Association Of Lectin-like Oxidized Low-density Lipoprotein Receptor-1 With Angiotensin Ⅱ Type 1 Receptor Induces Cardiac Hypertrophy

Cardiac hypertrophy is an adaptive response of the heart that occurs in various cardiovascular diseases, but prolonged hypertrophy typically culminates in chronic heart failure or sudden cardiac death. It is thus very important to understand the molecular mechanism that underlies the development of cardiac hypertrophy. Increasing data suggest that ox-LDL is associated with cardiac structure and function. As is known to all, oxidized low-density lipoprotein (ox-LDL) is considered to involve in inducing smooth muscle cell migration and proliferation and play a fundamental role in the entire process of atherogenesis. However, Cardiac growth induced by stimuli is characterized mainly by the enlargement of individual myocytes; that is, hypertrophy, not an increase in myocyte number. Hence, in addition to atherogenic effects, ox-LDL may also affect cardiac myocytes leading to myocardial hypertrophy. Recent study suggests that Ox-LDL impacts cardiac structure and function which was independent of classic risk factors such as lifestyle, inflammation, and prevalent vascular damage. Clinic study also shows that ox-LDL is a risk marker for early ventricular remodeling. Statins, the 3-hydroxy-3-methylglutaryl-CoA (HMG CoA) reductase inhibitors, widely prescribed cholesterol-lowering agents, were reported that they can prevent cardiac hypertrophy in cultured neonatal rat cardiac myocytes treated with Angiotensin II.There is evidence from experimental and animal studies for an interaction between ox-LDL and angiotensin II or AT1 receptor (Ang II). ox-LDL upregulated the expression of AT1 R in cultured human coronary endothelial cells. Angiotensin II (Ang II), an important risk factor in the development of both cardiac remodeling and atherosclerosis, upregulates the expression of Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) receptor in the cultured human coronary artery endothelial cells through the activation of AT1 receptor. LOX-1, a type II membrane protein functioned as a receptor of ox-LDL, is believed to play a crucial pathogenetic role in the ox-LDL induced atherosclerosis and the other cardiovascular disease. Stimulating with ox-LDL can markedly increase the expression of LOX-1 receptor in the neointima of aortas of rabbits. Treatment with losartan, an AT1 receptor blocker, markedly decreases this enhanced LOX-1 expression. This indicates crosstalk between AT1 receptor and LOX-1 receptor may modulate the ox-LDL induced cardiac remodeling. Although LOX-1 receptor hardly expressed in the normal cardiomyocytes, It had been found that the LOX-1 receptor markedly up-regulated in the cardiomyocytes of failure heart. Few researches focused on LOX-1 receptor play in the process of cardiac hypertrophy.Part One Ox-LDL induces cardiac hypertrophy in vivoAlthough oxidized low-density lipoprotein (ox-LDL) is a risk factor for atherosclerosis. Recently, it had been reported that ox-LDL may also be involved in cardiac injury including heart failure and cardiac hypertrophy. We proposed that ox-LDL could induce cardiac hypertrophy.We first confirmed the relationship between ox-LDL and cardiac hypertrophy. Eight-week-old male wild type (WT, C57BL/6) and apolipoprotein E gene null mice (ApoE-/-), fed with a standard or a high fat diet, were subjected to transverse aorta constriction (TAC), a commonly used pressure overload model which induced a similar elevation of blood pressure (BP) among all mice, or to sham operation which did not elevate BP. Two weeks later, the plasma concentration of ox-LDL was much more increased in ApoE-/-mice fed with high fat diet than those fed with standard diet, whereas the ox-LDL levels were not elevated in the WT mice no matter whether they were fed with high fat diet or not. Although cardiac hypertrophy, characterized by increases in left ventricular (LV) wall thickness, global heart size, heart weight/body weight ratio (HW/BW) and cross-sectional area (CSA) of cardiomyocytes in LV section and decreases of LV cavity, was obviously formed and fractional shortening (FS) was preserved in all mice 2 weeks after TAC, consisting with the levels of plasma ox-LDL, the degree of cardiac hypertrophy was severer in ApoE-/-mice than in WT ones fed with either high fat or standard diet. Interestingly, cardiac hypertrophy was markedly formed in high fat diet-fed ApoE-/-mice even without TAC, and TAC induced a more significant cardiac hypertrophy in these mice than in the ApoE-/-mice fed with standard diet, whereas cardiac hypertrophy similarly developed between high fat diet-fed and standard diet-fed WT mice after TAC. Especially, we could not find any atherosclerosis, which might be involved in cardiac injury, in thorax aorta and coronary artery of the WT or ApoE-/-mice used in the present study, consisting with the previous data that atherosclerotic