Migration of OxLDL + Macrophage Phenotypes from Pericoronary Adipose Tissue to Coronary Intima: A Possible Mechanism for Human Coronary Atherosclerosis

Background: We previously found that oxidized low-density lipoprotein (oxLDL), a core atherogenic protein, is stored in human pericoronary adipose tissue (PCAT) and macrophages residing in PCAT accumulate oxLDL. Here, we aimed to identify whether and how the macrophage phenotypes transport oxLDL from PCAT to the coronary intima. Methods and Results: Coronary arteries and their surrounding PCAT were excised from human autopsy subjects and examined using immunohistochemical techniques to investigate macrophage-mediated oxLDL transport from PCAT to the coronary intima. OxLDL + CD68 + and oxLDL + CD206 + macrophages were observed in PCAT of both normal coronary segments (normal group) and adjacent coronary segments with plaques (plaque group). External elastic lamina (EEL) was loosened, or fragmented and internal elastic lamina (IEL) was disrupted in the plaque group, and oxLDL + CD68 + and oxLDL + C206 + macrophages extending pseudopod forward were frequently observed to pass through these portions into the intima. OxLDL + CD11c + macrophages were not found in PCAT, EEL and the media but were found in disrupted IEL and plaques. Conclusions: The results suggest that CD206 + or CD68 + macrophages transport oxLDL from PCAT into the media and they and CD11c + macrophages transformed at the site of IEE into plaque via loosened or fragmented EEL and disrupted IEL and could participate in initiation and acceleration of human coronary atherosclerosis.

However, this process is largely dependent on animal experiments and it is not known whether it is the sole mechanism underlying atherosclerosis development in humans. Previously, we found that oxLDL is stored in human pericoronary adipose tissue (PCAT) and accumulated in macrophages residing in the PCAT [5]. However, whether macrophage acquire oxLDL from PCAT and through what route(s) are oxLDL transported to the intima remained unclear in our previous study. Thus, the present ex vivo study aimed to examine which of the macrophages namely those expressing CD68 (a nonspecific marker of macrophage), CD11c (a marker of atherogenic M1-macrophage) [6,7] or CD206 (a marker of anti-inflammatory and antiatherogenic M2-macrophage) [6], participate in this transport process and the routes they follow using immunohistochemical techniques and by excising the normal coronary segments (normal group) or segments containing plaques (plaque group) to-gether with their surrounding PCAT from human autopsy subjects who had suffered from ischemic heart disease.

Immunohistochemical Studies of Excised Human Pericoronary Adipose Tissue (PCAT) and Its Adjacent Coronary Artery Ethics
This ex vivo study was conducted after obtaining approval from the Ethical Committees of the Japan Foundation for Cardiovascular Research, Funabashi-Futawa Hospital, Chiba-kensei Hospital and Toho University, and after obtaining written informed consent from the families involved regarding the use of excised coronary artery and its surrounding adipose tissue for histological studies to clarify the mechanisms underlying atherosclerosis [8][9][10][11].

Selection of plaques and normal segments
A 4 -5 mm long section of proximal or middle segments of the anterior descending coronary artery or right coronary artery and their surrounding PCAT were isolated by transecting its proximal and distal ends at the shorter axes. The normal segments and those with plaques were excised (one segment from each subject). Thus, 7 normal segments (normal group) and 7 segments with plaques (plaque group) were obtained and used for further examination (Table 1). They were embedded in O.C.T. Compound (Sakura Finetck USA Inc., Torrance, CA) before being stored at -20 ○ C. Before embedding these segments, a 0.5 mm section was obtained from each segment and fixed with 5% glutaraldehyde solution for electron microscopic study.
at -20°C, were cut into successive 10 µm sections on a cryostat (Tissue Tec 3D, SakuraFinetec Japan, Tokyo). Such relatively thick and frozen sections were used to prevent leaking from the PCAT.
Sections were fixed with 4 % paraformaldehyde for 7 min at 4°C, and incubated with a mixture of 1% hydrogen peroxide in methanol for 30min. Successive sections were processed at first by single immunohistochemical as follows: oxLDL, CD68 for nonspecific macrophages [12], CD11c for M1-inflammatory and atherogenic macrophages [13] and CD206 for M2-anti-inflammatory and anti-atherogenic macrophages [14]. for the green fluorescence of oxLDL, a 555 nm BPF and a 575 nm BAF for the red fluorescence of macrophages, and a 345 nm BPF and 420 nm BAF for the blue fluorescence of cell nuclei as previously reported [16].

