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Abstract

Background:

Renalase is a protein that can regulate sympathetic nerve activity by metabolizing catecholamines, while redundant catecholamines are thought to contribute to atherosclerosis (As). Catecholamine release can be facilitated by angiotensin (Ang) II by binding to Ang II type 1 (AT1) receptors. Valsartan, a special AT1 antagonist, can dilate blood vessels and reduce blood pressure, but it remained unclear whether valsartan can promote the stability of atherosclerotic plaque by affecting renalase.

Objective:

This study examined the tissue distribution of renalase in ApoE^−/− mice fed with a high-fat diet and the effect of valsartan on expression of renalase.

Methods:

ApoE^−/− mice were fed with a high-fat diet for 13 or 26 weeks. As a control, 10 C57BL mice were fed with a standard chow diet. After 13 weeks on the high-fat diet, the ApoE^−/− mice were randomized (10 mice/group) and treated with valsartan, simvastatin, or distilled water (control group) for an additional 13 weeks accompanied by a high-fat diet.

Results:

Knockout of ApoE caused a dramatic increase in expression of renalase in mice adipose tissue. With the disturbance of lipid metabolism induced by a high-fat diet, renalase expression decreased in the liver. Renalase can be expressed in smooth muscle cells and M₂ macrophages in atherosclerotic plaque, and its expression gradually decreases in the fibrous cap during the transition from stable to vulnerable atherosclerotic plaque. Valsartan, an AT1 receptor antagonist, promotes the stabilization of atherosclerotic plaque by increasing the levels of renalase in serum and the expression of renalase in the fibrous cap of atherosclerotic plaque. It also reduces triglyceride levels in serum and increases the expression of renalase in the liver.

Conclusions:

Renalase may be a potential-related gene of lipid metabolism and As, and it may be the possible molecular target of valsartan to help stabilize atherosclerotic plaque.

Keywords

renalase lipid metabolism atherosclerosis atherosclerotic plaque stabilization angiotensin II

Introduction

Atherosclerosis (As) is a chronic inflammatory disease of the arterial wall that arises from an imbalance between lipid metabolism and inflammatory response.¹ In recent decades, As-related arterial diseases have become the leading cause of death and morbidity worldwide.² A vulnerable atherosclerotic plaque located in the blood vessels is a predisposition for the occurrence and development of acute cardiovascular events³; therefore, it has become crucial for the treatment of As-related arterial diseases to promote the conversion of unstable plaque to stable plaque. The complex molecular and cellular mechanisms underlying plaque destabilization remain largely obscure.

Among the complex molecular and cellular mechanisms for plaque destabilization, angiotensin (Ang) II, the main hormone of the renin–angiotensin system (RAS), is known to play a central role in the pathophysiology of As through Ang II type 1 (AT1) receptor.⁴ There is clear evidence that Ang II might influence As by increasing vascular permeability, leukocyte infiltration, and low-density lipoprotein (LDL) oxidation and uptake.^5
–7 Moreover, Ang II has been shown to increase plaque size and promote destabilization of established atheromas by directly modulating macrophage trapping, oxidative stress, and activation of matrix metalloproteinase.^8
–10

Renalase, first discovered in 2005, is a protein that is secreted into the blood by the kidneys and can regulate sympathetic nerve activity by metabolizing catecholamines in circulation.¹¹ Whether renalase is an amine oxidase or not is presently debating. Aliverti et al failed to demonstrate the catalytic activity of renalase,^12,13 which is contradictory to research reported by Xu et al.¹¹ Thus, additional evidence is needed to confirm whether renalase is an amine oxidase or not. Clinically, excess catecholamine in the blood is believed to contribute to the development of hypertension and tachycardia and to aggravation leading to conditions of heart failure. In addition, previous study data have shown that catecholamines cause vascular injury and, in the presence of hyperlipidemia, accelerate and aggravate As.^14

–17 In addition, recent studies have shown that renalase exerted important effects on As-related cardiovascular diseases. In rodents, parenteral administration of renalase lowered blood pressure, heart rate, and cardiac contractility.¹⁸ During cardiac ischemia in rats, infusion of recombinant renalase reduced the size of a myocardial infarct, whereas neonatal nephrectomy led to elevated sympathetic nervous system activity, renalase deficiency, and cardiac hypertrophy^19,20; therefore, renalase is considered to be a therapeutic target for cardiovascular disease because of its metabolizing effect on catecholamines.²¹ Until now, the role of renalase in the development of As was unknown.

