*Article* **Associations between High-Density Lipoprotein Functionality and Major Adverse Cardiovascular Events in Patients Who Have Undergone Coronary Computed Tomography Angiography**

**Hiroko Inoue 1, Yuhei Shiga 2,\*, Kenji Norimatsu 1,2, Kohei Tashiro 2, Makito Futami 1,2, Yasunori Suematsu 2, Makoto Sugihara 2, Hiroaki Nishikawa 1, Yousuke Katsuda <sup>1</sup> and Shin-ichiro Miura 1,2,\***


**Abstract:** The present study aimed to investigate the associations between high-density lipoprotein (HDL) functionality and major adverse cardiovascular events (MACE) in patients who have undergone coronary computed tomography angiography (CCTA). We performed a prospective cohort study and enrolled 151 patients who underwent CCTA and had a follow-up of up to 5 years. We measured cholesterol efflux capacity (CEC), caspase-3/7 activity and monocyte chemoattractant protein-1 (MCP-1) secretion as bioassays of HDL functionality. The patients were divided into MACE(−) (*n* = 138) and MACE(+) (*n* = 13) groups. While there was no significant difference in %CEC, caspase-3/7 activity or MCP-1 secretion between the MACE(−) and MACE(+) groups, total CEC and HDL cholesterol (HDL-C) in the MACE(+) group were significantly lower than those in the MACE(−) group. Total CEC was correlated with HDL-C. A receiver-operating characteristic curve analysis showed that there was no significant difference between the areas under the curves for total CEC and HDL-C. In conclusion, total CEC in addition to HDL-C, but not %CEC, was associated with the presence of MACE. On the other hand, HDL functionality with regard to anti-inflammatory and anti-apoptosis effects was not associated with MACE.

**Keywords:** high-density lipoprotein; cholesterol efflux capacity; major adverse cardiovascular events

#### **1. Introduction**

In patients who are treated for atherosclerotic cardiovascular disease (ASCVD), there is a possibility of some residual risks even when the low-density lipoprotein cholesterol (LDL-C) level has been significantly reduced [1,2]. Such residual risks include high levels of the triglyceride (TG), a low level of high-density lipoprotein cholesterol (HDL-C) and other uncontrolled risk factors [1–6]. A recent study in a Japanese cohort indicated that extremely high HDL-C (≥90 mg/dL) had an adverse effect on ASCVD mortality [7]. HDL mainly enhances reverse cholesterol transport, such as the cholesterol efflux capacity (CEC), as well as having anti-oxidative, anti-inflammatory and anti-apoptosis functions [8–10]. Recently, it has been considered that both HDL quality and HDL quantity are important for preventing CVD. Prospective studies revealed that CEC was inversely correlated with the incidence of CV events [11]. We also reported that the restenosis rates after coronary stent implantation were associated with CEC [12]. Thus, HDL functionality is a critical residual risk factor for ASCVD.

**Citation:** Inoue, H.; Shiga, Y.; Norimatsu, K.; Tashiro, K.; Futami, M.; Suematsu, Y.; Sugihara, M.; Nishikawa, H.; Katsuda, Y.; Miura, S.-i. Associations between High-Density Lipoprotein Functionality and Major Adverse Cardiovascular Events in Patients Who Have Undergone Coronary Computed Tomography Angiography. *J. Clin. Med.* **2021**, *10*, 2431. https://doi.org/10.3390/ jcm10112431

Academic Editor: Sandro Gelsomino

Received: 25 April 2021 Accepted: 26 May 2021 Published: 30 May 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Coronary computed tomography angiography (CCTA) has become more widely available in many general hospitals and enables the accurate non-invasive assessment of coronary artery stenosis for screening of coronary artery disease (CAD). In our previous cross-sectional study, high levels of HDL-C at the time of CCTA were associated with a reduced presence and severity of CAD [13]. In addition, total CEC and HDL-C were associated with the presence of CAD, while %CEC was not [14]. However, that study did not analyze the associations between various HDL functionalities, including CEC and the prognosis. Therefore, we determined the associations in this study.

#### **2. Materials and Methods**

In our previous study, 204 consecutive subjects who underwent CCTA for screening of CAD and were either clinically suspected to have CAD or had at least one cardiovascular risk factor were enrolled, and CEC was measured [14]. In this study, we excluded 53 of those patients due to the absence of follow-up (*n* = 8) and an insufficient volume of blood samples for further analysis of HDL functionality (*n* = 45), and finally analyzed HDL functionality in 151 patients.

We followed the patients for up to 5 years (average, 3.7 ± 0.7 years) and divided them into those with ((+) group, *n* = 13) and without ((−) group, *n* = 138) a major adverse cardiovascular event (MACE), where MACE was defined as all-cause deaths, acute myocardial infarction, coronary revascularization and ischemic stroke as a composite primary endpoint. When the patients had significant coronary stenosis as assessed by CCTA and received coronary intervention immediately after CCTA, the intervention was not included in MACE as coronary revascularization. We measured HDL functionality including CEC, caspase 3/7 activity associated with apoptosis and the secretion of monocyte chemotactic protein-1 (MCP-1) associated with inflammation. The protocol in this study was approved by the ethics committee of the Fukuoka University Hospital (# 09-10-02). All subjects gave their written informed consent to participate.

#### *2.1. Evaluation of Coronary Stenosis Using CCTA*

We assessed coronary stenosis using CCTA [13,14]. All patients were scanned by 64-multidetector row CT on an Aquilion 64 (TOSHIBA, Tokyo, Japan). The region of interest was placed within the ascending aorta. The scan was started when the CT density reached 100 Hounsfield Units higher than the baseline density. The scan was performed between the tracheal bifurcation and the diaphragm. All segments were evaluated according to the 15-segment American Heart Association coronary artery model. Fifteen segments of coronary arteries were evaluated. CAD was defined as any narrowing of the normal contrast-enhanced lumen to more than 50% in at least one major coronary artery that could be identified in multi-planar reconstructions or cross-sectional images. The number of significantly stenosed coronary vessels (0, 1, 2 and 3VD) was determined. In addition, the atherosclerotic severity of coronary artery disease was assessed in terms of the Gensini score.

#### *2.2. Evaluation of CAD Risk Factors and Left Ventricular Ejection Fraction (LVEF)*

Age, gender, body mass index (BMI), systolic blood pressure (SBP), diastolic BP (DBP), smoking status (current vs. nonsmoker), family history (myocardial infarction (MI), angina pectoris or sudden death) and chronic kidney disease (CKD) were collected as risk factors for CAD. Data of serum levels of LDL-C, HDL-C, triglycerides (TG), hemoglobin A1c (HbA1c) and fasting blood glucose (FBG) were also collected. LVEF was assessed by transthoracic echocardiography. BMI was calculated as weight (kg)/height (m2). BP was determined as the mean of two measurements obtained in an office setting by the conventional cuff method using a mercury sphygmomanometer after at least 5 min of rest. The presence of dyslipidemia (DL), hypertension (HTN), diabetes mellitus (DM) and use of medication were obtained from medical records. Patients who had SBP ≥ 140 mmHg and/or DBP ≥ 90 mmHg at present or who were taking antihypertensive treatment were

considered to have HTN. Patients with LDL-C ≥ 140 mg/dL, TG ≥ 150 mg/dL and/or HDL-C < 40 mg/dL or who were taking lipid-lowering treatment were considered to have DL. Patients with FBG ≥ 126 mg/dL, HbA1c ≥6.5% or who were receiving a glucoselowering drug were considered to have DM. We calculated the estimated glomerular filtration rate (eGFR) from the data of serum creatinine, age, body size and gender. We defined CKD as when eGFR was less than 60 mL per minute per 1.73 m<sup>2</sup> body surface area.