Microscopic observation of EEL and IEL
EEL and IEL exhibit a strong fluorescence when stained with fluorescein. One section from each sample was stained with fluorescein, after which EEL and IEL were examined using fluorescent microscopy. Normal EEL is composed of tight continuous elastin fibers that form a band. In case any loosening or fragmentation was noted, EEL was defined as loosened or fragmented EEL, and the number of loosened or fragmented portions in the circumference of EEL was compared between the normal and plaque groups.
IEL is composed of elastin plates arranged in series connected side by side with thin filaments. In a normal coronary artery, the distance between the plates is approximately ≦5 µm [8]. Therefore, widening of the distance by 10 µm, fractured or decaying portions of IEL were defined as disruption. The number of disrupted portions across the entire circumference of IEL was compared between the normal and plaque groups.

The Number of OxLDL + Macrophage Phenotypes Passing through EEL or IEL:
The number of oxLDL-containing macrophage phenotypes passing through the entire circumference of EEL or IEL was count-ed and compared between the normal and plaque groups. Macrophages are amoeboid cells and extend pseudopod toward the direction of crawling. Therefore, pseudopod side was considered as the direction of their crawling.

Electron Microscopic Study
Because macrophages are ameboid in shape, cells with an amebic configuration and passing through IEL were examined using an electron microscope (InTouchScope TM , JSM-IT200, Nihondenshi Co., Tokyo, Japan). Pseudopod was defined as a thin and low-density protrusion from the body.

Statistical analysis
Fisher's exact test was used for statistical analysis of data. The data obtained were expressed as mean± standard deviation (SD), A p value of < 0.05 was considered to be statistically significant.
Because of a large number of comparisons, Bonferroni correction was used [17].  . Dotted oxLDL is observed in the interstitial space between the adipocytes (arrowhead in A). CD68 + and CD206 + but not CD11c + macrophages were observed in the interstitial space (arrowhead in B, C, D). The macrophages co-insides with the dotted oxLDL (arrowhead in E -E-2). Scale bars= 5 µm.  A. No significant difference in density of oxLDL + CD68 + and oxLDL + CD206 + macrophages in PCAT is observed between normal group and plaque group. OxLDL + CD11c + macrophage is not observed in PCAT in both the groups.

OxLDL in PCAT
B. CD68 + and CD206 + macrophages density in the media is significantly higher in the plaque group than in the normal group. OxLDL + CD11c + macrophage is not observed in the media.

Am J Biomed Sci & Res Copy@ Yasumi Uchida
OxLDL was present in adipocyte cytoplasm AC; arrow in (Figure1a-A). Dotted oxLDL is observed in the interstitial space between adipocytes (arrowhead in Fig 1a-A), which is contained in macrophage phenotypes (arrowhead in (Figure 1a-E -E-2). These changes were observed in all samples of the normal and plaque groups.

Macrophage phenotypes in PCAT, media and intima
OxLDL + CD68 + and OxLDL + CD206 + macrophages were observed in the interstitial space between PCAT adipocytes arrows in (Figure 1a-B, C, Figure 1b

OxLDL + Macrophage phenotypes
Approximately, a half of the CD68 + macrophage population and about a quarter of CD206 + population contained oxLDL in PCAT, but there was no significant difference in oxLDL occurrence between the normal and plaque groups. CD68 + and CD11c + macrophages in plaques more frequently contained oxLDL than CD206 + macrophages.