Angiotensin II is known to facilitate catecholamine release from peripheral sympathetic neurons by both binding to AT1 receptors and by direct ganglionic excitation²²; therefore, it is possible that expression of renalase may be affected by blockading Ang II from binding to AT1 receptors in order to interfere with the development of As. Valsartan, as an Ang II receptor antagonist, can dilate blood vessels and reduce blood pressure; therefore, it can be used to confirm whether expression of renalase may be affected by blockading Ang II from binding to AT1 receptors, which would interfere with the development of As. Therefore, in this study, we preliminarily explored the possible role of renalase in the development of As by examining the expression of renalase in ApoE^−/− mice fed with a high-fat diet and the effect of valsartan on expression of renalase.

Material and Method

Animals

Male ApoE^−/− mice (n = 50, 8 weeks of age, weighing18-20 g) with the C57BL/6J background and 10 C57BL mice were introduced and bred by the Department of Laboratory Animal Science of Peking University Health Science Center. The housing and care of the study animals and all procedures performed on them were in strict accordance with the guidelines and regulations of the Peking University Health Science Center.

Materials and Reagents

A TRIzol kit was purchased from Invitrogen Company (California), polymerase chain reaction (PCR) primers were obtained from Sangon Biotech Co, Ltd (Shanghai, China), and an M-MLV RT kit and a real-time (RT)-PCR kit were purchased from Takara Company (Otsu, Shiga, Japan). Valsartan (H20040217) was purchased from Beijing Novartis Pharmaceutical Co, (Beijing, China) and simvastatin (20120224) was purchased from Hangzhou MSD Pharmaceutical Company Ltd (Hangzhou, China). The blood lipid kits were purchased from Zhongsheng Beikong Biotechnology Co, Ltd (Beijing, China) to measure total cholesterol (TC), triglycerides (TGs), LDL, and high-density lipoprotein (HDL-C). A renalase kit was purchased from R&D Systems, Inc (Minneapolis, Minnesota).

Establishment of As Model

Forty ApoE^−/− mice were fed with a high-fat diet containing 21% (wt/wt) fat from lard supplemented with 0.15% (wt/wt) cholesterol²³ from Beijing Ke’ao Xieli Feed Co Ltd (Beijing, China) for 13 or 26 weeks. For the control, 10 C57BL mice were fed with a standard chow diet containing 4.0% fat.

Drug Treatment

After 13 weeks on a high-fat diet, 10 ApoE^−/− mice were randomly chosen and euthanized. The remaining ApoE^−/− mice were randomized (10 mice per group) and treated with 84.09 mg/kg/d valsartan by intragastric administration, 9.01 mg/kg/d simvastatin (positive control drug), or distilled water (control group) for an additional 13 weeks accompanied by a high-fat diet. The choice of medical doses was based on the clinically relevant doses in humans (the conversion coefficient between human and mice is 9.01, the medical doses in mice is 9.01 × the clinically medical doses in humans).²⁴ In the clinic, the human dose of valsartan is 9.33 mg/kg/d and that of simvastatin is 1.0 mg/kg/d; therefore, the valsartan dose for mice is 84.09 mg/kg/d and that of simvastatin is 9.01 mg/kg/d. Distilled water was used to dilute the medicine. Distilled water consumption was monitored twice weekly, and drug concentration was adjusted as required.

Histology

After 13 weeks of drug therapy, all mice from both the groups were euthanized with 0.1% pentobarbital sodium. The heart from each mouse was removed, and one-third of the heart was fixed in 10% formaldehyde to determine the morphology of any atherosclerotic plaque by hematoxylin and eosin (HE) staining. The aorta, heart, liver, abdominal fat, kidney, testis, and brain from each mouse from both the high-fat diet group and the normal diet group were removed and stored in −80°C.

Plaque Composition and Morphometry

To quantitatively evaluate atherosclerotic lesions, a 5-μm thick section was selected and quantified. A morphometric analysis was performed using Image Pro Plus (Media Cybernetics, Rockville, Maryland). The mean thickness of the fibrous cap in As and the percentage of extracellular lipids in the entire atherosclerotic plaque were used to evaluate the stability of the plaque.