#### *2.3. Measurement of HDL CEC*

We examined HDL CEC with an ex vivo system that used J774 macrophages and HDL isolated from plasma of the study patients by ultracentrifugation [14]. Briefly, J774 macrophages were cultured and radiolabeled with 2 μCi/mL of 3H-cholesterol for 24 h. The day after labeling, the cells were washed and incubated with 8-Br-cAMP to upregulate ATP-binding cassette A1 transporter. Efflux medium containing isolated HDL (15 μg) was added for 4 h. Radiolabeled cholesterol counts were analyzed for the cell compartment and media. Percentage (%) of CEC was calculated as follows: (radioactivity in the medium/total radioactivity (radioactivity in medium + cells extracted with NaOH/NaCl)) × 100-CEC in sample-free medium. Total CEC was also calculated as the percentage of cholesterol efflux capacity/100 × HDL-C level.

#### *2.4. Measurement of Secretion of Monocyte Chemotactic Protein 1 (MCP-1)*

We evaluated the HDL-induced secretion of MCP-1 with an ex vivo system using human coronary endothelial cells (HCECs, Clonetics, San Diego, CA, USA) [15] and apo-Bdepleted plasma from the study participants as samples. HCECs were cultured and grown in media. In the experiments, HCECs were washed with medium. The cells were incubated with 5 μg/mL of samples under the same conditions for 24 h. After 24 h, the secretion of MCP-1 in the medium from HCECs was measured by a Human CCL2/MCP-1 Quantikine ELISA Kit (R & D Systems, Minneapolis, MN, USA). The relative secretion of MCP-1 in each sample was calculated by the ratio of the secretion in each sample to the secretion in standard HDL (EMD Millipore Corp., Billerica, MA, USA). The relative total secretion of MCP-1 was also calculated as the relative secretion of the MCP-1/100 × HDL-C level.

#### *2.5. Measurement of Caspase 3/7 Activity*

We analyzed the HDL-suppressed caspase 3/7 activity with an ex vivo system that used the H9C2 cell line of embryonic rat cardiomyoblasts (ATCC®, CRL-1446T, Manassas, VA, USA) and apo-B-depleted plasma from the study participants as samples. We used cardiomyoblasts to analyze anti-apoptosis by HDL because HDL may prevent the progression of cardiac dysfunction related to apoptosis. H9C2 cells were cultured and grown in media. In the experiments, H9C2 cells were grown under serum-free conditions for 2 h. After 2 h, the cells were incubated with 5 μg/mL of samples for an additional 6 h. The caspase 3/7 activities in the H9C2 cells were measured by the Caspase-Glo® 3/7Assay System (Promega Corp., Madison, WI, USA). Relative caspase 3/7 activity in each sample was calculated by the ratio of the activity in each sample to the activity in standard HDL. Relative total caspase 3/7 activity was also calculated as the relative caspase 3/7 activity/100 × HDL-C level.

#### *2.6. Statistical Analysis*

The statistical analysis was performed using IBM SPSS statistics software, version 22 (SPSS Inc., Chicago, IL, USA) and EZR, which is used in R (The R Foundation for Statistical Computing, Vienna, Austria). More precisely, it is a modified version of R Commander designed to add statistical functions frequently used in biostatistics [16]. Continuous variables are shown as mean ± standard deviation. Continuous and categorical variables were compared between the groups by the *t* test and a Chi-square analysis, respectively. We performed a Wilcoxon rank-sum test when continuous variables did not show a normal distribution expressed as a median value and interquartile range. The Spearman rank correlation coefficient was used to evaluate associations between the groups. A receiver-operating characteristic (ROC) curve analysis was used to determine the cut-off of the total CEC or HDL-C to distinguish between with and without MACE at the highest possible sensitivity and specificity levels. Area under the curve (AUC) values were compared between total CEC and HDL-C by a Chi-square analysis. A value of *p* < 0.05 was considered significant.

#### **3. Results**

*3.1. Patient Characteristics in All Patients and in the MACE(+) and MACE(*−*) Groups*

Table 1 shows the patient characteristics in all patients and in the MACE(+) and MACE(−) groups. The mean age was 65 (58–71) years and BMI was 23 ± 3 kg/m<sup>2</sup> in all patients. The frequencies of HTN, DL and DM in all patients were 78%, 71% and 23%, respectively. The MACE(+) group showed a higher level of %smoking and a lower level of HDL-C than the MACE(−) group. There were no significant differences in other factors, including %CAD, the number of VD, Gensini score, left ventricular ejection fraction (LVEF), %CKD, eGFR and medications between the MACE(+) and MACE(−) groups.

**Table 1.** Patient characteristics in all patients, the MACE(+) group and the MACE(−) group.


*MACE:* major adverse cardiovascular events, *BMI:* body mass index, *HTN:* hypertension, *SBP:* systolic blood pressure, *DBP:* diastolic BP, *DL:* dyslipidemia, *LDL-C:* low-density lipoprotein cholesterol, *HDL-C:* high-density lipoprotein cholesterol, *TG:* triglyceride, *DM:* diabetes mellitus, *HbA1c* hemoglobin A1c, *FBG:* fasting blood glucose, *CKD: chronic kidney disease, eGFR:* estimated glomerular filtration rate, *LVEF:* left ventricular ejection fraction, *CAD:* coronary artery disease, *the number of VD:* the number of significant stenosed coronary vessels, *ARB/ACE-I:* angiotensin II receptor blocker/angiotensin converting enzyme inhibitor, *CCB:* calcium channel blocker, *EPA:* eicosapentaenoic acid, *DPP4-I:* dipeptidyl peptidase-4-inhibitor. \* *p* < 0.05 vs. MACE(−) group.

### *3.2. %CEC, Total CEC and HDL-C in the MACE(+) and MACE(*−*) Groups*

As shown in Figure 1A–C, the MACE(+) group showed significantly lower total CEC

**Figure 1.** (**A**) %CEC, (**B**) total CEC and (**C**) HDL-C in the MACE(+) and MACE(−) groups. *A.U*: arbitrary unit.