Differences in the changes of EEL and IEL between normal and plaque groups
In the normal group, EEL was wavy and continuously arranged arrowheads in (Figure 2a -A). IEL was wavy and its plates were almost continuously arranged in series arrow in (Figure 2a -A). Contrastingly, in the plaque group, EEL was fragmented (arrowheads in (Figure 2a -B) and IEL was frequently disrupted asterisk in (Figure 2a -B). The incidence of loosened or fragmented portions in EEL and disrupted portions in IEL was significantly higher in the plaque group than in the normal group (Figure 2b   The incidence of oxLDL + CD68 + and oxLDL + CD206 + macrophages passing through disrupted portions of IEL was also higher in the plaque group than in the normal group. OxLDL + CD11c + macrophages were also observed in the plaque group (Figure 2c-B). b) Number of oxLDL + CD68 + and oxLDL + CD206 + macrophages that pass through EEL is significantly higher in the plaque group. OxLDL + CD11c + macrophages are not found. SD: Standard Deviation. ** p<0.01. *** p<0.001. b) Number of oxLDL + macrophage phenotypes passing through IEL. OxLDL + CD68 + and oxLDL + CD206 + macrophages as well as oxLDL + CD11c + macrophages are observed. The number of such macrophage phenotypes is significantly higher in the plaque than in the normal group. **p<0.01. ***p<0.001.

Electron microscopy of macrophage-like cells passing through IEL
Cells that were amoebic in configuration, characteristic of macrophages, and had only began to pass through protruding pseudopod into the intima (Figure 4-A) or had been traversing through the disrupted portion of IEL (Figure 4-B) were observed using an electron microscope.

OxLDL + Macrophage Phenotypes in PCAT
Storage of oxLDL was observed in PCAT not only in the plaque group but also in the normal group, indicating that oxLDL storage begins before atherosclerosis development. Percentages of oxLDL + CD68 + and oxLDL + CD206 + macrophages in PCAT were not different between the normal and plaque groups, indicating that oxLDL containing capacity of these macrophages was not influenced by the presence or absence of plaques in the adjacent coronary intima.

OxLDL + Macrophage Phenotypes in the Intima
In the plaque, the percentage of oxLDL + macrophage phenotypes was significantly higher than that in the intima of normal group. Their accelerated migration through the EEL and IEL is considered to be the reason for this difference.

Mechanism(s) for Damages in EEL and IEL
Fragmented EEL and disrupted IEL were frequently observed in the coronary segments with plaques (plaque group). There are at least three possible mechanisms for such changes: a) Plaques induce distension of EEL or IEL and subsequently damage them. In a normal coronary artery, IEL is composed of elastin plates in series, which are connected side by side with thin filaments, and the space between the plates is within 5 µm [18].
Probably, the filaments connecting IEL plates were disrupted and the plates were separated owing to plaque distension. b) Because EEL and IEL are composed mainly of elastin, elastases that were diffused from the lumen or adventitia eventually damaged EEL and IEL [19]. c) Macrophages excreted cytokines during invasion and caused EEL and IEL damage [20,21].

Migration of OxLDL + Macrophage Phenotypes through EEL and IEL
The incidence of invasion of oxLDL + macrophage phenotypes through EEL and IEL increased in the plaque group, suggesting that their influx into the intima contributed to plaque formation. EEL and IEL lost their function as a mechanical barrier, and may have enhanced macrophage migration.

Direction of crawling of OxLDL + Macrophages
Ameba and amoeboid cells such as white blood cell and macrophages protrude a pseudopod forward, retracts the pseudopod, change body shape and crawls, indicating that the pseudopod shows the direction of crawling [22][23][24][25]. In this study, oxLDL + macrophage phenotypes in the loosened EEL or disrupted IEL protruded their pseudopod toward the media and toward the intima, respectively, strongly suggesting that they crawled toward the media and then to the intima.

