CMKLR-1 Expression in Human Aorta Is Dominated by Smooth Muscle Cell Positivity

Abstract

Chemokine-like receptor 1 (CMKLR-1) is a G-protein-coupled receptor that functions as the binding site of chemerin. It induces chemotaxis in various immune cells, and it has recently been linked to vascular remodeling in inflammation. In this study, we investigated CMKLR-1 expression in human aorta samples, focusing on its distribution across different cell types and its potential association with clinical and histomorphological data, particularly concerning aortic dissection and aneurysms. We analyzed 62 aorta samples taken from patients undergoing dissection surgery (n = 27) or aneurysm/dilatation (n = 35) using immunohistochemical CMKLR-1 staining. CMKLR-1 expression was primarily observed in the smooth muscle cells (SMCs) of the aortic media layer, and band-like coloring appeared in the unstained center section. CMKLR-1 positivity in the inner and outer parts of the media layer was observed in only a few cases. One-third of the vasa vasorum exhibited staining. Staining in lymphocytes, macrophages, and endothelia was rare. No significant differences in CMKLR-1 expression were found between the dissection and aneurysm cases, and the clinical or histomorphological data. Although CMKLR-1 expression did not distinguish between the studied conditions, its presence in the aortic media, especially in SMCs, is noteworthy.

Introduction

The aorta is an organ that delivers oxygenated blood from the heart to the end organs. The thoracic aorta and abdominal aorta are separated by the diaphragm. The thoracic aorta comprises the root, ascending aorta, arch, and descending aorta. ¹ The aorta can be affected by many congenital and acquired aortic diseases, which can be roughly categorized into thoracic aortic aneurysm (TAA), abdominal aortic aneurysm (AAA), and acute aortic syndrome (AAS). These conditions are marked by distinctive clinical symptoms, but quick and precise diagnoses are essential for underpinning appropriate interventional strategies.^1–3

An aneurysm increases the diameter of the aorta by 50%. ³ TAA may involve one or more segments of the thoracic aorta, usually due to environmental and genetic factors that intrinsically weaken the wall, including hypertension and Marfan syndrome.^4–6 Inflammatory disorders, such as Takayasu disease and giant cell aortitis, may also be associated with TAA. ⁶

Abdominal aortic aneurysm includes a range of fatal conditions, such as acute aortic dissection (AAD), intramural hematoma, aortic pseudoaneurysm, and traumatic aortic injury, which impact the aortic wall. ¹ Patients with AAS are usually divided into two categories. Those with lesions beginning in the ascending aorta are classified as Standford type A, and otherwise, as Standford type B. This classification provides the prognostic guidance needed for treatment. ⁷ AAS involves many genetic and environmental risk factors, including hypertension and obesity, the prevalence of which has increased in the past 50 years.^8,9 The association between obesity and AAS may be explained by inflamed peripheral vascular adipose tissue inducing vascular dysfunction. ¹⁰

Chemokine-like receptor 1 (CMKLR-1) is a G-protein-coupled receptor that functions as the binding site of chemerin. Originally, when activated, it was thought to induce chemotaxis in dendritic cells, macrophages, and natural killer cells. ¹¹ CMKLR-1 is also expressed in endothelial cells, and its expression is regulated by inflammatory cytokines, such as interleukin-1β, interleukin-6, and tumor necrosis factor α. ¹² Thus, it is possible that chemerin may contribute to changes in vascular reactivity in inflammatory states and that various biomarkers may play a role in AAS pathogenesis and outcomes. Some recent studies indicated that CMKLR-1 led to vascular structural remodeling, increased systolic blood pressure, and promoted the development of atherosclerosis in mice.^13,14

The objective of this study was to topographically investigate CMKLR-1 expression and staining intensities in various aortic layers and cell types. We compared the positivity to clinical dissection data, focusing on histomorphological features (i.e., degeneration and inflammation). In our study, we used commercially available antibody that was validated by a purchasing company. In our laboratory, we verified the antibody staining protocol and, in all staining, both positive and negative controls were used.

