OD280ratio at1.8-2.0was used to test. At the same time, detect RNA concentration.(3) Synthesizing cDNA with reverse transcription:Take2ug RNA as the template to prepare the mixture of reverse transcription, according to the instruction of PrimeScript(?)1st Strand cDNA Synthesis Kit (TAKARA). The total volume of mixture was20ul.(4) real-time PCR:Dissolve primers of ACE, ACE2, and GAPDH with1%oDEPC insuring concentration10uM, low temperature storage. Prepare the mixture of real-time PCR, according to the instruction of SYBR(?) Premix Ex Taq kit (TAKARA). The total volume of mixture was25ul. (5) Analysis of the results:Based on real-time PCR, detected the CT value and calculated2-△△CT that reflects the relative mRNA expression. The formula of△△CT as following:△△CT=(CT, Target-CT, reference)intervention-(CT, Target-CT, reference)Control3. Statistical AnalysisAll the data are expressed as the mean±SD. Statistical significance was determined with one-way analysis of variance as homogeneity of variance and t-tests were used for multiple comparisons by LSD when P values less than0.05. Welch method was used when equal variances is not assumed, and multiple comparisons was analyzed by Games-Howell method when P values less than0.05. Differences were considered statistically significant at value of P<0.05. Statistical analysis was conducted using SPSS13.0.[Results]1. Cells morphology:By inverted phase contrast microscope, Cell morphology was observed. The shape of cell appeared oval, Fusiform, polygon, stones of paving the road. Cells were full of cytoplasm, and the fringe of cells was clear.2. RNA purity:The ratio of OD260/OD280was at1.8-2.0, and RNA was of high purity.3. The effect of different concentration Ang Ⅱ on the mRNA expression of ACE, ACE2in cultured HUVEs (Human umbilical vein endothelial cells):The level of the mRNA expression of ACE in cultured HUVEs in Middle concentration of Ang Ⅱ group, and High concentration of Ang Ⅱ group were significantly higher than that in control group, and Low concentration of Ang Ⅱ group (p<0.05). In High concentration of Ang Ⅱ group, the level of the mRNA expression of ACE in cultured HUVEs was significantly higher than that in Middle concentration of Ang Ⅱ group (p <0.05). It was similar in the level of expression of ACE mRNA in cultured HUVEs between Low concentration of Ang Ⅱ group and control group (p>0.05). The level of the mRNA expression of ACE2in cultured HUVEs in control group was higher than that in three different concentration of Ang Ⅱ groups (p<0.05). The level of the mRNA expression of ACE2in cultured HUVEs was significantly lower than that in low concentration of Ang Ⅱ group (p<0.05). It was no difference in the level of the mRNA expression of ACE2in cultured HUVEs between High concentration of Ang Ⅱ group and Middle concentration of Ang Ⅱ group (p>0.05).4. The effect of different antagonist of Ang Ⅱ receptor on the mRNA expression of ACE, and ACE2in cultured HUVEs:In Ang Ⅱ group and Ang Ⅱ+AT2R blocked group, the level of the mRNA expression of ACE in cultured HUVEs were higher than that in Control group and Ang Ⅱ+irbesartan group (p<0.05). It was not different in the level of the mRNA expression of ACE in cultured HUVEs to compare Ang Ⅱ group with Ang Ⅱ+AT2R blocked group (p>0.05). The level of the mRNA expression of ACE in cultured HUVEs in Control group was similar to that in Ang Ⅱ+irbesartan group (p>0.05). In Ang Ⅱ group and Ang Ⅱ+AT2R blocked group, the level of the mRNA expression of ACE2in cultured HUVEs were lower than that in Control group and Ang Ⅱ+irbesartan group (p<0.05). It was not different in the level of the mRNA expression of ACE2in cultured HUVEs to compare Ang Ⅱ group with Ang Ⅱ+AT2R blocked group (p>0.05). The level of the mRNA expression of ACE2in cultured HUVEs in Control group was similar to that in Ang Ⅱ+irbesartan group (p>0.05).[Conclusions]1. Ang Ⅱ could upregulate the expression of ACE, and downregulate the expression of ACE2in vascular endothelial cells in dose-dependent manner.2. It was Mediated by AT1R that Ang Ⅱ upregulate the expression of ACE, and downregulate the expression of ACE2vascular endothelial cells. Chapter2The mechanism of AngⅡ regulating the mRNA expression of ACE and ACE2in vascular endothelial cells.