Determination of Plasma Lipid Concentration

Blood samples were drawn from the left ventricle of a cohort of all male ApoE^−/− mice that had received a high-fat diet for 26 weeks. The TC and TGs were determined by enzyme studies in serum. The LDL and HDL were determined by immunoturbidimetry. Finally, all indices were determined using the RX-2000 radiometer (Technicon Instruments Company, Tarrytown, New York). The levels of renalase in the mice serum were determined using an enzyme-linked immunosorbent assay kit (R&D Systems).

Real-Time PCR

The total RNA of mice tissues was extracted using a TRIzol kit according to the manufacturer’s instructions. The primers of renalase and glyceraldehyde 3-phosphate dehydrogenase are shown in Table 1. The protocol for RT-PCR is according to our previous method.²⁵ The messenger RNA (mRNA) expression values were shown in the folds of the mRNA expression in tissues of C57 mice fed with a standard chow diet.

Table 1.

The Primer Sequences of Renalase and GAPDH.

Primer Sequences	Probes	Sizes of PCR Products, bp
Renalase forward primer	5′-GAAGATTGGTGTCCCTTGGT-3′	138
Renalase reverse primer	5′-AATGGGACAGTGGTTTGGAT-3′	138
GAPDH forward primer	5′-GCAAGTTCAACGGCACAG-3′	141
GAPDH reverse primer	5′-CGCCAGTAGACTCCACGAC-3′	141

Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase; PCR, polymerase chain reaction.

Immunocytochemistry

Serial 5-μm paraffin-embedded tissue sections were dewaxed and rehydrated. Endogenous peroxidase activity was inhibited by incubating the sections in 3.0% hydrogen peroxide (H₂O₂). After blocking the sections with 20% (v/v) goat serum in phosphate-buffered saline (PBS), the sections were incubated overnight at 4.0°C with renalase (Abcam, USA 1:200) antibody. Then, the sections were incubated with the appropriate secondary antibodies. A negative control, in which the primary antibody was replaced with the same dilution of either mouse or rat immunoglobulin G (IgG), was always included. Blinded analysis of positive immunostained sections was performed using Image Pro Plus (Media Cybernetics, Warrendale, Pennsylvania).

Immunofluorescence

Serial 5-μm paraffin-embedded tissue sections were dewaxed and rehydrated. Endogenous peroxidase activity was inhibited by incubating the sections in 3.0% H₂O₂. After blocking sections with 20% (v/v) goat serum in PBS, the sections were incubated overnight at 4.0°C with renalase (Abcam, USA 1:200), CD68 (Proteintech, USA 1:200), α-actin (Proteintech, USA 1:200), or arginase 1 (Arg-1; Proteintech, USA 1:200) antibody. Then, the sections were incubated with fluorescein isothiocyanate-conjugated goat antirabbit IgG and tetraethyl rhodamine isothiocyanate (TRITC)-AffiniPure goat antirabbit IgG purchased from Beijing Zhongshan Golden Bridge Biotechnology Co (Beijing, China). A negative control, in which the primary antibody was replaced with the same dilution of either mouse or rat IgG, was always included.

Statistical Analysis

Mean values and standard deviations were calculated for each variable studied. All statistical procedures were performed using SPSS 11.5. Normally distributed data were analyzed using 1-way analysis of variance with a Bonferroni post hoc test to evaluate the statistical significance of intergroup differences in all the tested variables. In all cases, statistical significance was P < .05.

Results

Feeding Fat Provokes the Formation of Vulnerable Plaque

Ripe atherosclerotic plaques can be clearly observed in the aortic roots of ApoE^−/− mice fed with a high-fat diet for 13 consecutive weeks. After feeding with a high-fat diet for another 13 weeks, the levels of the blood lipids in serum, especially TC, TGs, and LDL-C, of the ApoE^−/− mice were significantly increased compared with those of the C57 mice fed with a standard chow diet (P < .05; Figure 1A), and the atherosclerotic plaques showed the distinct morphologic features of vulnerable plaque, such as the large lipid core and the thin fibrous cap (Figure 1B).