Materials and Methods

Study Cohort

We collected 62 surgical aortic samples from patients aged 22–85 years, with a mean age of 63.8 years (SD 13.6). Twenty-two (35.5%) patients were female, and 40 (64.5%) were male. Of the study population, 27 (43.5%) patients had dissection, and the rest had undergone surgery for aneurysm/dilatation. All specimens were fixed in 10% formalin. At least six samples of resected ascending aorta were embedded in paraffin, cut to 4 μm thick sections, and stained with Hematoxylin and Eosin, Verhoeff-van Gieson elastic stain, and Alcian Blue-Periodic Acid-Schiff. Histological analysis confirmed that all samples contained various amounts of inflammatory cells in the aortic walls.

For the width measurements in the media layer, we reduced the study population to ensure that clinically diagnosed dissection and histomorphologically observed dissection correlated in the dissection group. We did this to rule out clinically nondissected samples with falsely observed dissection sites (i.e., clinically dissected samples categorized as nondissected due to a lack of observable dissection sites). This reduced the total group to 43 patients: 20 patients in the dissection group (46.5%), and 23 patients in the nondissected group (53.5%). The mean age in this group was 62.6 years (SD 14.8); 15 (34.9%) of the patients were female, and 28 (65.1%) were male.

Immunohistochemical and Morphometric Analyses

We performed immunostaining for CMKLR-1 with Rabbit polyclonal anti-CMKLR-1 (N-terminal) antibody (product no. AB150491; Abcam Ltd., Cambridge, UK; dilution 1:75) using a Ventana BenchMarkGX immunostainer (Roche Diagnostics International AG, Basel, Switzerland) with an ultraView DAB detection kit (Roche Diagnostics International AG, Basel, Switzerland). The antibody was verified before use and all runs included both positive (tonsil) and negative (primary antibody omitted) controls.

We scanned the stained sections using a NanoZoomer-XR scanner (Hamamatsu Photonics, Hamamatsu, Japan) at 40× magnification and conducted the sample visualization and measurements using QuPath 0.3.0 open-source software for bioimage analysis. ¹⁵

Analysis of the CMKLR1-stained Samples

We performed the following measurements on each media layer. We measured the overall thickness of the media layer at its thickest point and the thicknesses of the CMKLR-1-stained and nonstained sections from both the intima and adventitia sides. If a sample was not dissected, we measured the overall thickness of the unstained section in the center of the media layer. For stained sections, we could directly compare the dissected and nondissected groups. However, we had to sum up the measurements from both sides of the lumen for the dissection samples before we could compare the unstained central sections to the unstained central sections of the nondissected samples.

We conducted semi-quantitative analyses separately for the intima, media, and adventitia layers of the aorta samples and conducted a visual assessment of the proportions of stained CMKLR-1-positive cell types within each entire sample. Subsequently, we categorized the staining according to specific numerical values: 0% cells stained = 0, 1–33% cells stained = 1, 34–66% cells stained = 2, and 67–100% cells stained = 3. In the intima layer, cell types included macrophages, fibroblasts, lymphocytes, smooth muscle cells (SMCs), and endothelial cells. In the media layer, they included SMCs, lymphocytes, and macrophages. In the adventitia layer, we evaluated the staining of lymphocytes and fibroblasts in the same way. In addition, we measured the thickness of the intima layer at the thickest point.

We assessed the vasa vasorum blood vessels located in the aortic adventitia layer by assigning a value of one to at least one clearly positively stained vessel observed. If no staining was present in the vessels, we assigned a value of zero.

We further compared the collected CMKRL-1 staining data to the patients’ clinical data and compared staining in the media layer to morphological data regarding the degeneration of the media layer. The categories of clinical and morphological data are shown in Appendix Tables A1 and A2.

Statistical Analyses

We combined the data from the CMKLR-1-stained samples with histomorphological and clinical data, using IBM SPSS Statistics 29.0.1.0 (Armonk, NY: IBM Corp.) for the statistical analyses. We used the Mann–Whitney nonparametric test for continuous variables, and the chi-squared test for categorical analysis. p-values <0.05 were considered statistically significant in all the statistical analyses.

Ethical Statement

The Pirkanmaa Health Care District Ethical Committee approved the study (permission number R15013), and Valvira approved the use of the samples for research. The study was conducted in accordance with the Declaration of Helsinki.

