S100a8 and s100a9 are elevated in aortic wall from patients with moderate-and-severe obstructive sleep apnea syndrome

Abstract

Purpose

Obstructive sleep apnea (OSA) is associated with systemic inflammation and cardiovascular disease, including atherosclerosis. S100A8 and S100A9 are pro-inflammatory proteins involved in atherogenesis, but their expression in the aortic walls of OSA patients remains unknown. This study aimed to determine the relationship between OSA severity and S100A8/S100A9 expression in aortic tissue from patients undergoing coronary artery bypass grafting (CABG).

Methods

This study included 46 patients who underwent CABG. OSA severity was assessed using the WatchPAT™ home sleep apnea test, classifying patients into control and mild (0 < AHI < 15), and moderate to severe (AHI ≥ 15) OSA groups. Aortic wall samples were collected intraoperatively, and S100A8 and S100A9 expression was evaluated using immunohistochemistry. Statistical analysis compared protein expression across OSA severity groups.

Results

Patients with moderate-to-severe OSA exhibited higher S100A8 and S100A9 expression in aortic tissue compared to control and mild OSA groups. The difference was statistically significant for S100A9 (p < 0.01), while S100A8 showed a non-significant increasing trend (p = 0.073).

Conclusion

This study provides novel evidence that S100A8 and S100A9 are overexpressed in the aortic walls of patients with moderate-to-severe OSA. These findings suggest a potential role for these proteins in OSA-related vascular inflammation and atherosclerosis. Further research is needed to explore their potential as biomarkers or therapeutic targets in OSA-related cardiovascular disease.

Introduction

Atherosclerosis is a chronic inflammatory disease of large and medium-sized arteries. Atherosclerosis-triggered diseases, such as ischemic heart disease, stroke, and peripheral vascular disease, are the number one cause of global fatalities [1]. Inflammatory processes within blood vessels contribute to the development of atherosclerosis by activating molecular and cellular pathways. They promote endothelial dysfunction, lipid accumulation, and smooth muscle cell proliferation within arterial walls. Key mediators such as cytokines, chemokines, and adhesion molecules drive this progression, ultimately leading to plaque formation and vascular remodeling [2]. Inflammation is pivotal in destabilizing atherosclerotic plaques, thus increasing the risk of rupture, which can result in stroke or myocardial infarction [3].

Obstructive sleep apnea (OSA) [4] is one of the most common sleep disorders among adults worldwide. It is characterized by recurrent episodes of partial or complete upper airway collapse during sleep [5]. Intermittent hypoxia, chronic inflammation, and other consequences of apneic episodes in OSA are clearly linked to the atherosclerosis [6]. The pathways activated during atherosclerosis heavily overlap with pathways activated by OSA.

Inflammatory processes induce atherosclerosis by activation of multiple receptors. One of the leading pathways is mediated by receptor for advanced glycation endproducts (RAGE) and Toll-like receptor 4 (TLR4) [7]. They activate NF-κB, which leads to an overexpression of cytokines, adhesive molecules, and matrix metalloproteinases (MMPs) linked to atherogenesis [8]. Our previous study demonstrated increased levels of these receptors in carotid plaques in patients with OSA [9].

RAGE and TLR4, on the other hand, are predominantly activated by S100A8 and S100A9 proteins [10]. S100A8 and S100A9 proteins belong to the S100 protein family, which are involved in various physiological and pathological processes, including cell cycle regulation, cell differentiation, and immune responses [11]. Elevated levels of S100A8/A9 were observed across a range of pathological conditions, reflecting its central role in inflammation. Infectious stimuli strongly induce S100A8/A9 expression in neutrophils, macrophages, and monocytes. In metabolic inflammatory states such as obesity, diabetes, and gout, S100A8/A9 levels are similarly increased. Moreover, autoimmune and degenerative disorders—including rheumatoid arthritis, psoriasis, and osteoarthritis—also show marked upregulation of S100A8/A9. These findings highlight the multifactorial regulation of S100A8/A9 and its relevance as a biomarker and potential therapeutic target in diverse inflammation-associated diseases [12].