Figure 1.

The change in blood lipid in serum and pathological morphology of aorta. A, The change in the level of blood lipid in serum of ApoE^−/− mice 26 weeks after feeding with high-fat diet. B, The change in pathological morphology of ApoE^−/− mice at 13 and 26 weeks after being fed with a high-fat diet.

The Tissue Distribution of Renalase in ApoE^−/− Mice and the ApoE^−/− Mice Fed With a High-Fat Diet for 26 Weeks

To define the effect of ApoE gene inhibition on expression of renalase, the tissue expression of renalase in ApoE^−/− mice at 8 weeks of age and the C57 mice at the same weeks of age was detected using RT-PCR. The folds of mRNA expression of renalase in the tissues from both kinds of the mice were converted. As shown in Figure 2A, compared with that of the C57 mice at the same weeks of age, the mRNA expression of renalase in the abdominal fat and brain of the ApoE^−/−mice at 8 weeks of age was also significantly increased 1.4-fold and 0.7-fold (P < .05), while there were no significant differences in the expression of renalase in the aortae, hearts, livers, and testes of the ApoE^−/− mice at 8 weeks of age (P > .05).

Figure 2.

The tissue expression profiles of renalase in ApoE−/− mice feeding without/with high-fat diet. A, Real-time PCR analysis showing the mRNA expression folds of renalase in the ApoE^−/− mice at 8 weeks of age compared with those of the wild-type C57 mice at the same week age. B, Real-time PCR analysis showing the mRNA expression folds of renalase in the ApoE^−/− mice at 26 weeks after having been fed with a high-fat diet compared with those of the wide-type C57 mice fed with a standard chow diet. NC group, normal control group; HF group, high-fat group. *P < .05 versus NC group (n = 5). C, The expressions of renalase in the adrenal gland and liver from the wide-type C57 mice and the ApoE^−/− mice 26 weeks after fed with chow diet or high-fat diet. Wide-type C57 mice, adrenal gland; ApoE^−/− mice, adrenal gland; wide-type C57 mice, liver; and ApoE^−/−mice, liver (×200). mRNA indicates messenger RNA; PCR, polymerase chain reaction.

To investigate the effect of a high-fat diet on the expression of renalase in As-related tissues of ApoE^−/− mice, the expressions of renalase in the tissues of the ApoE^−/− mice fed with a high-fat diet for 26 weeks and the C57 mice fed with a standard chow diet for the same period was detected by RT-PCR. The folds of mRNA expression of renalase in both kinds of mice were converted. As shown in Figure 2B, compared with those of C57 mice fed with a standard chow diet for 26 weeks, the mRNA expressions of renalase in the kidneys, testes, and brains of the ApoE^−/− mice were increased 4.41-, 1.62-, and 0.66-folds after 26 weeks on a high-fat diet (P < .05), while the expression of renalase in the livers of these same ApoE^−/− mice was decreased by 0.67-folds (P < .05). There were no significant differences in the expression of renalase in the aortae and hearts of the ApoE^−/− mice fed with a high-fat diet compared with those of the C57 mice fed with a standard chow diet for 26 weeks (P > .05). In addition, after 26 weeks of a high-fat diet, the results of immunohistochemistry showed that the expression of renalase in the cortex and medulla of the adrenal glands and livers of ApoE^−/− mice was significantly reduced compared with that of C57 mice fed with a standard chow (Figure 2C).

Localization of Renalase in Atherosclerotic Plaques

To understand the cellular localization of renalase in atherosclerotic plaque, the expression of renalase and α-actin, renalase and CD68, and renalase and Arg-1 was also detected by immunofluorescence. This finding showed that there were colocalizations in the expression of renalase and α-action and in the expression of renalase and Arg-1 in atherosclerotic plaque, especially in the fibrous cap of ApoE^−/− mice 26 weeks after having been fed a high-fat diet (Figure 3A).

Figure 3.