Results

Measurements in the Aortic Media Layers: Dissected vs. Nondissected

In many cases with visible dissection sites, the CMKLR-1-negative sections were on the luminal side of the walls, and positively stained SMCs were on the intimal or adventitial sides of the media layer (Fig. 1A). Only 5 of the 20 nondissected samples showed band-like coloring that allowed clear unstained center sections of otherwise CMKLR-1-positive media to be seen (Fig. 1B). When comparing this phenomenon between dissected and nondissected aortas, we found no statistical difference. Only the thickness of the entire media layer showed a statistically significant difference between dissected and nondissected aortas. The dissection group had thicker medial walls (mean 1.82 mm [SD 0.58]) than the nondissected group (1.32 mm [SD 0.59, p-value 0.002]) (Appendix Table A3).

Figure 1.

CMKLR-1 immunostaining in the dissected and nondissected aortic media layers. (A) Dissected aorta with a band-like unstained area in the middle of the media layer. Wine-red circle locates the dissection lumen. The red bar highlights the CMKLR-1-stained parts of the media wall, and the green bar the unstained parts. Blue bar highlights an atherosclerotic intima layer. The yellow star symbol locates the adventitia layer. (B) The nondissected aortic wall with undisturbed CMKLR-1 immunostaining in the media layer. The red bar highlights the CMKLR-1-stained aortic media layer. The blue bar indicates the intima layer with moderate atherosclerosis. The yellow star symbol locates the adventitia layer. Scale bar represent 500 µm.

CMKLR-1 in the Aortic Media Layers

The most important finding regarding the localization of CMKLR-1 positivity was related to the SMCs in the media (Figs. 1A–B and 2A–C). Of the 62 samples, SMCs were stained with the following intensities: intensity 1 in 33 cases (53.2%), intensity 2 in 19 cases (30.6%), intensity 3 in 8 cases (12.9%), and no staining in 2 cases (3.2%). In lymphocytes, the following intensities were observed: intensity 1 in 14 cases (22.6%), intensities 2 and 3 in zero cases, and no staining in 48 cases (77.4%). The macrophages exhibited no intensity in 59 cases (95.2%), intensity 1 in 2 cases (3.2%), and intensity 2 in 1 case (1.6%). In summary, almost all samples showed positive SMCs of varying intensities. A few samples also showed staining in lymphocytes and macrophages (Appendix Table A4).

Figure 2.

A panel figure shows examples of the CMKLR-1 immunostaining in the smooth muscle cells (SMCs) of the aortic wall. Purple circles highlight CMKLR-1 positive SMCs. (A) Aortic media layer with border between CMKLR-1-stained and unstained SMCs. (B) Aortic media with band-like CMKLR-1-unstained region in the middle, surrounded with CMKLR-1-positive SMCs in intima and adventitia sides of the media wall. (C) CMKLR-1-positive SMCs within the atherosclerotic plaque in the intima layer. Scale bar represents 100 µm.

CMKLR-1 in the Aortic Intima Layers

In the intima layers, SMCs, showed CMKLR-1 positivity in 16 out of 62 samples, with intensity 1 in 11 cases (17.7%) and intensity 2 in 5 cases (8.1%). Fibroblasts showed CMKLR-1 positivity in 3 cases, all with an intensity of 1 (4.8%). Endothelial cells showed CMKLR-1 positivity in 2 cases, with an intensity of 1 (3.2%). Lymphocytes showed positivity in only one case. In summary, most CMKLR-1-stained cells in the intima layer were CMKLR-1 negative (Appendix Table A5).

CMKLR-1 in the Aortic Adventitia Layers

In the adventitia layers, lymphocytes were stained positively in 21 out of 62 samples, with intensity 1 in 18 cases (29.0%) and intensity 2 in 3 cases (4.8%). Some samples showed positively stained lymphocyte cells, either as individual cells or in clusters (Fig. 3A and D). Fibroblasts were completely CMKLR-1 negative. Vasa vasorum displayed positivity in 19 cases (30.6%) (Fig. 3B and E) (Appendix Table A6).

Figure 3.

