Hyperlipidemia inhibits the protective effect of lisinopril after myocardial infarction via activation of dendritic cells

Abstract To investigate the prevention of cardiac remodelling and inflammatory immune response after myocardial infarction (MI) via ACEI regulating dendritic cells (DCs), we explored whether the protective effect of ACEI was repressed under hyperlipidemic environment. In vivo, the survival rate and left ventricular function of the mice were recorded on day 7 after MI. Tissue samples of the myocardium, spleen, bone marrow and peripheral blood were assessed for Ang II concentration, inflammatory cytokines and DCs expression. In vitro, DCs were treated with ox‐LDL + Ang II, simulating the internal environment of MI in ApoE−/− mice to explore the mechanism involved in the DCs maturation and inflammation. Under hyperlipidemic circumstances, we found that the cardioprotective effect of ACEI was attenuated through regulating DCs maturation and inflammation after MI, affecting survival rate and left ventricular function. Effects of lisinopril on the release of spleen‐derived DCs and myocardial infiltration were also reduced under hyperlipidemic conditions. In vitro, immune maturation and inflammation of DCs were further induced by ox‐LDL on the basis of Ang II treatment, as indicated by the upregulation of CD83, CD86, and the expressions of cytokines and chemokines. Furthermore, ox‐LDL could activate TLR4‐MyD88 signalling pathway, promoting IRAK‐4 and NF‐κB. The present study demonstrated that ACEI reduced the recruitment of DCs to the infarct site, leading to a higher survival rate and improved function. However, this effect was inhibited under hyperlipidemic environment. TLR4‐MyD88 signalling pathway may be responsible for the molecular mechanism involved in the immune maturation and inflammation of DCs induced by ox‐LDL.


| INTRODUC TI ON
Angiotensin-converting enzyme inhibitor (ACEI) can act directly on myocardial tissue through inhibition of angiotensin II (Ang II) formation, which improves myocardial hypertrophy and fibrosis, 1 reducing the overall morbidity and mortality of acute myocardial infarction (MI). 2 However, the protective effects of ACEI may not be beneficial for all patients in clinical practice. 3 The two most important risk factors associated with the occurrence and development of MI include hyperlipidemia and hypertension. Together, the two can have a strong synergistic effect, while oxidized low-density lipoprotein (ox-LDL) and Ang II also play a pivotal role. Cytotoxic ox-LDL is a risk factor for early ventricular remodelling, affecting cardiac structure and function. 4,5 Previous studies have shown that the local myocardial tissue concentration of ox-LDL may be much higher than that of the peripheral circulation, which can directly damage the endothelium and cardiomyocytes, leading to hypertrophic changes. 6 Other studies have also suggested that serum levels of ox-LDL antibodies are significantly elevated in patients with MI and are positively associated with MI-associated mortality. 7 Similarly, the level of Ang II in the peripheral blood is increased after MI, 8 which also affects myocardial remodelling in both infarcted and non-infarcted areas.
Ang II and ox-LDL have an inseparable relationship. 9 Ang II and ox-LDL can increase the expression of ACE in coronary artery endothelial cells. Based on previous research, we have been suggested that ox-LDL is an important pathological stimulating factor leading to the RAS activation. The aggregation of dendritic cells (DCs) has been observed in arteriosclerotic plaques and infarcted myocardium. Our research team had previously established that ox-LDL and Ang II 10 can induce DCs maturation.
Toll-like receptors (TLRs) are mainly expressed on the surface of monocytes, macrophages and DCs, which can result in the release of inflammatory mediators. TLR4 is the most distinctive member of the TLR family, due to its ability to transduce signals through both the MyD88 signalling pathway and the TRIF signalling pathway during inflammatory response. Previous studies have shown that ox-LDL induced circulatory and inflammatory response is achieved partially through TLR4, 11 which suggested its potential role in lipid molecules and inflammation during MI.
Based on the above results, we can envisage that a hyperlipidemic state triggers immune maturation and migration of DCs, thereby affecting the myocardial protective effects of ACEI.

| Animals
C57BL/6 and ApoE −/− mice, with an average age of 8-10 weeks, fed on a control diet and high-cholesterol diet, respectively, were obtained from the Animal Administration Center of Fudan University.
Mice were treated with lisinopril at a dose of 100 mg/L 12 via drinking water, which was initiated 2 days before MI and continued for 7 days thereafter.
Myocardial infarction was induced by permanent coronary ligation at 8-10 weeks of age. All procedures and protocols were approved by the Institutional Review Board of Zhongshan Hospital, Fudan University and Shanghai Institutes for Biological Sciences-CAS (A5894-01) and were conducted in conformity with the Public Health Service Policy on Humane Care and Use of Laboratory Animals. Splenectomy was performed at the time of MI as well.
Necrotic cells were used as control. In the inhibitor experiment, the cells were exposed to several inhibitors for 1 hour.