The cellular localization and the expression difference of renalase. A, The immunofluorescence results showed that the colocalization of renalase and α-actin, renalase and CD68, and renalase and arginase 1 (Arg-1) in atherosclerotic plaque. White arrow indicated fibrous cap of atherosclerosis plaque (×200). B, Immunohistochemical staining showing the protein expressions of renalase in fibrous caps of atherosclerotic plaque of ApoE^−/− mice 13 or 26 weeks after feeding with high-fat diet. The ApoE^−/− mice 13 weeks after feeding with high-fat diet; the ApoE^−/− mice 26 weeks after feeding with high-fat diet. Black arrow indicated fibrous cap of atherosclerosis plaque (×200). C, The statistical analysis of the percentage of renalase expression in the fibrous caps of the ApoE^−/− mice at 13 weeks and the ApoE^−/− mice at 26 weeks after feeding with high-fat diet. #, P < .01 versus at 13 weeks in the HF group (n = 10).

To define the possible role of renalase in both stable and vulnerable atherosclerotic plaque, the expression of renalase in the fibrous cap of atherosclerotic plaque of ApoE^−/− mice 13 and 26 weeks after feeding with a high-fat diet was detected by immunohistochemistry. As shown in Figure 3B, renalase is predominately expressed in the fibrous cap of atherosclerotic plaque in the aortae of ApoE^−/− mice. Moreover, the expression of renalase in the fibrous cap of atherosclerotic plaque from ApoE^−/− mice 26 weeks after feeding with a high-fat diet was significantly decreased compared with that of the mice 13 weeks after feeding with a high-fat diet (P < .05; Figure 3C).

Effect of Valsartan on Plaque Composition and the Thickness of Fibrous Cap From Vulnerable Atherosclerotic Plaque

To investigate the effect of valsartan, an antagonist of the Ang II receptor, on atherosclerotic plaque stability of mice with As, the differences between the percentage of extracellular lipid and atherosclerotic plaque and the thicknesses of the fibrous caps of the atherosclerotic plaque in the control, simvastatin, and valsartan groups were further evaluated using HE staining and IPP software analysis. As shown in Figure 4, valsartan can significantly increase the thickness of the fibrous cap in the atherosclerotic plaque of ApoE^−/− mice and decrease the ratio of extracellular lipid to atherosclerotic plaque (P < .05), but there was no significant difference in the effect of valsartan compared with that of simvastatin (P > .05). In addition, to investigate the effect of valsartan on smooth muscle cells (SMCs) and M₂ macrophages in atherosclerotic plaque, the expression of Arg-1 and α-actin in atherosclerotic plaque in the control, simvastatin, and valsartan groups was further detected using immunohistochemistry. As shown in Figure 5, valsartan can significantly increase the expression of Arg-1 and α-actin in the fibrous caps in atherosclerotic plaque (P < .05).

Figure 4.

The effect of valsartan on the stability of atherosclerotic plaque. A, Hematoxylin and eosin (HE) staining showing the morphology of atherosclerosis plaque in the aortae of ApoE^−/− mice at 26 weeks after having been fed a high-fat diet and treated by saline, simvastatin, and valsartan. Control group, simvastatin group, and valsartan group (×200). B, The statistical analysis of the comparison of the thickness of fibrous cap of atherosclerosis plaque in arota of ApoE^−/− mice at 26 weeks after high-fat diet and treated by saline, simvastatin, and valsartan. C, The statistical analysis of the comparison of the percentage of extracellular lipid in atherosclerotic plaque in the aortae of ApoE^−/− mice at 26 weeks after having been fed a high-fat diet and treated by saline, simvastatin, and valsartan. *P < .05 versus control group (n = 10).

Figure 5.

The effect of valsartan on the expressions of Arg-1 and α-actin in fibrous caps of atherosclerotic plaque. A, Immunohistochemical staining showing the expressions of Arg-1 in fibrous caps of atherosclerotic plaque in aorta of ApoE^−/− mice at 26 weeks after having been fed a high-fat diet and treated by saline, simvastatin, and valsartan. Black arrow indicated fibrous cap of atherosclerosis plaque (×200). B, The statistical analysis of the comparison of the percentage of the expressions of Arg-1 in the fibrous caps of atherosclerotic plaque in aorta of the ApoE^−/− mice 26 weeks after having been fed a high-fat diet and treated by saline, simvastatin, and valsartan. #, P < .05 versus control group, *, P < .01 versus control group (n = 10). C, Immunohistochemical staining showing the expressions of α-actin in fibrous caps of atherosclerotic plaque in aorta of ApoE^−/− mice 26 weeks after high-fat diet and treated by saline, simvastatin, and valsartan (×200). D, The statistical analysis of the comparison of the percentage of the expressions of α-actin in fibrous caps of atherosclerotic plaque in aorta of ApoE^−/− mice 26 weeks after high-fat diet and treated by saline, simvastatin, and valsartan. #, P < .05 versus control group, *, P < .01 versus control group (n = 10). Arg-1 indicates arginase 1.