*3.3. Relative Caspase 3/7 Activity, Relative Total Caspase 3/7 Activity, Relative Secretion of MCP-1 and Relative Total Secretion of MCP-1 in the MACE(+) and MACE(*−*) Groups*

Figure 2 shows caspase 3/7 activity and the secretion of MCP-1. There were no differences in relative caspase 3/7 activity (*p* = 0.819), relative total caspase 3/7 activity (*p* = 0.745), the relative secretion of MCP-1 (*p* = 0.235) or relative total secretion of MCP-1 (*p* = 0.307) between the groups.

**Figure 2.** (**A**) Relative caspase 3/7 activity, (**B**) relative total caspase 3/7 activity, (**C**) relative secretion of MCP-1 and (**D**) relative total secretion of MCP-1 in the MACE(+) and MACE(−) groups. *A.U*: arbitrary unit.

#### *3.4. Correlations between %CEC, Total CEC and HDL-C in All Patients*

Total CEC was positively correlated with HDL-C in all patients (*r* = 0.793, *p* < 0.001), whereas %CEC showed no correlation (*r* = 0.024, *p* = 0.769) (Figure 3A,B).

**Figure 3.** Correlations between (**A**) %CEC, (**B**) total CEC and HDL-C in all patients. *A.U*: arbitrary unit.

*3.5. Cut-Off Values of Total CEC or HDL-C Levels for the Diagnosis of MACE in All Patients*

A ROC curve analysis showed that the AUC for total CEC and HDL-C were 0.682 and 0.696, respectively, in all patients (Figure 4A,B). The cut-off levels of total CEC and HDL-C that gave the greatest sensitivity and specificity for the presence of CAD were 12.4 (sensitivity 0.572, specificity 0.692) and 47 mg/dL (sensitivity 0.659, specificity 0.692), respectively. There was no significant difference between the AUC for total CEC and HDL-C (*p* = 0.656), which indicated that these two factors contributed to MACE to a similar extent.

**Figure 4.** Cut-off values of (**A**) total CEC or (**B**) HDL-C levels for the diagnosis of MACE in all patients.

#### **4. Discussion**

In this study, we hypothesized that HDL functionality, CEC in particular, was associated with MACE. The main finding was that total CEC and HDL-C in the MACE(+) group were significantly lower than those in the MACE(−) group. In addition, total CEC was correlated with HDL-C. These two factors contributed to MACE to a similar extent. On the other hand, HDL functionality with regard to anti-inflammatory and anti-apoptosis effects was not associated with MACE.

We showed that total CEC and HDL-C in the MACE(+) group were significantly lower than those in the MACE(−) group. We measured %CEC using isolated HDL by ultracentrifugation to eliminate the effects of other lipoproteins as much as possible, since several studies have reported that other lipoproteins might influence cholesterol efflux capacity [17,18]. By this method, we could estimate the efflux capacity for a fixed amount of isolated HDL. This reflects the effect of the per unit capacity of HDL, but not total CEC in the bloodstream. Therefore, %CEC was normalized to total CEC in blood by the HDL-C concentration. Although we used HDL without the effects of other lipoproteins to measure pure CEC, %CEC values alone could not predict the occurrence of MACE. The involvement of other lipoproteins, other than Apo-AI, was also considered. Since the MACE (+) group had significantly lower HDL levels and the total CEC, which is the cholesterol uptake rate multiplied by the HDL-C value, was associated with MACE, the HDL-C value itself was at least related to MACE. In addition, since the correlation coefficient between HDL-C and total CEC was 0.793, which is a relatively strong correlation, it may be possible to predict MACE from the HDL-C value without measuring total CEC. However, the HDL-C value is 20–30% of the weight of HDL, and it is not clear whether the HDL-C value alone directly reflects the functionality of HDL itself. In any case, these results show that both HDL quality and quantity are important. In this study, the cut-off levels of total CEC and HDL-C for the presence of MACE according to a ROC curve analysis were 12.4 and 47 mg/dL, respectively. To the best of our knowledge, only our previous report has addressed the cut-off levels of the total CEC for the diagnosis of CAD [14]. In that study, the total CEC in the presence of CAD was 12.2, which is similar to the value observed here. Next, the cut-off level of HDL-C for the presence of MACE was 47 mg/dL, which is neither high nor low compared to values in the literature [6,19]. The cut-off level of HDL-C for the diagnosis of CAD was 48 mg/dL [14], which is similar to the cut-off for the presence of MACE. In addition, after adjusting for demographics, co-morbidities, lipid profile, statin use and date of procedure, our model demonstrated a U-shaped association between HDL-C and overall mortality, with HDL-C levels of 30–50 mg/dL associated with the most favorable outcomes, and HDL-C levels <30 mg/dL or >50 mg/dL associated with worse outcomes [20]. Decreased HDL-C levels were associated with a significantly increased risk of CV events in women (<49 mg/dL in women) but not in men (<42 mg/dL in men) [21]. According to the Japan Atherosclerosis Society Guidelines for Prevention of Atherosclerotic Cardiovascular Diseases 2017, serum HDL-C levels should be maintained ≥40 mg/dL for the primary and secondary prevention of CVD [6]. Thus, the cut-off level of HDL-C at 47 mg/dL, while relatively low, seems to be reasonable. On the other hand, higher levels of HDL-C have not been found to be associated with atheroprotection [7,22–24]. In NIPPON DATA90, high HDL-C (60–79 mg/dL) was associated with a significantly reduced risk of CAD, whereas very high HDL-C (≥80 mg/dL) was not [24]. Recently, extremely high levels of HDL-C (≥90 mg/dL) were significantly associated with an increased risk of ASCVD mortality, an increased risk of CAD and ischemic stroke in a pooled analysis of Japanese cohorts (EPOCH-JAPAN) [7]. In any case, our results suggest that when patients show a total CEC less than around 12.4 and/or HDL-C less than around 47 mg/dL at the time of CCTA, they might develop MACE in the future.

HDL functionality with regard to anti-inflammation and anti-apoptosis was not associated with MACE in this study. Many studies have shown that vascular inflammation is associated with adverse events and C-reactive protein is a critical biomarker of CVD [25–30]. HDL mainly acts as a scavenger, removing deposited cholesterol from macrophages. It

also provides anti-inflammation and anti-apoptosis effects. Inflammation and apoptosis are associated with not only HDL function, but also with many other inflammation factors (MCP-1 [30], interleukin-6 and -8 [31], etc.) and apoptosis factors (tumor necrosis factor-a, B-cell lymphoma 2 [32], etc.). Thus, anti-inflammation and anti-apoptosis by HDL were not associated with MACE.

This study has several important limitations. First, although the sample size was relatively small, which limited our ability to determine significance, such as in the ROC analysis, including the cut-off levels of total CEC and HDL-C, which may be affected by gender-specific differences, we found that total CEC clearly had a significant correlation with MACE. Second, the population was only selected from Japan and the findings may not be applicable to other populations. Third, the CEC assay itself has several limitations because cell-based assays are labor-intensive. We did not analyze the anti-oxidative function of HDL. Fourth, we divided patients according to the presence of MACE and the duration of follow-up was only up to five years. A large-scale survey with a longer follow-up and further analysis will be needed.