Macrophage Transformation
There are a number of macrophage phenotypes. Among them, M 1 -macrophages(CD11c + macrophages) are considered as atherogenic and M 2 -macrophages (CD206 + macrophages) are considered as anti-atherogenic, mostly based on the findings in animals or cultured cells [26]. In our study, CD11c + macrophages were not found in PCAT, adventitia and media, but were found at the site of disrupted IEL, suggesting that CD68 + and/or CD206 + macrophages were switched over to CD11c + macrophages while passing through IEL. In the present study, not only CD11c + but also CD206 + macrophages frequently contained oxLDL. This finding suggested that CD11c + and CD206 + macrophages (so-called M 1and M 2 -macrophages, respectively) are not well differentiated with respect to oxLDL carriage. Switching of M 2 -to M 1 -macrophages seen in animals, [27]   CD68 + or CD206 + macrophages in PCAT acquired oxLDL from adipocytes, moved to adventitia, passed though loosened or fragmented portions of EEL into the media, and subsequently passed through disrupted portions of IEL, whereas a portion of such a macrophage population transformed into CD11c + macrophages, entered into the intima and secreted cytokines [4,7] to induce atherosclerosis [1]. EC: Endothelial Cell. MPO: Myeloperoxidase. HOCL: Hypochlorous Acid. MMP: Matrix Metalloprotease.

Possible mechanisms for underlying migration of OxLDL + macrophages phenotypes from PCAT to the Intima
Based on the findings of the present study and previously reported articles by other investigators [1,4], we propose a possible mechanism for human coronary atherosclerosis. The oxLDL stored in PCAT is transferred to CD68 + and/or CD206 + macrophages that reside in the interstitial space between adipocytes; oxLDL + CD68 + and /or oxLDL + CD206 + macrophages pass through the adventitia and loosened or fragmented EEL into the media; Subsequently, these macrophages pass through the disrupted portions of IEL, while a part of them transform into CD11c + macrophages, and enter into the intima; these oxLDL + macrophage phenotypes thus enter into the intima, release a number of cytokines [1,4,7] and cause atherosclerosis ( Figure 5).

Study Limitations
1. Because a majority of the patients were admitted for a serious condition at terminal stage, lipid plasma levels and other substances examined at this stage were not considered to reflect the levels during stable conditions, therefore it was difficult to examine the association of plasma lipid levels and other substances with macrophages.
2. Although oxLDL storage in human PCAT was confirmed, the following mechanisms remain to be elucidated. a) Despite the findings of the present study strongly suggesting that oxLDL is transported by macrophages, the real-time movement of oxLDL + macrophages was not examined, and should be clarified by tracing labelled native oxLDL or macrophages in vivo.
b) The factor(s) that regulate oxLDL storage in PCAT as well as macrophage movement into the intima (plaque) remain to be elucidated.
c) It is still unknown whether oxLDL is synthetized in PCAT, or from where and how oxLDL is transported to the PCAT.

Clinical Perceptives
Therapies targeting PCAT and/or oxLDL + macrophage phenotypes could prevent human coronary atherosclerosis. Transthoracic or transcardiac administration via the right atrium [29] of anti-oxLDL substances could be used for this purpose.

Conclusion
OxLDL is stored in human PCAT. CD68 + and CD206 + macrophage acquire oxLDL from PCAT and transport it through the adventitia, loosened and fragmented EEL, media, and disrupted IEL into the intima, however a portion of these macrophages get converted to CD11c + macrophages while passing through IEL before entering into the intima. Thus, our findings suggest that such an process would contribute to the initiation and progression of human coronary atherosclerosis and targeting therapy on oxLDL in PCAT or oxLDL + macrophages could suppress coronary atherosclerosis.

Disclosure
The authors have no conflicts of interest to declare. Relationships with Industry: The authors have no relationships with industry.

Funding
No external funding was received for this study.

Author contributions
Yasumi Uchida and Yasuto Uchida conceived and designed the study, performed the in vitro study and wrote the manuscript; Ei Shimoyama and Nobuyuki Hiruta performed autopsy and immunohistochemical staining and conducted microscopic studies; and Tsuyoshi Tabata performed statistical analysis. All authors played a role in editing the manuscript.