Effect of Valsartan on the Expression of Renalase in Atherosclerotic Plaque in ApoE^−/−Mice

To investigate the effect of valsartan on the expression of renalase in atherosclerotic plaque and whether the RAS can regulate the expression of renalase, the levels of renalase in the serum of ApoE^−/− mice and the expression of renalase in the fibrous caps of atherosclerotic plaque were detected in the control, simvastatin, and valsartan groups using immunohistochemistry. As shown in Figure 6, valsartan can significantly increase the level of renalase in the serum of ApoE^−/− mice compared with that in the control group (P < .05) and can significantly increase the expression of renalase of the fibrous caps in atherosclerotic plaque (P < .05).

Figure 6.

The effect of valsartan on the protein expression of renalase in ApoE−/− mice. A, The statistical analysis of the comparison of the level of renalase in serum of ApoE^−/− mice, detected by enzyme-linked immunosorbent assay (ELISA) method, at 26 weeks after having been fed a high-fat diet and treated by saline, simvastatin, and valsartan. NC group, normal control group; HF group, high-fat group. #, P < .05 versus control group, *, P < .01 versus control group (n = 10). B, The statistical analysis of the comparison of the percentage of renalase expression in fibrous cap in aorta of ApoE^−/− mice at 26 weeks after having been fed a high-fat diet. #, P < .05 versus control group, *, P < .01 versus control group (n = 10). C, Immunohistochemical staining showing the expressions of renalase in the fibrous cap of atherosclerotic plaque of ApoE^−/− mice 26 weeks after having been fed a high-fat diet and treated by saline, simvastatin, and valsartan. Blue square frame indicated fibrous cap of atherosclerosis plaque (×200).

Effect of Valsartan on Blood Lipid Levels and Expression of Renalase in Liver and Abdominal Fat

To investigate the effect of valsartan on the levels of blood lipids, blood lipids in the serum of the ApoE^−/− mice were detected in the control, normal, simvastatin treatment and valsartan treatment groups. The results showed that valsartan can significantly decrease the serum level of TGs (P < .05) but not significantly change the serum levels of TC, LDL-C, and HDL-C in ApoE^−/− mice (P > .05; Figure 7A). The liver and fat tissues are the main and most important components involved in lipid metabolism. The expression of renalase in liver and fat tissues in the treatment groups was further detected using RT-PCR. The results showed that the mRNA expression of renalase in liver tissue can be increased by treating with valsartan, while the mRNA expression of renalase in fat tissue cannot be significantly changed compared with that of the control group (Figure 7B).

Figure 7.

The effects of valsartan on the levels of blood lipids and the expressions of renalase in liver and abdominal fat. A, The blood lipids levels of high-fat fed ApoE^−/− mice at 13 weeks after valsartan or simvastatin treatment. B, The messenger RNA (mRNA) expression of renalase in liver and fatty tissue of high-fat fed ApoE^−/− mice at 13 weeks after valsartan or simvastatin treatment. *, P < .01 versus control group, #, P < .05 versus control group (n = 5).

Discussion

Renalase, a protein synthesized by the kidneys and expressed in the glomeruli and proximal tubules, is secreted into the blood and metabolizes catecholamines into the circulation,¹¹ while redundant catecholamine can contribute to As.^14

–17 Catecholamine release can be facilitated by Ang II by binding to AT1 receptors.²² Valsartan, a special AT1 antagonist, can dilate blood vessels and reduce blood pressure, but it remained unclear whether valsartan can promote the stability of atherosclerotic plaque by affecting renalase. In the present study, the data show that renalase may be a gene potentially related to lipid metabolism and As. Valsartan can promote the stability of atherosclerotic plaque by upregulating renalase expression.