#### **5. Conclusions**

Total CEC was correlated with HDL-C. Total CEC in addition to HDL-C, but not %CEC, was associated with the presence of MACE. These two factors contributed to MACE to a similar extent. On the other hand, HDL functionality with regard to anti-inflammatory and anti-apoptosis effects was not associated with MACE.

**Author Contributions:** Conceptualization, S.-i.M.; Data curation, Y.S. (Yuhei Shiga), K.T. and M.F.; Formal analysis, H.I. and Y.S. (Yuhei Shiga); Investigation, H.I., K.N., K.T. and M.F.; Methodology, Y.S. (Yuhei Shiga); Project administration, S.-i.M.; Resources, K.N. and K.T.; Supervision, H.N., Y.K. and S.-i.M.; Validation, H.I., Y.S. (Yuhei Shiga) and K.N.; Visualization, M.S., H.N. and Y.K.; Writing original draft, H.I.; Writing—review and editing, Y.S. (Yasunori Suematsu) and S.-i.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study has not received funding.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the ethics committee of Fukuoka University Hospital.

**Informed Consent Statement:** Written informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** The data that support the findings of this study are available from the corresponding author upon reasonable request.

**Conflicts of Interest:** The authors declare that there is no conflict of interest.

#### **References**


## *Article* **Increased Plasma Levels of Myosin Heavy Chain 11 Is Associated with Atherosclerosis**

**Lisa Takahashi 1,2, Tomoaki Ishigami 3, Hirofumi Tomiyama 1, Yuko Kato 2, Hiroyuki Kikuchi 4, Koichiro Tasaki 5, Jun Yamashita 1, Shigeru Inoue 4, Masataka Taguri 6, Toshitaka Nagao 5, Taishiro Chikamori 1, Yoshihiro Ishikawa <sup>7</sup> and Utako Yokoyama 2,7,\***


**Abstract:** Many studies have revealed numerous potential biomarkers for atherosclerosis, but tissuespecific biomarkers are still needed. Recent lineage-tracing studies revealed that smooth muscle cells (SMCs) contribute substantially to plaque formation, and the loss of SMCs causes plaque vulnerability. We investigated the association of SMC-specific myosin heavy chain 11 (myosin-11) with atherosclerosis. Forty-five patients with atherosclerosis and 34 control subjects were recruited into our study. In the atherosclerosis patients, 35 patients had either coronary artery disease (CAD) or peripheral artery disease (PAD), and 10 had both CAD and PAD. Coronary arteries isolated from five patients were subjected to histological study. Circulating myosin-11 levels were higher in the CAD or PAD group than in controls. The area under the receiver operating characteristic curve of myosin-11 was 0.954. Circulating myosin-11 levels in the CAD and PAD group were higher than in the CAD or PAD group, while high-sensitivity C-reactive protein concentrations did not differ between these groups. Multinomial logistic regression analyses showed a significant association of myosin-11 levels with the presence of multiple atherosclerotic regions. Myosin-11 was expressed in the medial layer of human atherosclerotic lesions where apoptosis elevated. Circulating myosin-11 levels may be useful for detecting spatial expansion of atherosclerotic regions.

**Keywords:** atherosclerosis; immunohistochemistry; biomarkers; smooth muscle cells; myosin heavy chain

#### **1. Introduction**

Atherosclerosis is the main pathological process underlying myocardial infarction, heart failure, peripheral artery disease (PAD), stroke, and cerebral infarction, and it has been the leading cause of morbidity and mortality globally [1]. Atherosclerotic plaque formation develops over long periods with chronic inflammation based on complex processes, including the oxidation of accumulated cholesterol-carrying low-density lipoprotein (LDL), immune cell infiltration, production of inflammatory mediators, endothelial dysfunctions,

**Citation:** Takahashi, L.; Ishigami, T.; Tomiyama, H.; Kato, Y.; Kikuchi, H.; Tasaki, K.; Yamashita, J.; Inoue, S.; Taguri, M.; Nagao, T.; et al. Increased Plasma Levels of Myosin Heavy Chain 11 Is Associated with Atherosclerosis. *J. Clin. Med.* **2021**, *10*, 3155. https://doi.org/10.3390/ jcm10143155

Academic Editor: Emmanuel Androulakis

Received: 10 June 2021 Accepted: 14 July 2021 Published: 16 July 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

and vascular smooth muscle cell (VSMC) proliferation and migration [2,3]. Plaque instability, in which the activation of proteases for extracellular matrices and the loss of VSMCs are involved, increases the risk of cardiovascular events [4]. Because these pathological changes in the vascular wall are mostly asymptomatic, the detection of atherosclerotic lesions by serological biomarkers before critical clinical features emerge has been extensively investigated [5–9].

In atherosclerotic plaques, differentiated VSMCs that are derived from the tunica media undergo phenotypic switching to proliferative synthetic cells that produce extracellular matrices (ECMs) and contribute to plaque stabilization [10]. In vulnerable plaques, previous studies indicated a high proportion of infiltrated monocytes/macrophages and extracellular lipids rather than VSMCs [4]. Thus, VSMCs have been thought to play relatively minor roles in the progression to rupture-prone atherosclerosis [1,4,10]. Over the past decade, however, studies that used fate-mapping and lineage-tracing revealed that VSMCs account for up to 70% of all plaque cells in murine models of atherosclerosis and that VSMCs contribute to multiple plaque cell phenotypes, i.e., macrophage-like cells, foam cells, osteochondrogenic cells, and mesenchymal stem cells alongside ECM-producing α-smooth muscle actin (αSMA)-positive cells [11–15], which highlight the importance of VSMCs. Thus, VSMCs are a major cell type in plaque formation and play a greater role in atherosclerosis than previously recognized [1].

Loss of VSMCs through cell death, including apoptosis and secondary necrosis, was shown to occur during the progression of atherosclerosis [16,17]. In studies using animal models, relatively acute VSMC apoptosis induced features of plaque vulnerability, such as fibrous cap thinning [18], and chronic apoptosis of VSMCs accelerated plaque growth necrotic core enlargement, plaque calcification, medial expansion and degeneration, elastin breaks, and failure of outward remodeling [19]. In humans, a decrease in the VSMC cell number was correlated with plaque instability [4]. On the basis of these findings, we hypothesized that SMC-specific proteins leak into the circulation from dying cells with the development and spread of atherosclerotic regions.

Some of myosin superfamily members were expressed in a cell-type-specific manner [20] and were reported to be increased in the blood of patients with several diseases, including autoimmune diseases [21], myocardial cell damages [22,23], and skeletal muscle injuries [24]. Myosin heavy chain 11 (myosin-11) is exclusively enriched in VSMCs [25]. A recent report demonstrated that patients with a non-ruptured abdominal aortic aneurysm (AAA), in which VSMCs undergo apoptosis, had significantly higher levels of circulating myosin-11 than normal controls, and its levels were correlated with the maximum aortic diameter [26]. These data indicated that circulating myosin-11 levels are associated with the loss of VSMCs in the vascular wall. We investigated the association of plasma levels of myosin-11 and atherosclerosis to identify a new tissue-specific serological biomarker for atherosclerosis.

