Study On The Mechanism Of Angiotensin Ⅱ Regulating The Expression Of Matrix Metalloproteinase - 2 In The Pathogenesis Of Ascending Aortic

Background and ObjectivesAscending thoracic aortic aneurysm (ATAA) is a severe life-threatening disease characterized by progressive dilatation and rupture of the ascending thoracic aorta. Once ATAAs rupture, mortality is exceedingly high, approximately 94% to 100%. The diagnosis of ATAA has increased because of improvement in imaging. As there are no validated medical therapies for ATAAs, treatment is confined to surgical repair. Thus, there is an urgent need to determine the pathogenic mechanisms of this disease for development of effective pharmacological therapies. Bicuspid aortic valve is the most common congenital malformation of the heart or great vessels, affecting nearly 1 to 2% of Americans. Aggressive management strategies are commonly recommended because of the increased rate of aortic expansion and increased prevalence of aortic dissection and aneurysm rupture in these patients. Recently it has been reported the pathogenesis of BAV is different from aneurysmal associated with TAV as dermined by proteomic and genomic studies.Matrix metalloproteinases are a family of zinc-dependent endopeptidases that are produced by leukocytes and smooth muscle cells (SMCs) within the aortic wall and are capable of degrading elastin, collagen, and other proteins that maintain aortic integrity.Several studies have demonstrated that matrix metalloproteinase expression, particularly matrix metalloproteinase 2 (MMP-2), was significantly elevated in the walls of ATAAs. Increased MMP-2 can degrade components of the extracellular matrix, such as elastin and collagen fibres, leading to loss of the normal structure of the aortic wall and progressive expansion of the aortic lumen, which is thought to be an important event in ATAA formation. However, the possible cellular and molecular mechanisms of MMP-2 upregulation in ATAAs were unknown. Moreover, it has been reported that MMP-2 activety is different between BAV and TAV patients.Mitogen-activated protein kinases (MAPK) are serine-threonine protein kinases that are involved in several processes important to cardiac surgery such as vascular permeability, cytokine production, vasomotor function, and reperfusion injury. Three major mitogen-activated protein kinase families were identified as the extracellular signal-regulated kinases, c-Jun NH2-terminal protein kinases, and p38 kinases. Extensive investigation has established roles for extracellular signal-regulated kinases, c-Jun NH2-terminal protein kinases, and p38 kinases in cardiovascular signal transduction pathways. JNK and ERK have involved the pathogenesis of abdominal aortic aneurysm, but up to date, whether MAPK participates in ATAA is unknown.Angiotensin Ⅱ (Ang Ⅱ) is a multifunctional octapeptide with diverse actions that modulates vasomotor tone, cell migration, cell growth, apoptosis and extracellular matrix deposition. Recently, accumulating evidence has demonstrated that Ang Ⅱ is strongly associated with the formation and progression of thoracic aortic aneurysms in humans and mouse models. To date, however, circulating and local Ang Ⅱ concentrations in non-syndrome ATAA have not been reported. Additionally, ACE (angiotensin-converting enzyme) insertion/deletion polymorphism is a risk factor for thoracic aortic aneurysm in patients with bicuspid or tricuspid aortic valves. Furthermore, imbalanced down-regulation of ACE and ACE2 mRNA expression levels may play an important role in the development and progression of thoracic aortic aneurysmal dilatation and subsequently dissection. There is increased expression of ATIR (angiotensin Ⅱ type 1 receptor) and AT2R (angiotensin Ⅱ type 2 receptor) in the tunica media of ascending aorta of Marfan syndrome and BAV. However, in the current studies of the effect of RAS on the pathogenesin of ATAA, there are some defects such as small sample size and most of reports about ATAA associated with connective tissue diseases.It has become evident that Ang Ⅱ in vitro can stimulate rat vascular smooth muscle cells (VSMCs) and human umbilical vein endothelial cells to produce, excrete and activate MMP-2. Recent studies have indicated that MAPK cascades are involved in Ang Ⅱ-mediated regulation of MMP-2 protein expression in human retinal pigment epithelium cells, human umbilical vein endothelial cells and rat aortic smooth muscle cells. In addition, Wang M et al showed that in vivo and ex vivo Ang Ⅱ significantly increased MMP-2 expression and activity in rat carotid arteries. However, it is not yet known whether Ang Ⅱ contributes to MMP-2 production and intracellular signalling pathways involved in aneurysmal smooth muscle cells (ASMCs) derived from ATAAs. Therefore we hypothesised that Ang Ⅱ stimulates MMP-2 upregulation by ASMCs via activation