Whether renalase is an amine oxidase or not is the present debate. Aliverti et al failed to demonstrate this catalytic activity of renalase,^12,13 which is contradictory to the study of Xu et al.¹¹ Thus, renalase requires further evidence to confirm whether renalase is an amine oxidase or not. Genes expressed selectively or specifically in a certain organ may play crucial physiological and/or functional roles in this particular tissue. Our previous study showed that renalase is predominantly expressed in mice steroidogenic/reproductive systems, including in the ovaries, testes, and adrenal glands,²⁶ and it is also expressed in mice livers.²⁷ Although these organs are also involved in cholesterol synthesis and/or metabolism, it is an interesting question whether renalase may be involved in lipid metabolism. The ApoE deficiency is associated with dyslipidemia and As.²⁸ Therefore, we first detected the expressions of renalase in the tissues of ApoE^−/− mice at 8 weeks of age to investigate the effect of ApoE knockout on the expression of renalase. The results show that knockout of ApoE causes a dramatic increase in renalase expression in adipose tissue, suggesting that ApoE can regulate expression of renalase in mice adipose tissue. The knockout of ApoE does not cause the expression of renalase in mice liver, a key tissue that is involved in cholesterol synthesis and/or metabolism. It is possible that ApoE can regulate renalase expression in a tissue-specific manner. Because a high-fat diet can aggravate dyslipidemia and the development of As in the aortae of ApoE^−/− mice, which is a basic animal model to study As and atherosclerotic plaque stabilization,²⁶ we then detected the tissue profile of renalase in ApoE^−/− mice 26 weeks after they had been fed with a high-fat diet. The results show that with the disturbance of lipid metabolism induced by a high-fat diet, the expression of renalase decreased in liver, while the expression of renalase in adipose tissue did not change. It shows that renalase might be involved in the disturbance of lipid metabolism and As is induced by a high-fat diet.

In addition, after feeding with a high-fat diet for 26 weeks, the data showed that the expressions of renalase in the cortex and medulla of the adrenal glands were significantly reduced compared with that in C57 mice. It is known that the medulla of the adrenal glands is the main tissue that secretes catecholamines, while the cortex of the adrenal glands is the extrahepatic tissue involved in cholesterol metabolism. The possible explanation on this change is that the reduced expression of renalase in the medulla of adrenal glands in mice fed a high-fat diet may result in an increased level of catecholamines that would promote the progression of As. Taken together, the results indicate that renalase might be involved in the disturbance of lipid metabolism and As induced by a high-fat diet and renalase may be a potentially related gene of lipid metabolism and As.

To further investigate the mechanism that functions in the renalase of As, we also detected the cellular localization of renalase in atherosclerotic plaque of the ApoE^−/− mice fed with a fat diet for 26 weeks by immunofluorescence. The data show that renalase is expressed in SMCs and macrophages, especially in M2 macrophages. Smooth muscle cells are important components of atherosclerotic plaques and are responsible for promoting plaque stability in advanced lesions.²⁹ M2 phenotypes can decelerate atherosclerotic disease progression through activation of attenuating inflammatory responses.³⁰ So it further suggests that renalase might be related to the stabilization of atherosclerotic plaques. To confirm this, we further detected the expression of renalase using immunohistochemistry in the fibrous cap of atherosclerotic plaques of ApoE^−/− mice 13 weeks (stable atherosclerotic plaque, shown in Figure 1B and after having been fed a high-fat diet for 26 weeks (unstable atherosclerotic plaque, shown in Figure 1B . The data show that the expression of renalase in the fibrous cap of unstable atherosclerotic plaque was decreased significantly compared with that in the stable atherosclerotic plaque. Therefore, this is an indication that renalase may be a gene with a potential relationship with atherosclerotic plaque stabilization.