#### **2. Materials and Methods**

#### *2.1. Study Subjects*

Forty-five patients with atherosclerosis were recruited from June in 2013 to January in 2015 at Yokohama City University Hospital. Atherosclerosis patients consisted of two groups: 35 patients with either coronary artery disease (CAD) or PAD (the CAD or PAD group) and 10 patients with both CAD and PAD (the CAD + PAD group). Plasma samples were taken within 1 week before the patients received percutaneous coronary artery intervention or endovascular treatment. Plasma samples from 34 control subjects were collected from age- and sex-ratio-matched healthy volunteers at Maebashi Hirosegawa Clinic who did not have a history of CAD, PAD, cerebrovascular disease, aortic aneurysm, diabetes mellitus, or renal insufficiency. Medical interviews were performed when the subjects were recruited into this study, and their systolic and diastolic blood pressure and body mass index (BMI) were recorded at that time. Plasma samples were stored at −80 ◦C until analysis. The proximal segments of the left coronary arteries from five patients

(2–5 segments per patient) were collected at autopsies in 2020 at Tokyo Medical University Hospital. Tissues were fixed in 4% paraformaldehyde for histological analysis.

#### *2.2. Clinical and Biochemical Analysis*

Plasma levels of myosin-11 were quantified in a specific sandwich enzyme-linked immunosorbent assay (ELISA) (Cusabio Biotech, College Park, MD, USA) in accordance with the manufacturer's instructions. Although the information on antibody-binding sites was not disclosed by the company, the ELISA was reacted to human recombinant coiled-coil domain of myosin-11 (data not shown). High-sensitivity C-reactive protein (hsCRP) was measured by SRL, Inc. (Tokyo, Japan).

#### *2.3. Tissue Staining and Immunohistochemistry*

Paraffin-embedded blocks containing the coronary artery tissues were cut into 4 μm–thick sections. Elastica van Gieson staining and Masson's trichrome staining were performed for morphological analysis. We used anti-CD68 (1:200 dilution, M0876, Dako, Carpinteria, CA, USA) to detect macrophages in immunohistochemical staining.

The 5 and 3 end of *Myh11* (myosin-11 gene) are alternatively spliced to generate COOH-terminal isoforms (SM1 and SM2) and NH2-terminal isoforms (SM-A and SM-B) [27]. Among all combinations of the four isoforms (SM1A, SM1B, SM2A, and SM2B) identified in humans [28], the artery expressed exclusively SM1A and SM2A [27]. We, therefore, used anti-SM1 (1:500 dilution, 7600, Yamasa, Chuo-ku, Tokyo, Japan), and anti-SM2 (1:200 dilution, 7601, Yamasa, Chuo-ku, Tokyo, Japan) antibodies to detect myosin-11 isoforms. Biotinylated horse anti-mouse IgG (Vectastain Elite ABC IgG kit, Vector Labs, Burlingame, CA, USA) was used as a secondary antibody. Negative staining of immunohistochemistry was confirmed by the omission of primary antibodies. TdT-mediated dUTP Nick End Labeling (TUNEL) (G7130, Promega Corporation, Madison, WI, USA) was performed to evaluate apoptotic cells.

#### *2.4. Statistical Analysis*

In Table 1, the data that are shown as values were statistically analyzed by using the Kruskal-Wallis test, and the data shown as ratios were analyzed by using the chi-square test. Both of these tests were followed by Fisher's least significant difference post hoc test, the Mann-Whitney U-test, or Fisher's exact test, as appropriate. In Table 2, the data that are shown as values were statistically analyzed by using the Mann-Whitney U-test, and the data shown as ratios were analyzed by Fisher's exact test. The data in Figure 1a,b and Figure 2a,b were statistically analyzed by using the Kruskal-Wallis test, followed by Fisher's least-significant-difference post hoc test and the Mann-Whitney U-test. The Mann-Whitney U-test was used in Figure 1c,d. Spearman's correlation analysis was used in Figure 1e. Data were analyzed by using Prism software (version 5.0; GraphPad, San Diego, CA, USA). Receiver-operating characteristic (ROC) analysis was performed by using a binary analysis of factors to evaluate the diagnostic performance. Multinomial logistic regression analyses were used to assess the difference of hsCRP (Model 1) and myosin-11 (Model 2) values within the three groups, after adjusting the Brinkman index (per 100), hypertension (dichotomous), and dyslipidemia status (dichotomous). Before the analysis, both myosin-11 and hsCRP values were transformed into the standardized score (z-score) for comparability. ROC analysis was examined by using SPSS (version 26; IBM Corp., Armonk, NY, USA), and multinomial logistic regression analyses were performed by using Stata (version 15; Stata Corp., College Station, TX, USA). The *p*-values < 0.05 were considered to be statistically significant.


**Table 1.** Clinical baseline characteristics of patients with atherosclerosis and controls.

Continuous variables are shown as the mean ± SD and categorical variables are expressed as the number (%). \* *p* < 0.05; n, number of subjects; CAD, coronary artery disease; PAD, peripheral artery disease; BP, blood pressure; HDL, high-density lipoprotein; LDL, low-density lipoprotein; Hb, hemoglobin; eGFR, estimated glomerular filtration rate; ACE, angiotensin-converting enzyme; ARB, angiotensin receptor blocker; SD, standard deviation.

**Table 2.** Clinical baseline characteristics of patients with CAD or PAD.


Continuous variables are shown as the mean ± SD, and categorical variables are expressed as the number (%). \* *p* < 0.05; *n*, number of subjects; CAD, coronary artery disease; PAD, peripheral artery disease; BP, blood pressure; HDL, high-density lipoprotein; LDL, low-density lipoprotein; Hb, hemoglobin; eGFR, estimated glomerular filtration rate; ACE, angiotensin-converting enzyme; ARB, angiotensin receptor blocker; SD, standard deviation.

**Figure 1.** Plasma myosin-11 (myosin heavy chain 11) levels in patients with atherosclerosis. (**a**,**b**) Myosin-11 and hsCRP (high-sensitivity C-reactive protein) concentrations in plasma samples of control subjects (Controls, *n* = 34), patients with CAD (coronary artery disease) or PAD (peripheral artery disease) (CAD or PAD, *n* = 35), and patients with both CAD and PAD (CAD + PAD, *n* = 11). Data are shown as the median with interquartile ranges. (**c**,**d**) Myosin-11 and hsCRP concentrations in plasma samples from CAD (*n* = 24) and PAD (*n* = 11) patients. Data are shown as the median with interquartile ranges. (**e**) Correlation between plasma concentrations of myosin-11 and hsCRP in all patients and controls (*n* = 80).