| Myocardial infarction protocol
Mice were anesthetized by inhalation of isoflurane and intubated with a 22-G intravenous catheter, followed by full anesthetization with 1.0%-2.0% isoflurane gas and mechanical ventilation with a positive pressure ventilator. The heart was exposed through a left thoracotomy, and MI was induced by ligating the left coronary artery with an 8-0 nylon suture. Successful ligation was confirmed when the anterior wall of the left ventricle turned pale. Mice that died within 24 hours after the operation were excluded from further analysis. Sham-operated animals underwent the same procedure without ligation of the coronary artery.

| Preparation of cardiac, splenic and peripheral blood cells
Mice were killed on day 7 after MI (n = 5-6 mice per group). Spleen was removed, triturated in HBSS (Mediatech, Inc) at 4°C with the end of a 3 mL syringe and filtered through a 100 μm nylon mesh (BD Biosciences). The cell suspension was centrifuged at 300 g for 10 minutes at 4°C. Heart tissue was harvested, minced and digested by collagenase II (Sigma) at a concentration of 0.5 mg/mL in 37°C for 30 minutes. Single-cell suspension was screened with 40 μm cell strainers (BD Biosciences). The cells were washed and resuspended by HBSS containing 2% BSA. Total spleen and cardiac cell numbers were determined with Trypan blue (Mediatech, Inc). Peripheral blood was drawn via cardiac puncture and subjected to red cell lysis with ACK lysing buffer (150 mmol/L NH 4 Cl, 10 mmol/L KHCO 3 , 10 mmol/L EDTA) and three washes with PBS buffer (PBS containing 1% FCS and 5 mmol/L EDTA).

| Flow cytometry
A subset of six mice per group was used for flow cytometry analysis. Cell suspensions were incubated with a mixture of antibodies (anti-CD11c-PE, anti-CD45-FITC, anti-CD83-PE, anti-CD86-FITC; BD Biosciences) at 4°C for flow cytometry analysis to determine the percentage of DCs in hearts, spleens and peripheral blood, respectively. Samples were loaded after two washes with PBS (2% BSA), and the raw data were analysed performed with a flow cytometer (Beckman).
The membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibody. Immunoreactive proteins were identified using Super Signal West Pico Chemiluminescence Substrate (Thermo). Densitometric analysis of Western blots was performed with the use of Image J software. GAPDH was used as loading control.

| Enzyme-Linked Immunosorbent Assay
The supernatant of the cultured BMDCs was harvested and stored at −70°C. The cytokine concentrations of TNF-α and IL-6 were analysed using enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems) according to the manufacturer's instructions.
Ang II concentration of the blood was determined with Ang II ELISA (Cayman Chemical) according to the manufacturer's instructions.
F I G U R E 1 Cardiac function measured by echocardiography on day 7 post-surgery. A, Comparison of lipid profiles 7 days after myocardial infarction. B, Kaplan-Meier survival analysis. Percentage of surviving mice after MI was plotted, comparison between-group difference was tested by the log-rank test. C, Representative M-mode images from individual groups. Measurements shown are LVEDD, LVESD and LVEF in the different treatment groups. The data are shown as the mean ± (SD) (n = 6). *P < .05 vs WT ACEI + MI

| Statistical analyses
All statistical analyses were performed with SPSS 22. Continuous data were presented as mean ± SD. Statistical comparisons between two groups were evaluated by Student's t test and corrected by ANOVA for multiple comparisons. Survival rates were compared by the Kaplan-Meier method and analysed with the log-rank test. A P-value of <.05 was considered statistically significant.

| Comparison of lipid profiles 7 days after myocardial infarction
Total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) were detected on day 7 post-MI. The results showed that the blood lipid levels of ApoE −/− mice were significantly higher than those of WT mice, including TC, LDL-C and TG, while HDL-C level was significantly lower ( Figure 1A, P < .05). Variables such as presence of MI, treatment with lisinopril and spleen resection did not affect the blood lipid levels of C57BL/6J mice.

| Effect of hyperlipidemia on survival after myocardial infarction
The survival rate on day 7 post-MI was significantly higher in the lisinopril and spleen resection group, compared to control (80% or 75% vs 52.4%, P < .05). The survival rate of the sham operation group was 100%. In comparison with the WT group, hyperlipidemia significantly reduced post-MI survival rate and inhibited the protective effect of lisinopril on mortality ( Figure 1B, 65% vs 80%, P < .05).