of MAPKs cascades. Therefore, in the present study, we investigated our experiments.Methods and ResultsSection 1. Expression of Matrix Metalloproteinase-2 in Ascending Aortic Aneurysms and Aortic Valves of Patients with Bicuspid or Tricuspid Aortic Valves:a Comparative StudyMethods:Ascending aortic aneurysmal wall specimens and valve samples were taken from TAV patients (n=20,51.5± 7.6 years) and BAV patients (n=20,47.4±6.3 years) when surgery repair. Normal ascending aortic specimens (n=20) were obtained from 20 patients with coronary heart diseases (CHD) undergoing CABG without any diagnosis of aortopathy. MMP-2 expression was examined by quantitative PCR, Western blot and immunohistochemistry in aortic and valve tissues. Some aneurysmal walls were stained with haematoxylin-eosin, elastin Van Gieson for microscopic examinations.Results:By histological examination all aortic aneurysm walls showed TAV aneurysms patients exhibited severe elastin fragmentation and markedly decreased elastin and much collagen deposition, compared with BAV aneurysms. Aortas from the BAV group displayed intact and regular arrayed elastic fibers in the intima and media and very few collagens. No specimen showed histological evidence of atherosclerosis. No important differences were seen between the groups with regard to age, gender, smoking, aneurysm size. The level of MMP-2 protein was elevated in ascending aortic wall specimens associated with BAV and TAV patients when compared with CHD patients (p<0.05). Furthermore, MMP-2 expression was greater both in valves and aortic aneurysm tissues from ATAAs associated with BAV when compared with those from TAV, irrespective of the level of mRNA and protein (p<0.05).Section 2. Expression of Mitogen-Activated Protein Kinases in Ascending Aortic Aneurysms and Aortic Valves of Patients with Bicuspid or Tricuspid Aortic Valves:a Comparative StudyMethods:Ascending aortic aneurysmal wall specimens and valve samples were taken from TAV patients (n=20,51.5±7.6 years) and BAV patients (n=20,47.4±6.3 years) when surgery repair. Normal ascending aortic specimens (n=20) were obtained from 20 patients with coronary heart diseases (CHD) undergoing CABG without any diagnosis of aortopathy. Total c-Jun amino kinase terminal kinase (t-JNK), phospho-JNK (p-JNK), total extracellular-signal regulated kinase (t-ERK) 1/2, phospho-ERK1/2 (p-ERK1/2), total p38 (t-p38) MAPK, phospho-p38 (p-p38) MAPK were measured by Western blot and immunohistochemistry in aortic and valvular specimens. Some aneurysmal walls were stained with haematoxylin-eosin, elastin Van Gieson for microscopic examinations. Results:By histological examination all aortic aneurysm walls showed TAV aneurysms patients exhibited severe elastin fragmentation and markedly decreased elastin and much collagen deposition, compared with BAV aneurysms. Aortas from the BAV group displayed intact and regular arrayed elastic fibers in the intima and media and very few collagens. No specimen showed histological evidence of atherosclerosis. No important differences were seen between the groups with regard to age, gender, smoking, aneurysm size. In the aneurysmal wall specimens, t-p38 MAPK was higher in TAV patients than that in BAV patients and CHD patients (p<0.05). The p-p38 level was also increased in TAV aneurysmal aortic walls compared with BAV group. However, there is no significant difference in t-p38 MAPK between BAV patients and CHD patients (p>0.05). In addition, t-p38 and p-p38 MAPK levels had also no significant differences between BAV and CHD patients. Moreover, for p-p38/t-p38 MAPK, there were no statistical differences between these three groups. Additionally, No statistical differences were found in p-ERK1/2, t-ERK1/2 and p-ERK/t-ERK1/2 between these three groups. Compared with normal aortic group, p-JNK, t-JNK and p-JNK/t-JNK were increased in two aneurysmal groups associated with BAV and TAV (p<0.05). Additionally, for t-p46JNK and p-p46JNK, there were no statistically differences between these two aneurysmal groups associated with BAV and TAV (p>0.05). Furthermore, the BAV patients have increased levels of t-p54JNK and p-p54JNK compared with TAV patients. In the valvular tissues, p-p38, t-p38 and p-p38/t-p38 MAPK were no significant differences between BAV and TAV patients. Additionally, t-ERK1/2 level was higher in TAV patients than in BAV patients while p-ERK1/2 and p-ERK/t-ERK1/2 levels had no statistical differences between TAV and BAV patients. Compared with BAV patients, the levels of p-JNK, t-JNK and p-JNK/t-JNK were increased in TAV patients (p<0.05).Section 3. Renin Angiotensin System Expression in Ascending Thoracic Aortic AneurysmsMethods Ascending aneurysmal aortic specimens (n= 40) were obtained at the time of operative aneurysm repair from 40 patients ranging in age from 43 to 74 years. Normal aortic specimens (n= 45) were