Studies suggest an important role for these two proteins in the development of atherogenesis [13]. Also, they were analyzed in OSA patients as the inflammatory biomarkers [14–16], but no study focused on their role in plaque formation in this patient population.

The aim of the current study was to determine if levels of expression of S100A8 and S100A9 in the aortic wall were associated with the severity of OSA.

Materials and methods

Patients and tissue samples

Sixty-two patients met inclusion criteria and were enrolled in the study. Inclusion criteria encompassed consecutive elective patients scheduled for coronary surgery with confirmed two-vessel disease, who underwent plaque sampling and had a preoperative sleep study available. The exclusion criteria were as follows: BMI > 35 kg/m2, smoking > 20 years, or previous treatment for OSA. Sixteen out of 62 patients were excluded post-enrollment because of calcified ascending aorta (“porcelain aorta”) or inadequate quality of sleep study. Forty-six patients qualified for surgical treatment of stable coronary artery disease and completed the entire study protocol. All surgeries involved coronary artery bypass grafting using the classic approach through a median sternotomy. Depending on the advancement of the disease, 1–3 bypasses were performed using the left internal mammary artery (LIMA) and the great saphenous vein. Intraoperatively, samples of the ascending aortas, using a full-wall fragment, were taken during venous bypass anastomosis. Aortic samples were collected from a standardized site - ascending aorta (in between the aortic bulb and the proximal part of an aortic arch). The surgical tool - standard size punch was used to collect a sample of a full thickness aortic wall with a surface ca. 7mm² (diameter of a specimen is ca. 3 mm). Before closing the chest, flow in coronary bypass grafts was measured using a Transition Time Flow Meter probe.

Sleep Study

A diagnosis of OSA was confirmed before surgery using a Home Sleep Apnea Test (HSAT) with the WatchPAT™ (Itamar Medical, Caesarea, Israel) portable sleep apnea diagnostic device. The WatchPAT system monitors peripheral arterial tonometry, oximetry, heart rate, actigraphy, body position, and snoring. OSA severity was classified by the apnea hypopnea index (AHI) as: control, AHI < 5; mild, AHI ≥ 5; moderate, AHI ≥ 15; and severe, AHI ≥ 30 events/hour. Sleep studies were performed within a range of 1 to 3 days prior to surgery.

Immunohistochemistry

Formalin-fixed, paraffin wax embedded tissue specimens were cut on 4 μm sections and mounted on adhesion microscope slides (SuperFrost^®Plus, Menzel Gläser). After deparaffinization in xylene and hydration, heat-induced epitope retrieval was carried out by cooking in high pH EnVision FLEX Target Retrieval Solution (Agilent/Dako) for 40 min at 97^oC in a water bath (except ICAM– in low pH EnVision FLEX Target Retrieval Solution, Agilent/Dako). At a later stage, the samples were machine-stained in Autostainer Link 48 (Agilent/Dako) using a visualization kit for immunohistochemical staining; EnVision Flex + System, High pH (Link) (Agilent/Dako). Santa Cruz Biotechnology mouse monoclonal antibodies were used to test antibody expression: S100A8 (clone C-10) [1:2500], S110A9 (clone B-5) [1:3000]. Immunohistochemical staining was evaluated by a single, experienced pathologist blinded to the samples (see the example on Fig. 1). Results were interpreted using a light microscope (Axio Observer Z1, Zeiss, Jena, Germany) equipped with Axiovision 4.8 software (Zeiss Microimaging GmbH, Jena, Germany) and an illumination system LUMEN200 (Prior Scientific, Ltd., Jena, Germany).

Fig. 1.

Immunohistochemical analysis of S100A8 and S100A9 in aortic wall in patients undergoing coronary artery bypass grafting. The patients were divided in 4 groups: control, mild, moderate and severe obstructive sleep apnea. Immunohistochemistry staining was performed with rabbit or mouse antibodies and with isotype control IgG. Original magnification Å~ 200. Scale bar = 200 μm

Evaluation of Immunohistochemistry

The results were presented as the percentage of positive cells within the field of view (FOV), based on an assessment of five randomly selected high-power fields (HPF x200 magnification). The number of positive cells was categorized using the following 4-tiered classification system: 0, negative reaction (less than 10% of stained cells); 1, low expression (11–50% of cells stained positive); 2, intermediate expression (51–75% of cells stained positive); 3, high expression (over 75% of cells stained positive). To eliminate bias, experimental groups were blinded to the pathologist.