plaque in aorta and coronary lesion is significantly developed in ApoE-/- mice fed with high-fat diet for at least 15 and 20 weeks, respectively. We also established ox-LDL-induced cardiac hypertrophy model in WT mice. These results suggested that elevation of ox-LDL not only enhances the development of cardiac hypertrophy during pressure overload but also initially induces cardiac hypertrophy.Part Two Ox-LDL induces cardiomyocyte hypertrophy in vitroWe have demonstrated that ox-LDL induces cardiac hypertrophy in vivo. We further studied whether ox-LDL can directly induce cardiomyocyte hypertrophy in vitro. At first, cardiomyocytes were cultured in the presence of ox-LDL (50μg/ml) and the cellular protein synthesis was evaluated by the [3H]-leucine incorporation assay. Ox-LDL incubation did induce a significant increase in protein synthesis. Secondly, cardiomyocyte size evaluated by surface area of□-myosin heavy chain (α-MHC)-stained cardiomyocytes was also obviously enlarged after stimulation with ox-LDL. Phosphorylation of protein kinases such as extracellular signal-regulated protein kinases (ERKs) and reprogramming of expression of some fetal genes such as atrial natriuretic peptide (ANP) and skeletalα-actin (SAA) are also important hypertrophic responses in cardiomyocytes. Ox-LDL treatment induced upregulation of not only phosphorylation of ERKs but also mRNA expression of ANP and SAA detected by Western blotting and real-time RT-PCR, respectively. These results collectively indicated that ox-LDL does directly initiate an in vitro cardiomyocyte hypertrophy.Part Three The roles of LOX-1 and AT1-R in cardiac hypertrophyWe have found that ox-LDL can induce cardiomyocyte hypertrophy both in vivo and in vitro models. We then assessed the role of LOX-1 in ox-LDL-elicited cardiac hypertrophy by using a LOX-1 neutralizing antibody as a blocker of LOX-1. In cultured cardiomyocytes, pretreatment with the LOX-1 neutralizing antibody completely abolished ox-LDL-induced increases in cell size, ERKs phosphorylation and mRNA expression of ANP and SAA. Additionally, administration of the LOX-1 neutralizing antibody in mice, which did not affect the BP, plasma lipid indexes and FS%, resulted in a great inhibition of the increases in LV wall thickness, global heart size, HW/BW and CSA of cardiomyocytes and the decrease of LV chamber size observed in the ApoE-/- mice fed with high fat diet. Hypertrophic signallings such as phosphorylation of ERKs, gene expression of ANP and SAA were also downregulated by the LOX-1 neutralizing antibody in hearts of high fat diet-fed mice. All these data suggest a mediation of ox-LDL-induced cardiac hypertrophy by LOX-1.RAS has been thought to be involved in regulation of LOX-1 in process of atherosclerosis. We therefore tested the roles of Angll and AT1-R in ox-LDL-induced cardiac hypertrophy using an AT1-R blocker, Losartan and an Ang converting enzyme inhibitor (ACEI), Enalapril, respectively. As did by the LOX-1 neutralizing antibody, both in vitro and in vivo development of cardiomyocyte hypertrophy resulted from ox-LDL were merely abrogated by Losartan, suggesting a crucial role of AT1-R, which was similar to LOX-1, in the ox-LDL caused heart event. However, Enalapril only partially attenuated the ox-LDL-related cardiac hypertrophy, which was significantly less than did by Losartan. Since neither Losartan nor Enalapril could affect the BP, plasma concentrations of ox-LDL and cardiac function of the mice, we conclude that AT1-R itself exerts more effects than dose AngⅡin ox-LDL/LOX-1-induced cardiac hypertrophy. Previous studies have indicated that ox-LDL upregulates the expression of angiotensin converting enzyme through LOX-1 pathway, which increases local AngⅡand then activities of ATl-R.Part four Association of lectin-like oxidized low-density lipoprotein receptor-1 with angiotensinⅡtype 1 receptor induces cardiac hypertrophyWe have found important roles of LOX-1 and AT1-R in our preliminary data. Here we further determine the relationship between the two receptors. The expression of LOX-1 and AT1-R either at protein or at mRNA levels were both upregulated not only in hearts of ApoE-/-mice fed with high fat diet but also in cultured cardiomyocytes incubated with ox-LDL. Pretreatment with the LOX-1 neutralizing antibody and Losartan significantly suppressed the ox-LDL-induced upregulation of the both receptors, whereas Enalapril only partially reduced the upregulation of the two receptors, suggesting that there must have a direct association of LOX-1 with AT1-R during ox-LDL stimulation. In agreement with the results from Western blotting and RT-PCR, immunofluorescent staining with LOX-1 (Red) and AT1-R (Green) revealed that the signals of LOX-1 and AT1-R locating nearly cellular membrane were stronger in cardiomyocytes incubated with ox-LDL than in those without the incubation, which became weakened when pretreated with the LOX-1 neutralizing antibody or Losartan.It