obtained from 40 patients (45 to 75 y) who underwent coronary artery bypass surgery and from 5 heart transplant donors. The concentration of angiotensin Ⅱ (Ang Ⅱ) in the plasm and aorta tissue samples was measured by radioimmunoassay. Additionally, the angiotensinogen (AGT), Ang Ⅱ type 1 receptor (AT1R), and Ang Ⅱ type 2 receptor (AT2R) were determined by Western blot. The angiotensin conversion enzyme (ACE) activity was measured using a synthetic substrate, hippuryl-His-Leu, specifically designed for ACE. Sections of each specimen were immunostained with antibodies for ACE, AT2R, AT1R and Ang Ⅱ.Results We found that compared with the normal aorta walls, Ang Ⅱ amount, AT1R and AT2R protein expression were significantly elevated in aneurysmal aorta (p<0.05). The circulating Ang Ⅱconcentration also significantly increased (p<0.05).The ACE activities in the aneurysmal and normal aortas were 0.96±0.15 and 0.19±0.08 mU/mg protein respectively (p<0.05). Increased expression of Ang Ⅱ, AT1R, AT2R and ACE was also confirmed by immunohistochemistry.Section 4. Angiotensin Ⅱ Stimulates Matrix Metallopeptidase-2 Upregulation in Human Aortic Smooth Muscle Cells of Thoracic Aortic Aneurysms by Activating of P38, ERK1/2, JNK SignalingMethods:Human aneurysmal smooth muscle cells (ASMCs) were cultured by explant outgrowth from ascending aortic aneurysms walls obtained from ATAA patients during surgery repair. We examined the effect of Ang Ⅱ upon MMP-2 production at different concentrations (0μM,0.01μM,0.1μM, 1μM,10μM) and time points (0h,12h,18h,24h, 48h) of Ang Ⅱ by western blot. HASMCs were treated with 1μM Ang Ⅱ for 0,2,5,10, 30,60 min to measure MAPKs pathway activation. ASMCs were treated with 1μM Ang II for 48h with or without pretreatment 60min by 1μM angiotensin Ⅱ type 1 receptor (AT1R) inhibitor Candesartan and by 10 μM angiotensin Ⅱ type 2 receptor (AT2R) inhibitor PD123319 respectively. HASMCs were treated with 1μM AngⅡ for 48 h with or without pretreatment 30 min by 20 μM p38 MAPK inhibitor SB203580,10 μM ERK1/2 inhibitor PD98059, and 10μM JNK inhibitor SP600125 respectively. Western blot was used for detecting MMP-2 protein expression and the phosphorylation of ERK1/2, p38 MAPK and JNK proteins. Additionally, the normal aortic smooth cells and ATAA aortic wall pieces was also cultured and performed above experiments.Results:Western blotting results showed that Ang Ⅱ increased MMP-2 expression in a dose-and time-dependant manner in aneurysmal smooth muscle cells derived from ATAA, normal smooth muscle cells derived from normal thoracic aorta, and ATAA tissue (Ang Ⅱ 0.01 and 0.1 μM versus 0 uM, p<0.05; Ang Ⅱ 1 μand 10 μM versus 0 μM,p<0.01) (Ang Ⅱ18 h versus 0 h,p<0.05; Ang Ⅱ 24 h and 48 h versus 0 h,p<0.01). Candesartan significantly blocked Ang Ⅱ-induced expression of MMP-2 as compared with only Ang Ⅱ group (p<0.05), whereas the AT2 receptor antagonist PD123319 did not block these responses. Candesartan also blocked the Ang Ⅱ-induced phosphorylation of ERK1/2 and JNK. Ang Ⅱ-induced MMP-2 expression in was significantly attenuated by MAPK inhibitors SB203580, PD98059, and SP600125 respectively (p<0.05) in aneurysmal smooth muscle cell. In normal smooth muscle cells, Ang Ⅱ-induced MMP-2 expression in was significantly attenuated by PD98059. Candesartan completely blocked Ang Ⅱ-induced MMP-2 expression in aneurysmal smooth muscle cells and normal smooth muscle cells.Conclusions1. The expression level of MMP-2 in ascending aortic aneurysm walls was higher than that of MMP-2 in normal aorta walls of CHD patients.2. The up-regulation of MMP-2 in aneurysms associated with bicuspid aortic valves may partly elucidate the predilection to aneurysm formation in these patients compared with tricuspid aortic valves.3. The up-regulation of JNK in aneurysmal wall specimens may play a potentially crucial role in the pathogenesis of ATAAs. Furthermore, in the BAV associated with ascending aortic aneurysms, increased p54JNK level was found, which may contribute to elucidate the elevated level of MMP-2 compared with TAV associated with ascending aortic aneurysms.4. MAPK molecules expresson in ascending thoracic aortic aneurysm associated with BAV and TAV were different between aortic and valvular tissues.5. The renin angiotensin system may be a critical component in the pathogenesis of ascending thoracic aortic aneurysms.6. Angiotensin Ⅱ can induce MMP-2-upregulation via JNK, ERK1/2 and p38 MAPK signaling pathwaysn human aortic smooth muscle cells from thoracic aortic aneurysms.7. Ang Ⅱ-induced MMP-2 expression in human aortic smooth muscle cells was significantly attenuated by MAPK inhibitors, which suggests MAPK inhibitors may contributed to the treatment of ATAA.8. Candesartan significantly blocked Ang Ⅱ-induced expression of MMP-2 in human aortic smooth muscle cells, which suggests ARB drugs may contribute to the treatment of ATAA.