[Objective]To investigate the mechanism of Ang II regulating the mRNA expression of ACE and ACE2in vascular endothelial cells.[Method]1. Cells culturing and distribution:The steps of culturing cells was same to that in chapter1. Cells distribution as following:Control group (DMEM/F12without FBS), Ang Ⅱ group (DMEM/F12without FBS+1.0μmol/L Ang Ⅱ), AngⅡ+irbesartan group (DMEM/F12without FBS+1.0μmol/L Ang Ⅱ+1.0μmol/L irbesartan), Ang Ⅱ+p38blocked group (DMEM/F12without FBS+1.0μmol/L Ang Ⅱ+10.0μmol/L SB203580(p38MAPK blocker)), Ang Ⅱ+JNK blocked group (DMEM/F12without FBS+1.0μmol/L AngⅡ+10.0μmol/L SP600125(JNK blocker)).After cells cultured in regular condition being trypsinized, cells were inoculated into the petri dish of35mm in accordance with the density of1×106/ml.2ml DMEM/F12with10%FBS was added to every petri dish. When cells reached to80%-90%confluency in the petri dish, cells medium (DMEM/F12with10%FBS) was placed by DMEM/F12without FBS. cells had cultured in DMEM/F12without FBS for24hours in order to insure cells growth state synchronized. While cells growth state was synchronized,different kind of reagent above was used to intervene cells.24hours after being intervened with different reagent, cells were collected to be used for experiment. Cells in Control group, Ang Ⅱ group and Ang Ⅱ+irbesartan group were detected for the mRNA expression of ACE, and ACE2. Cells in Ang Ⅱ+p38blocked group was detected for the mRNA expression of ACE2only. Cells Ang Ⅱ+JNK blocked group was detected for the mRNA expression of ACE only. Cells in Control group, Ang Ⅱ group and Ang Ⅱ+irbesartan group were detected for the mRNA expression of JNK, and p38MAPK. 2. Detection of ACE mRNA, ACE2mRNA, JNK mRNA, p38MAPK mRNA with realtime fluores-cence quantitative PCR. The procedure and the analysis of result were same to chapter1. At last,2-△△CT reflects the relative mRNA expression.3. Statistical AnalysisAll the data are expressed as the mean±SD. Statistical significance was determined with one-way analysis of variance as homogeneity of variance and t-tests were used for multiple comparisons by LSD when P values less than0.05. Welch method was used when equal variances is not assumed, and multiple comparisons was analyzed by Games-Howell method when P values less than0.05. Differences were considered statistically significant at value of P<0.05. Statistical analysis was conducted using SPSS13.0.[Results]1. The effect of p38MAPK blocker on the mRNA expression of ACE2in cultured HUVEs:In Ang Ⅱ group, the level of the mRNA expression of ACE2in cultured HUVEs were lower than that in Control group, Ang Ⅱ+irbesartan group and Ang Ⅱ+p38blocked group (p<0.05). It was not different in the level of the mRNA expression of ACE2in cultured HUVEs among Control group, Ang Ⅱ+irbesartan group and Ang Ⅱ+p38blocked group (p>0.05).2. The effect of JNK blocker on the mRNA expression of ACE in cultured HUVEs:In Ang Ⅱ group, the level of the mRNA expression of ACE in cultured HUVEs were lower than that in Control group, Ang Ⅱ+irbesartan group and Ang Ⅱ+JNK group (p<0.05). It was not different in the level of the mRNA expression of ACE in cultured HUVEs among Control group, Ang Ⅱ+irbesartan group and Ang Ⅱ+JNK blocked group (p>0.05).3. The effect of Ang Ⅱ on the mRNA expression of JNK and p38MAPK in cultured HUVEs:The level of the mRNA expression of JNK and p38MAPK in cultured HUVEs in Ang Ⅱ group was higher than that in Control group and Ang Ⅱ+irbesartan group (p<0.05). It was similar in the level of the mRNA expression of JNK and p38MAPK in cultured HUVEs between Control group and Ang Ⅱ+irbesartan group (p>0.05).[Conclusions]1. Ang Ⅱ activates JNK to upregulate ACE expression by AT1R in vascular endothelial cells.2. Ang Ⅱ activates p38MAPK to downregulate ACE2expression by AT1R in vascular endothelial cells.

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