A panel figure shows examples of CMKLR-1-positivity and negativity in different cell types. Purple arrows show positive cells and green arrows negative cells. (A) CMKLR-1-stained lymphocytes in the aortic adventitia. (B) CMKLR-1-positive vasa vasorum vein in aortic adventitia. (C) CMKLR-1-positivity in atherosclerotic plaque. CMKLR-1-positivity was observed in the smooth muscle cells, to a lesser extent, in the macrophages and lymphocytes. (D) CMKLR-1-negative lymphocyte cluster in the aortic media. (E) CMKLR-1-negative vasa vasorum veins in aortic adventitia. (F) CMKLR-1-positivity in atherosclerotic plaque debris and single lymphocytes. Scale bar represents 100 µm.

Differences in CMKLR-1 Staining Between the Dissection and Aneurysm/Dilatation Groups

When we compared the dissection and aneurysm/dilatation groups in the previously described manner, we found no statistically significant differences in the prevalence or intensity of CMKLR-1 staining in different layers or cell types (Appendix Tables A4–A6).

Analysis of CMKLR-1 Staining Compared With Clinical and Morphological Data

When we compared CMKLR-1 staining to clinical data, we obtained no significant results for any of the categories studied. The results are summarized in Appendix Table A1. When we compared CMKLR-1 staining in the media layer to morphological media degeneration data, we again found no significant differences. These results are summarized in Appendix Table A2.

Discussion

Summary of the Results

In our study, we examined the staining of the CMKLR-1 protein in various cells in human aorta specimens removed during dissection or aortic aneurysm/dilation surgery. To our knowledge, few researchers have studied CMKLR-1 staining in human aortic walls.

Among the different cells, the most significant staining was observed in the SMCs of the aortic media layer. In almost all samples, the media-layer SMCs showed some degree of staining. In the intima layer, especially when atherosclerotic plaque was present (Fig. 2C), SMC staining was also observed. The staining of macrophages was minimal, with only a few samples showing individual CMKLR-1-positive macrophages (Fig. 3C). Most lymphocytes were negative for CMKLR-1; nevertheless, in the adventitia layers, some samples showed notable staining of lymphocytes (Fig. 3A). Only one sample showed CMKLR-1 positivity in lymphocytes in the intima layer, meaning that almost all samples were negative for lymphocyte staining in the intima layers. Fibroblast staining was almost entirely negative in both the intima and adventitia layers, as was endothelial staining in the intima layer. In the vasa vasorum vessels, approximately one-third of the samples showed a degree of staining (Fig. 3B and E).

A comparison of the dissection and aneurysm/dilation groups revealed no statistically significant differences in CMKLR-1 staining between the different cell types.

During the study, we observed a segmental staining pattern in several dissected aortic samples (Figs. 1A and 2B). The medial walls on the lumen side were often completely CMKLR-1 negative. We investigated this phenomenon by measuring the stained and unstained sections of the media layer but obtained no statistically significant data. Nonetheless, the phenomenon itself is noteworthy.

No statistically significant results were obtained when we compared the clinical and morphological data to CMKRL-1 staining. The results are summarized in Table 1.

Table 1.

Summary of CMKLR Staining in the Aortic Wall.

	Intima Layer	Media Layer	Adventitia Layer
SMC	+ AS (atherosclerotic plaque)	+++	N/A a
Endothelium	–	N/A a	+ (vasa vasorum)
Fibroblasts	–	N/A a	–
Lymphocytes	–	+	+
Macrophages	–	–	N/A a

a

Cells in named layer of aorta were not observed and therefore not studied.

CMKLR-1 Staining in SMCs

In our study, almost all human aortic samples showed some CMKLR-1 positivity in SMCs in the media layer, agreeing with Weng et al., ¹¹ who studied the effects of chemerin and CMKRL-1 in obesity-induced hypertensive rat aortic samples. They showed that CMKRL-1 staining was higher in aortic samples taken from rats with obesity-induced hypertension. The same study also showed that high chemerin might induce vasoconstriction and therefore be a risk factor for hypertension. ¹¹ Hypertension is considered one of the greatest risk factors for aortic dissection, ¹⁶ and 10% of hypertensive patients have some degree of proximal aortic dilatation. ¹⁷ Kostopoulos et al. studied chemerin and CMKLR-1 expression in human arteries and periadventitial fat. According to their data, most of the samples showed CMKLR-1 negativity in vascular SMCs, in contrast to our findings. It is worth mentioning that chemerin expression was detected in 100% of the vascular smooth muscle cells (VSMCs) of the samples studied. ¹⁸ However, Kostopoulos et al. studied arteries in their cohort rather than aortas. Our study revealed no differences in staining intensity between the dissection and aneurysm/dilatation groups. Obesity could be one of the factors increasing CMKLR-1 intensity in aortic media, potentially leading to hypertension in patients. Our study showed no differences in media-layer SMC staining compared with known hypertension; however, clinical data may not always be complete, especially when hypertension is undiagnosed. In addition, hypertension is usually presented as a categorial variant. Perhaps a study performed with hypertension as an absolute value would show significance.