| Effects of lisinopril on the release of spleenderived dendritic cells and myocardial infiltration under hyperlipidemic conditions
We wanted to explore to what extent the impact on the DCs flux contributes to the overall benefits of lisinopril. We thus neutral-

| Effects of lisinopril on the secretion of Ang II and inflammatory cytokines in the peripheral circulation under hyperlipidemic conditions
In order to establish that lisinopril inhibited the release of spleenderived DCs by decreasing Ang II levels and is not associated with Ang II, the level of Ang II in peripheral circulation after MI was measured. The concentration of Ang II in the peripheral circulation of the MI group was significantly higher than that of the sham-operated group, while intervention with lisinopril significantly reduced Ang II levels (P < .05). Under hyperlipidemic state, the concentration of Ang II in the peripheral circulation was not affected, in comparison to WT mice ( Figure 3E

| ox-LDL further enhanced DCs migration, maturation and secretion of inflammatory cytokines
The migration ability of DCs is dependent on the infiltration of in-   Figure 6A,B).

| ox-LDL + Ang II can induce DCs activation of TLR4/MyD88-NF-κB signalling pathway
Simultaneously, the inhibition of TLR4 and MyD88 significantly decreased the phosphorylation of NF-κB and IκB, and the NF-κB signalling pathway was also inhibited (P < .05). The degree of phosphorylation of NF-κB and IκB was not affected by TRIF inhibitor Resveratrol. This indicated that TLR4 is achieved via the MyD88 pathway rather than the TRIF pathway during activation of NF-κB pathway via DCs after treatment with ox-LDL + Ang II ( Figure 7A,B, P < .05). TLR4/MyD88-NF-κB pathway is involved in the process of DCs maturation and inflammatory response in the development and progression of myocardial infarction with concurrent hyperlipidemia.

| Relationship between TLR4 and MyD88
TLR4 inhibitor EFCG was used to interfere with ox-LDL + Ang IIinduced DCs, which resulted in inhibition of MyD88 expression F I G U R E 4 A, Quantitation of CD45 + CD11c + DCs by flow cytometry in the heart, spleen and blood after MI. The data are shown as mean ± (SD) (n = 5-6); *P < .05 vs Sham; &P < .05 vs Saline MI; #P < .05 vs WT ACEI + MI; B, ox-LDL enhances the migration of DCs on the basis of Ang II. C, D, ox-LDL further induces DCs maturation and secretion of inflammatory cytokines. *P < .05 vs medium alone; &P < .05 vs Ang II F I G U R E 5 ox-LDL + Ang II can induce DCs activation of TLR4/MyD88-NF-κB signalling pathway. A, Representative immunoblots and the results of quantitative analysis of IκB and NF-κB phosphorylation in the infarcted heart. B, Western blot shows the expression of TLR4, IRAK-4, phospho-IRAK-4, MyD88 and TRIF after intervention with Ang II alone and ox-LDL + Ang II. Data were represented as mean ± SD (n = 3). *P < .05 ( Figure 7C, P < .05). Conversely, when switched to MyD88 inhibitor ST2825, phosphorylation of IRAK-4 was reduced (P < .05), while TLR4 expression showed a downward trend, but the difference was not statistically significant ( Figure 7D, P > .05).
This indicated that MyD88 is located downstream of TLR4 and is regulated by TLR4. The phosphorylation of IRAK-4 is regulated by TLR4 regulates via the MyD88 pathway, thereby promoting NF-κB nuclearization and transcription of inflammatory cytokines.

| D ISCUSS I ON
Inflammatory response is an important part of ventricular remodelling after myocardial infarction. 15 As a full-time antigen-presenting cell and initiator of immune-inflammatory response in vivo, 16  In this study, we used a high-fat diet ApoE −/− mice for coronary artery ligation, so MI is induced in a chronic low-inflammatory environment, which can be used to simulate the internal environment of AMI patients. The mechanism of coronary atherosclerotic plaque rupture is often due to hyperlipidemia.
In the present study, we found that treatment with lisinopril as well as splenectomy reversed the number of recruited DCs and the quan- suggest that the expression of MyD88 was inhibited. On the contrary, MyD88 inhibitor ST2825 decreased the expression of TLR4 is, but the difference was not statistically significant. MyD88 is located downstream of TLR4 and is regulated by TLR4. The expression of TRIF was not significantly different under the action of two inhibitors. These results indicate that TLR4 is achieved by the MyD88 pathway rather than the TRIF pathway during activation of the NF-κB pathway via DCs induced by ox-LDL + Ang II. Therefore, we conclude that TLR4-MyD88-NF-κB pathway is involved in the process of DCs immune maturation and inflammatory response.

ACK N OWLED G EM ENT
We express our sincere appreciation to all participants in this study.

CO N FLI C T O F I NTE R E S T
The authors declare no competing financial interests.

D I SCLOS U R E
The abstract of this manuscript has been presented at 2019 ESC Congress, Paris, France, August 31-September 4.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.