**Figure 2.** Plasma myosin-11 (myosin heavy chain 11) levels in subjects without renal insufficiency. (**a**,**b**) Myosin-11 and hsCRP (high-sensitivity C-reactive protein) concentrations in plasma samples of control subjects (Controls, *n* = 29), patients with CAD (coronary artery disease) or PAD (peripheral artery disease) (CAD or PAD, *n* = 14), and patients with both CAD and PAD (CAD + PAD, *n* = 3). Data are shown as the median with interquartile ranges.

#### **3. Results**

#### *3.1. Characteristics of Patients with Atherosclerosis*

Clinical baseline characteristics of patients with atherosclerosis (*n* = 45) and control subjects (*n* = 34) are shown in Table 1. Patients with atherosclerosis were divided into two groups: the CAD or PAD group (*n* = 35), in which patients had either CAD or PAD, and the CAD + PAD group (*n* = 10), in which patients had both CAD and PAD. There were no significant differences in age, frequencies of male gender, or BMI between the three groups. Both the CAD or PAD group and the CAD + PAD group had a higher frequency of hypertension, dyslipidemia, smokers, renal dysfunction, and diabetes mellitus than the control group, and there were no differences in the frequencies of these populations between the CAD or PAD and the CAD + PAD groups. Use of statins, angiotensinconverting enzyme (ACE) inhibitors, angiotensin receptor blockers (ARBs), β-blockers, and acetylsalicylic acid was also more common among patients with atherosclerosis compared to control subjects. With the exception of statins, there were no differences in the usage of these medications between the CAD or PAD and the CAD + PAD groups.

#### *3.2. Myosin-11 Plasma Levels Were Upregulated in Patients with Atherosclerosis*

We measured plasma myosin-11 concentrations in patients with atherosclerosis patients and control subjects using an ELISA that was specific for myosin-11. Plasma myosin-11 levels were higher in the CAD or PAD group (median (25th–75th percentiles): 139.2 (89.3–200.0) pg/mL) and the CAD + PAD group (median (25th–75th percentiles): 252.1 (208.6–386.3) pg/mL) than in control subjects (median (25th–75th percentiles): 30.0 (13.1–53.6) pg/mL) (Figure 1a). In addition, myosin-11 levels were significantly higher in the CAD + PAD group than in the CAD or PAD group (Figure 1a). In this sample set, we analyzed the plasma concentrations of hsCRP, which is the most extensively studied potential biomarker for atherosclerosis [29,30]. Moreover, hsCRP plasma levels were significantly higher in the CAD or PAD group (median (25th–75th percentiles): 0.44 (0.10–1.58) pg/mL) than in control subjects (median (25th–75th percentiles): 0.07 (0.03–0.16) pg/mL) (Figure 1b), while there was no difference in hsCRP between the CAD or PAD group and the CAD + PAD group (median (25th–75th percentiles): 0.42 (0.17–0.85) pg/mL) (Figure 1b).

#### *3.3. Circulating Myosin-11 Levels in Patients with CAD or PAD*

Next, we examined whether there was a difference in circulating myosin-11 levels between patients with CAD and PAD. The patient information is shown in Table 2. There were no differences in patient characteristics between CAD and PAD patients except for the frequencies of diabetes mellitus and usage of acetylsalicylic acid. Plasma levels of myosin-11 were similar between CAD patients (median (25th–75th percentiles: 153.5 (82.2–238.8 pg/mL) and PAD patients (median (25th–75th percentiles): 129.7 (96.1–200.0) pg/mL) (Figure 1c). Similar to myosin-11, there was no difference in hsCRP between CAD (median (25th–75th percentiles): 0.52 (0.11–1.41] pg/mL) and PAD (median (25th–75th percentiles): 0.35 (0.10–1.69) pg/mL) patients (Figure 1e). There was no positive association of plasma myosin-11 levels with hsCRP (Figure 1e).

#### *3.4. Circulating Myosin-11 Levels and Clinical Parameters*

We analyzed the values of myosin-11 or hsCRP together with traditional clinical risk factors. We performed multinomial logistic regression analyses by using hypertension, dyslipidemia, and the Brinkman index as risk factors. Because we recruited control subjects from among individuals who did not have a history of diabetes mellitus or renal insufficiencies, we did not include these factors in this analysis. The significant association between increased circulating myosin-11 levels and the presence of either CAD or PAD compared to control subjects persisted after adjustment for the risk factors (Table 3, Model 1). Similarly, hsCRP levels had a significant association with the presence of either CAD or PAD (Model 2). When we set the CAD or PAD group as a reference, the significant association between

circulating myoin-11 levels and the presence of multiple regions of atherosclerosis (CAD and PAD) (Table 3, Model 1) remained after the adjustment. However, traditional risk factors, i.e., smoking history, hypertension, and dyslipidemia, and plasma levels of hsCRP were not associated with the presence of both CAD and PAD (Models 1 and 2).


**Table 3.** Association between standardized score of myosin-11/hsCRP and atherosclerosis.

\* *p* < 0.05. <sup>1</sup> Values were transformed into a standardized score (z-score) before this analysis. <sup>2</sup> The AORs (adjusted odds ratio) were calculated using a per-100 change to the index. Myosin-11, myosin heavy chain 11; hsCRP, high-sensitivity C-reactive protein; CAD, coronary artery disease; PAD, peripheral artery disease; AOR, adjusted odds ratio; 95%CI, 95% confidence interval.

#### *3.5. The Effects of Renal Function on Circulating Myosin-11 Levels*

Although the differences in creatinine levels and estimated glomerular filtration rate (eGFR) between the CAD or PAD group and the CAD + PAD group did not reach significance, atherosclerosis patients had a higher frequency of renal insufficiency. We subsequently investigated the plasma levels of myosin-11 in atherosclerosis patients and control subjects who did not have a renal insufficiency, which was defined by less than 60 mL/min/1.73 m2 of eGFR. In accordance with the results that are presented in Figure 1, plasma myosin-11 levels were significantly higher in the CAD or PAD group (median (25th–75th percentiles): 84.6 (72.77–140.9) pg/mL) and the CAD + PAD group (median (25th–75th percentiles): 170.9 (152.8–238.0) pg/mL) than in control subjects (median (25th–75th percentiles): 27.5 (12.3–50.1) pg/mL) (Figure 2a). Circulating myosin-11 levels were also higher in the CAD + PAD group than in the CAD or PAD group (Figure 2a). In this dataset, plasma levels of hsCRP were significantly higher in the CAD or PAD group (median (25th–75th percentiles): 0.17 (0.07–1.03) pg/mL) than in control subjects (median (25th–75th percentiles): 0.07 (0.03–0.19) pg/mL) (Figure 2b), while hsCRP levels did not differ between the CAD or PAD group and the CAD + PAD group (median (25th–75th percentiles): 0.21 (0.09–1.38) pg/mL) (Figure 2b).