The RAS are hormone systems that regulate blood pressure and water (fluid) balance. Angiotensin II, the main hormone of RAS, can play a central role in the pathophysiology of As by binding to AT1 receptors.⁴ Angiotensin II contributes to atherogenesis mainly through oxidative stress and inflammation.³¹ Angiotensin II is also known to facilitate the release of catecholamines from peripheral sympathetic neurons by binding to AT1 receptors.³² Furthermore, renalase can regulate sympathetic nerve activity and affect cardiovascular diseases by metabolizing circulating catecholamines. Valsartan is an Ang II receptor antagonist with a particularly high affinity for the AT1 angiotensin receptor. By blocking the action of angiotensin, valsartan dilates blood vessels and reduces blood pressure.³³ From our results, we can clearly see that valsartan can increase the levels of renalase in serum and the expression of renalase in the fibrous cap of atherosclerotic plaques to stabilize a vulnerable plaque, including increasing the percentage of M2 macrophages and SMC in the fibrous cap of atherosclerotic plaques, which suggests that Ang II regulates renalase expression by binding to AT1 receptors. These results imply that functional inhibition of Ang II by valsartan interferes with RAS and leads to the upregulation of renalase expression.

The disturbance of lipid metabolism in the liver is predominantly a result of the abnormal aggregation of TGs in hepatocytes. Previous studies have shown that the AT1 receptor blocker was potent in reducing plasma TG levels,³³ and, conversely, Ang II infusion increased plasma TG levels in rats by stimulating hepatic TG production.³⁴ In this study, the data show that valsartan, an Ang II receptor antagonist, also reduces the level of TGs in serum and increases the expression of renalase in the livers of ApoE^−/− mice 26 weeks after feeding a high-fat diet. The above-mentioned data show that the expression of renalase decreases in the liver of ApoE^−/− mice at 26 weeks after having been fed a high-fat diet compared with that of the C57 mice that had been fed with a chow-diet for the same period. Taken together, it indicates that valsartan might reduce TGs by reducing the expression of renalase in the liver.

Summary

Our data provide some insights on renalase that may be a gene with a potential relationship with lipid metabolism, As, and valsartan (the Ang II receptor antagonist) and can promote the stability of atherosclerotic plaque by upregulating renalase expression. This result hints at a novel understanding into the function of renalase and the mechanism of valsartan, which promotes atherosclerotic plaque stabilization.

Footnotes

Author Contributions

Mingxue Zhou contributed to conception and design, contributed to acquisition, analysis, and interpretation drafted manuscript, gave final approval and agrees to be accountable for all aspects of work ensuring itegrity and accuracy. Chao Ma ontributed to conception, contributed to analysis and interpretation and drafted manuscript. Weihong Liu contributed to conception, contributed to interpretation, and critically revised manuscript. Hongxu Liu, contributed to design, contributed to analysis and critically revised manuscript. Ning Wang contributed to conception and contributed to analysis. Qunfu Kang contributed to conception and contributed to acquisition. Ping Li contributed to design, contributed to interpretation and critically revised manuscript.

Authors’ Note

Mingxue Zhou and Chao Ma equally contributed to this work.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by grants from Beijing Natural Science Foundation (7133233), National Natural Science Foundation of China (Grant No. 81303086), and Beijing Nova Program (No. Z131107000413026).

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Valsartan Promoting Atherosclerotic Plaque Stabilization by Upregulating Renalase

Abstract

Background:

Objective:

Methods:

Results:

Conclusions:

Keywords

Introduction

Material and Method

Animals

Materials and Reagents

Establishment of As Model

Drug Treatment

Histology

Plaque Composition and Morphometry

Determination of Plasma Lipid Concentration

Real-Time PCR

Immunocytochemistry

Immunofluorescence

Statistical Analysis

Results

Feeding Fat Provokes the Formation of Vulnerable Plaque

The Tissue Distribution of Renalase in ApoE−/− Mice and the ApoE−/− Mice Fed With a High-Fat Diet for 26 Weeks

Localization of Renalase in Atherosclerotic Plaques

Effect of Valsartan on Plaque Composition and the Thickness of Fibrous Cap From Vulnerable Atherosclerotic Plaque

Effect of Valsartan on the Expression of Renalase in Atherosclerotic Plaque in ApoE−/−Mice

Effect of Valsartan on Blood Lipid Levels and Expression of Renalase in Liver and Abdominal Fat

Discussion

Summary

Footnotes

Author Contributions

Authors’ Note

Declaration of Conflicting Interests

Funding

References

The Tissue Distribution of Renalase in ApoE^−/− Mice and the ApoE^−/− Mice Fed With a High-Fat Diet for 26 Weeks

Effect of Valsartan on the Expression of Renalase in Atherosclerotic Plaque in ApoE^−/−Mice