is worth asking how LOX-1 and AT1-R interact after stimulation with ox-LDL. Do they interact directly or through other mediators? To answer this question we first employed the method of co-immuneprecipitation using both anti LOX-1 and anti AT1-R antibodies. Western blot analyses of immunoprecipitate with anti-AT1-R antibody from heart tissue of mice and cultured cardiomyocytes for LOX-1 expression showed a combination of LOX-1 with AT1-R, which was much more significant in the ApoE-/- mice and ox-LDL stimulated cardiomyocytes than in WT mice and vehicle treated cardiomyocytes, respectively. Treatment with losartan or the LOX-1 neutralizing antibody almost completely abolished the combination. Enalapril, however, had no markedly inhibitory effect on this association. Similar results were obtained in the analysis of immunoprecipitate with anti LOX-1 antibody for AT1-R expression. All these findings suggest that there exists a binding of AT1-R and LOX-1 initiated by ox-LDL stimulation.We also explored whether the two proteins bind directly or indirectly. To address this issue, we performed bimolecular fluorescence complementation assay, which provides a novel approach for identification of the direct combination of two proteins modified by connecting to a specific protein construct in cultured cells. The following fusion plasmids were constructed:Subunit C of green fluorescent protein (GFP) tagged the C terminals of AT1-R (AT1/KGC), subunit N of GFP tagged the C terminals of AT1-R (AT1/KGN), subunit N of GFP tagged N terminals of LOX-1 (LOX-1/KGN) and subunit C of GFP tagged N terminals of LOX-1 (LOX-1/KGC). Since green fluorescence can be detected only when subunit N and C of GFP were bound together, we co-transfected AT1/KGC and LOX-1/KGN or AT1/KGN and LOX-1/KGC into the COS7 cells, respectively and stimulated the cells with ox-LDL Green fluorescence was observed under the confocal microscopy in COS7 cells either co-transfected with AT1/KGC and LOX-1/KGN or with AT1/KGN and LOX-1/KGC after stimulation with ox-LDL, suggesting an association of the C-terminal of AT1-R with the N-terminal of LOX-1. These findings clearly indicate that LOX-1 and AT1-R can directly bind together under the presence of ox-LDL. It is usually believed that the receptors are expressed on the surface of cells. However, LOX-1 and AT1-R have been indicated existing also in cellular organelles. We here also observed expression and therefore interaction of the two receptors within the cells.Because of a presumed interaction between LOX-1 and AT1-R that is independent of AngII, COS7 cells, which lack expression of endogenous AngII and AT1-R were used. We transiently transfected the plasmids of LOX-1 and AT1-R into cultured COS7 cells and then stimulated the cells with ox-LDL. Although ox-LDL did not induce phosphorylation of ERKs in COS7 cells without any transfection or with transfection of LOX-1 or AT1-R alone, the phosphorylation of ERKs was largely upregulated by ox-LDL in those doubly transfected by LOX-1 and ATl-R. We also confirmed the role of AT1-R in LOX-1 activation genetically. Three AT1-R mutants were constructed whose binding sites for Angll or ARB were mutated by the replacement of Lys 199 with Glutamine (K199Q), Glu 257 with Alanine (Q257A), Cys 289 with Alanine (C289A) respectively. The wild type AT1-R or three mutants of AT1-R was co-transfected into COS7 cells with LOX-1, and the cells were then stimulated with ox-LDL. Ox-LDL induced significant increases in phosphorylation of ERKs in ATl-R-co-transfected COS7 cells, and the increases were disappeared in Q257A-co-transfected cells but maintained in either K199Q-or C289A-co transfected ones. We found that expression of LOX-1 with AT1-R provides COS7 cells with the ability to respond to ox-LDL even without involvement of Angll, and Glu 257 in AT1-R plays a critical role in interaction of the receptor with LOX-1. These data indicate that Association of lectin-like oxidized low-density lipoprotein receptor-1 with angiotensin II type 1 receptor induces cardiac hypertrophy.Conclusions1. Stimulation by ox-LDL can induce cardiomyocyte hypertrophy in both in vivo and in vitro models.2. That the direct binding of LOX-1 to AT1-R is crucially involved in the ox-LDL induced cardiac hypertrophy. 3. Inhibition of either LOX-1 or AT1-R declined ox-LDL-induced hypertrophic responses whereas an angiotensin converting enzyme inhibitor only partially inhibited the responses stimulated by ox-LDL.The potential application of this study1. This work develops the cardiac hypertrophy theory and constitutes a satisfactory platform for cardiac hypertrophy research.2. Development of a LOX-1 antagonist and its application to the treatment of myocardial hypertrophy would be worth attempting in the future.The novelty of this study1. Ox-LDL-induced cardiac hypertrophy model has been established.2. Association of LOX-1 with AT1-R is critical for ox-LDL-induced cardiac hypertrophy...