#### *3.6. Efficacy of Myosin-11 for Diagnosis*

We evaluated the diagnostic value using ROC analysis of myosin-11 to detect the presence of either CAD or PAD. The area under the curve (AUC) of myosin-11 was 0.954 (95% confidence interval [CI]: 0.909–0.998, *p* < 0.001), with a specificity of 88% at a sensitivity of 90% (Figure 3a), and the positive predictive value, negative predictive value, accuracy, and cutoff value were 89%, 91%, 90%, and 72.5 pg/mL, respectively. The AUC of hsCRP was 0.771 (95% CI: 0.657–0.884, *p* < 0.001), with a specificity of 29% at a sensitivity of 90%, and the positive predictive value, negative predictive value, and accuracy were 56%, 71%, and 59%, respectively. The AUC of myosin-11 was significantly greater than that of hsCRP (*p* < 0.025) (Figure 3a).

**Figure 3.** ROC (receiver-operating characteristic) analysis of myosin-11 (myosin heavy chain 11) and hsCRP (high-sensitivity C-reactive protein). (**a**) ROC curves of myosin-11 and hsCRP in patients with CAD (coronary artery disease) or PAD (peripheral artery disease) and control subjects. (**b**) Receiveroperating characteristic curves of myosin-11 and hsCRP in patients with CAD + PAD and CAD or PAD. AUC: the area under the curve.

To further investigate the efficacy of myosin-11 to detect the presence of multiple regions of atherosclerosis, we compared circulating myosin-11 levels between the CAD or PAD group and the CAD + PAD group. The AUC of myosin-11 was 0.814 (95% CI: 0.691–0.938, *p* = 0.002), with a specificity of 63% at a sensitivity of 91% (Figure 3b), and the positive predictive value, negative predictive value, accuracy, and cutoff value were 43%, 96%, 70%, and 168.4 pg/mL, respectively. The AUC of hsCRP was 0.522 (95% CI: 0.339–0.705, *p* = 0.827), with a specificity of 20% at a sensitivity of 91%. The positive predictive value, negative predictive value, and accuracy were 26%, 88%, and 37%, respectively. The AUC of myosin-11 was significantly greater than that of hsCRP (*p* = 0.007) (Figure 3b). These results suggest that circulating myosin-11 levels were increased in patients with atherosclerosis, and its levels may reflect the spatial expansion of atherosclerotic regions.

#### *3.7. Expression of Myosin-11 Isoforms in the Coronary Arteries*

Finally, we investigated myosin-11 expression and apoptosis during atherosclerosis progression in humans. The patient information is shown in Table 4.


**Table 4.** Patient characteristics for histological analyses.

HCC, hepatocellular carcinoma; CHF, chronic heart failure.

Proximal regions in human left coronary arteries were evaluated based on the American Heart Association (AHA)-recommended classification of atherosclerotic lesions [31]. Expression of SM1 and SM2 was greatly decreased for both in the intimal layer in Type II through Type V lesions, except in the shoulder region of atheromatous plaque in Type IV lesion and in intraplaque neovasculatures in Type V lesion defined as those in which major parts of the fibromuscular cap represent replacement of tissue disrupted by accumulated lipid and hematoma or organized thrombotic deposits (Figure 4). These findings were consistent with a previous report [28]. Expression of both SM1 and SM2 was gradually de-

creased in the medial layer as atherosclerosis progressed, while both expression levels were relatively maintained in the medial layer compared to that in the intimal layer (Figure 4).

**Figure 4.** Histological analysis of human coronary arteries. Representative images of EVG (Elastica van Gieson staining), MT (Masson's trichrome staining), and immunohistochemistry for CD68 (a marker of macrophages), SM1 and SM2 (myosin heavy chain 11 isoforms), and TUNEL (TdTmediated dUTP Nick End Labeling) stain in Type II (patient 4), Type III (patient 1), Type IV (patient 3), and Type V (patient 3) atherosclerotic lesions. \* Magnified images of black boxes in the intimal layer. \*\* Magnified images of black boxes in the medial layer. Scale bars for EVG and MT: 200 μm. Scale bars for immunohistochemistry and TUNEL stain: 50 μm.

TUNEL-positive apoptotic cells were present in the intimal layer in Type II lesion and more advanced atherosclerotic lesions where macrophages were accumulated, as reported previously [32]. In the medial layer, apoptosis was increased in Types III, IV, and V lesions, in which moderate expression of both SM1 and SM2 proteins was observed (Figure 4). These apoptotic cells seemed originate from SMCs, because CD68-positive cells were rarely seen in these areas.

#### **4. Discussion**

During the past decade, the importance of VSMCs in atherosclerosis pathology has been re-evaluated [1]. Considerable efforts to identify subjects who are at a higher risk for cardiovascular events have been made, and numerous biomarkers were proposed [5,9]. The associated pathological processes are complex, and proteins related to lipid, inflammation, oxidative stress, coagulation, neurohumoral activity, and myocardial damage have been shown to be associated with the presence of atherosclerosis and vulnerability of atherosclerotic plaques [33]. Although recent studies identified micro-RNA enriched in VSMCs as a potential biomarker for atherosclerosis [5,34], vascular-tissue-specific circulating biomarkers are not currently available. In the present study, we focused on the VSMC-enriched protein myosin-11 and demonstrated increased plasma levels of myosin-11 in patients with CAD or PAD compared to control subjects. Plasma myosin-11 levels did not differ between patients with CAD and PAD, and circulating myosin-11 levels were higher in the CAD + PAD group than in the CAD or PAD group, suggesting that a higher level of circulating myosin-11 is associated with the presence of multiple atherosclerotic regions.

Pathological intimal thickening, which is the first stage of atherosclerosis, progresses to fibroatheromas, which are characterized by the formation of a fibrous cap and a necrotic core [1]. Differentiated mature contractile VSMCs express contractile genes, such as αSMA and myosin-11 [35], and VSMCs in the early stage fibrous cap, which protects against plaque rupture, also express αSMA and myosin-11 [36]. Recent lineage-tracing studies involving the use of *Myh11* (myosin-11 gene)-CreERT2 have demonstrated that VSMCderived cells contributed substantially to the generation of a plaque core that is composed of αSMA-positive cells, macrophage-like cells, osteochondrogenic cells, and mesenchymal stem-cell-like cells [1,11,13–15]. Most αSMA-positive cells within the fibrous cap are positive for the VSMC-lineage label [37–39]. In the late stage of atherosclerosis, apoptosis is a hallmark of advanced atherosclerotic regions in humans [40]. It was reported that VSMC uptake of oxidized LDL and VSMC-derived foam cell formation seemed to induce VSMC apoptosis [17]. Induction of VSMC apoptosis using SM22α-hDTR/ApoE−/− mice induced fibrous cap thinning, necrotic core enlargement, plaque calcification, and medial degeneration, indicating that VSMC apoptosis accelerates atherosclerosis progression [19]. These data suggest that VSMCs proliferate during the early stage of atherosclerosis, and their loss in the arteries promotes plaque instability. Because myosin-11 is the component of smooth muscle myosin that is a major cytoskeletal/contractile protein of VSMCs and is theoretically not secreted from cells, increased circulating myosin-11 levels were thought to reflect dying VSMCs. In the present study, we measured plasma concentrations of myosin-11 in patients with advanced atherosclerosis in the coronary and peripheral arteries, so we do not know how early a stage of atherosclerosis we could possibly detect. The histology data in the present study demonstrated that expression the of both SM1 and SM2 was greatly decreased in the intimal layer, as reported previously [28], but were relatively maintained in the medial layer of advanced atherosclerotic lesions of human coronary arteries. Apoptosis in VSMCs seemed to be elevated in the medial layer of Type III lesions. Although the immunohistochemistry did not demonstrate that apoptotic SMCs in the medial layer are the source of circulating myosin-11, myosin-11 derived from VSMCs in Type III, as well as in Types IV and V, lesions may possibly affect circulating myosin-11 levels. Further study investigating the association of circulating myosin-11 levels with circulating apoptosis markers, such as cytokeratin-18 M30 antigen [41] and nucleosomes [42], at an asymptomatic early stage in atherosclerosis patients would clarify the timing at which circulating myosin-11 is elevated during atherosclerosis progression.