CMKRL-1 Staining in Lymphocytes and Macrophages

CMKLR-1 expression was detected in various immune cells. CMKLR-1 expression was detected in various immune cells. CMKLR-1 positivity was found with monoclonal antibodies in macrophages and immature dendritic cells. CMKLR-1 positivity was found with monoclonal antibodies in macrophages and immature dendritic cells,^19,20 but these studies did not consider aortic samples. Kostopoulos et al. ¹⁸ also observed staining in aortic foam cells when studying CMKLR-1 in aortic atherosclerosis. In our study, we found very little CMKRL-1 staining in macrophages in any of the layers of the aortic wall; overall, very few macrophages were identified in our samples. The thoracic aorta has less atherosclerosis than the abdominal aorta.

In our study, we studied lymphocytes as a single group, although some previous researchers have categorized lymphocytes more precisely. Approximately one-fourth of our samples showed some level of lymphocyte positivity, but in CMKLR-1 positive samples, the staining intensity was mild. Ballet et al. ²¹ recently observed CMKLR-1 expression in a subset of CD8 T cells in mice. Previous studies have shown that human blood circulating plasmacytoid dendritic cells express CMKRL-1, but memory lymphocytes, monocytes, and granulocytes do not. ²² In addition, natural killer cells have exhibited CMKLR-1 expression. ²³ Chemerin is believed to be a chemoattractant recruiting natural killer and dendritic cells to tissue affected by lichen planus, ²³ increasing dendritic cell migration across endothelial cells ¹⁹ and stimulating higher migration of macrophages to inflamed tissues. ²⁴ It is possible that, in the aortic wall, lymphocytes are recruited by different chemoattractants due to a lack of CMKLR-1 positivity.

Similar CMKLR-1 Staining in the Dissection and Aneurysm/Dilatation Groups

As mentioned previously, no statistical differences were observed between the dissection and aneurysm/dilatation groups, possibly for several reasons. First, both diseases (dissection and aneurysm) have similar risk factors, including hypertension, smoking, and hyperlipidemia. In addition, both diseases tend to occur in older people over 60 years of age. ¹⁷ Degenerative processes in the aortic wall play an important role in both diseases. Damaged intralaminar elastic fibers, the cytotoxicity of SMCs, and increased amounts of matrix metalloproteinases have been reported in both diseases. ¹⁶ In addition, an increased aortic diameter increases the risk of aortic dissection, further linking these two diseases. ²⁵

Limitations of the Study

We based the assessment of CMKLR-1 staining in various cells solely on cell morphology, comparing dissected aortas with aortas affected by aneurysm/dilation. It would have been beneficial to include samples of healthy aortas as a control group to reveal potential differences. However, normal aortas are not provided as surgical resection materials. Obesity has been noted to increase inflamed peripheral adipose tissue, but in our study, we did not include BMI as a variable as the weight of the patients was not readily available in all subjects. This variable should be included in future studies.

In general, CMKLR-1 and chemerin have rarely been studied in human aortic samples. We found no differences in histological CMKRL-1 staining between the dissection and dilatation/aneurysm samples, but the study showed that CMKLR-1 was present in SMCs in the media layers of the human aortic walls. The clinical significance is unclear, but differences in CMKLR-1 intensity may play a role in the development of chemerin-based hypertension. In addition, only a small number of immune defense cells were positive. This slightly contradicts previous studies that revealed CMKLR-1 positivity in various immune defense cells, but the studies considered different species, vascular compartments, and diseases.