In a previous study, we conducted a secretome-based analysis of human AAA tissues and found that myosin-11 was abundantly detected in the supernatants of an organ culture of advanced AAA tissues [26]. In addition to the supernatants, circulating myosin-11 levels were increased in patients with AAA, and its levels were correlated with maximum aortic diameter [26]. It is widely recognized that aortic aneurysms that are localized at the abdominal region develop based on atherosclerotic pathological remodeling, and an advanced aneurysmal wall exhibits VSMC apoptosis and medial degeneration [43,44]. Another line of study also demonstrated that circulating myosin-11 levels were significantly increased in the blood immediately after aortic dissection occurred [45,46]. Together with the present study, these findings support the concept that elevated circulating myosin-11 levels seem to reflect the degree of VSMC loss.

An increasing amount of proteomic evidence has identified several circulating protein markers for atherosclerosis [9]. Although contractile genes are downregulated when VSMCs undergo phenotypic switching during atherosclerosis progression [47], VSMCrelated cytoskeleton and contracting proteins were identified in tissue extracts and secretome analyses of human atherosclerotic samples, such as carotid or coronary plaques [9]. To the best of our knowledge, however, molecular signatures of circulating myosin-11 in blood derived from patients with atherosclerosis remain uncertain.

VSMCs in the tunica media arise from local progenitor cells, and multiple distinct cell lineages are distributed across the arterial tree [48]. Coronary arteries are derived from pro-epicardium (lateral plate mesoderm) [49]. The infrarenal abdominal aorta and its distal peripheral arteries are derived from the splanchnic mesoderm [50]. A study using liquid chromatography/tandem mass spectrometric analysis (LC–MS/MS) analyses of the human AAA tunica media that is located in infra-renal regions demonstrated that the number of segments in a coiled-coil domain was larger than that of a motor domain in human AAA tissue secretion [26]. Because the infrarenal abdominal aorta and the peripheral arteries share a similar cell lineage, a coiled-coil domain of myosin-11 may be elevated in the blood of PAD patients. The present study, however, did not reveal molecular signatures of myosin-11 detected in the blood of CAD and PAD, because the ELISA used in this study could detect both fragments of myosin-11 containing a coiled-coil domain and full-length myosin-11. Identification of circulating myosin-11 by using mass spectrometric analysis and Western blotting in patients with atherosclerosis of each region, i.e., the coronary and peripheral arteries, would clarify whether myosin-11 in the blood is fragmented or full-length, and provide further information for atherosclerosis pathologies and developing a detection system.

There are several limitations to the present study. The present work was conducted by using a small number of samples, all with advanced atherosclerosis, and therefore, it lacks clinical background variety. Myosin-11 is abundantly expressed in arterial smooth muscle cells, but it is also found in the bladder, intestine, stomach, and uterus [27]. It is theoretically possible that circulating myosin-11 levels are elevated in patients with diseases of these organs. In addition to AAA and dissection of the aorta, circulating myosin-11 levels can be elevated in other vascular diseases, such as aortitis. The effect of gender on circulating myosin-11 levels remains unknown, because the number of female patients in this study was too small for statistical analysis.

The present study involved a higher frequency of renal insufficiency in atherosclerosis patients. A previous study reported that renal function did not increase circulating myosin-11 levels [46], and an analysis involving subjects without renal insufficiency in the present study demonstrated a similar tendency as that seen in the results using all subjects (Figure 2a,b). However, the effect of renal function on circulating myosin-11 levels should be considered, and the clearance of circulating myosin-11 needs to be examined in future studies. Because the progression of atherosclerosis consists of multiple pathological molecular processes, it is unlikely that a single molecule could be used to detect relatively early phase asymptomatic atherosclerosis and to monitor plaque vulnerability.

#### **5. Conclusions**

In conclusion, together with other biomarkers, circulating levels of myosin-11, which seem to reflect VSMC loss or damage, may help to detect the presence of atherosclerosis and spatial expansion in atherosclerosis regions.

**Author Contributions:** Conceptualization, U.Y.; methodology, U.Y.; validation, T.I., H.T., T.C., and Y.I.; formal analysis, L.T., H.K., and S.I.; investigation, L.T., T.I., Y.K., K.T., and U.Y.; resources, T.I., K.T., and T.N.; data curation, L.T., Y.K., H.K., S.I., M.T., and U.Y.; writing—original draft preparation, L.T., Y.K., and U.Y.; writing—review and editing, L.T., Y.K., and U.Y.; visualization, L.T., Y.K., and U.Y.; supervision, H.T., J.Y., T.C., and Y.I.; project administration, U.Y.; funding acquisition, L.T., Y.I., and U.Y. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by AMED (U Yokoyama and Y Ishikawa, JP21ek0210117) and was also partially supported by JSPS (L Takahashi, JP20K16533; U Yokoyama, JP20H03650; JP20K21638), and Kitsuen Research Foundation (Y Ishikawa, 1971000014).

**Institutional Review Board Statement:** The study protocols were approved by the Research Ethics Committee of Yokohama City University Hospital (approval number: B170800045) and Tokyo Medical University (approval number: T2020-0423). The studies were conducted in accordance with the principles outlined in the Declaration of Helsinki.

**Informed Consent Statement:** Written informed consent was obtained from all the subjects after they received a full explanation of the study of Yokohama City University Hospital. Histological analyses of coronary artery tissues were performed after the public announcement of the study protocol at Tokyo Medical University Hospital.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy.

**Acknowledgments:** The authors are grateful to Yuka Sawada and Fumiko Kato for their technical assistance.

**Conflicts of Interest:** Jun Yamashita and Lisa Takahashi are affiliated with an endowed department sponsored by Abbott Vascular Japan Co., Ltd. However, Abbott Vascular Japan had no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the article for publication. The other authors declare no conflict of interest.

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