Statistical analysis

The results are expressed as the mean ± standard deviation. Mann– Whitney U test was used to evaluate the differences between the groups, which were considered statistically significant at p < 0.05. Statistical analyses were performed with SPSS 23.0 software (IBM, Armonk, NY, USA).

Results

The mean age was 67.4 +/- 8.3 years. The control group (n = 4) included those patients with an AHI < 5 events/h. There were 18 patients (39.1%) with mild OSA, 11 (23.9%) with moderate OSA, and 13 (28%) with severe OSA (Table 1). Patients with moderate-and-severe OSA had larger BMIs (29.8 ± 4.7 kg/m²) compared with those with control and mild OSA (25.9 ± 2.6, respectively) (p < 0.05). Data regarding sleep study results are also shown in Table 1. Expression of S100A8 and S100A9 was increased in the walls of aorta in moderate-and-severe OSA with a significant difference for S100A9 (p < 0,01) (Table 2, Figs. 2 and 3). Since it has been reported that the S100A8 and S100A9 levels may be related to obesity, we modelled the four-level ordinal outcome S100A8 and S100A9 with a proportional-odds (cumulative-logit) regression fitted in R (MASS::polr), first without and then with BMI added. Including BMI in the model caused the significance of the S100A9 to lose statistical significance (p = 0.172). The BMI factor itself was also not statistically significant (p = 0.128). Nested models were compared with a likelihood-ratio test, to check if adding continuous BMI variable did improve the fit. Comparing both models for S100A8 and S100A9, adding BMI did not significantly improve the model (p = 0.968 and p = 0.106, respectively), therefore there was no justification for adjusting the relationship for BMI.

Table 1.

Patient demographic data and home sleep apnea test (HSAT) results in 2 groups of patients divided according to pAHI

	control + mild	moderate + severe
Cases, n	22	24
Age, years	66.8 ± 12.6	68 ± 11.2
Female/Male, n	4/18	4/20
BMI, kg/m2	25.9 ± 2.6	29.8 ± 4.7 *
pAHI, events/h	9.3 ± 5	35.8 ± 16 *
ODI	4 ± 4.5	22.4 ± 15 *
% time SaO2 < 90%	0.1 ± 4.6	2.7 ± 6.2
ESS score	6.5 ± 4.7	8.1 ± 5.1

BMI, body mass index; pAHI, peripheral arterial tone apnea/hypopnea index; ODI, oxygen desaturation index; ESS, Epworth sleepiness scale; Mean ±SD (standard deviation).; * p < 0.05 (Mann-Whitney U test)

Table 2.

Differences between OSA groups: control + mild and moderate + severe in terms of the level of S100A8 and S100A9

	control + mild					moderate + severe
Variable	N	M	SD	Me	Mrang	N	M	SD	Me	Mrang	U	p
S100A8	22	0.59	0.91	0.00	20.18	24	1.12	1.03	1.50	26.54	191.00	0.073
S100A9	22	0.09	0.29	0.00	18.45	24	0.67	0.82	0.50	28.12	153.00	0.003

_{Mrang = mean rank; U = U Manna−Whitney’s test; p = significance value}

Fig. 2.

Differences between OSA groups: control + mild and moderate + severe in terms of the level of S100A8. On the Y axis: the number of positive cells categorized using the following 4-tiered classification system: 0, negative reaction (less than 10% of stained cells); 1, low expression (11–50% of cells stained positive); 2, intermediate expression (51–75% of cells stained positive); 3, high expression (over 75% of cells stained positive)

Fig. 3.

Differences between OSA groups: control + mild and moderate + severe in terms of the level of S100A9

On the Y axis: the number of positive cells categorized using the following 4-tiered classification system: 0, negative reaction (less than 10% of stained cells); 1, low expression (11–50% of cells stained positive); 2, intermediate expression (51–75% of cells stained positive); 3, high expression (over 75% of cells stained positive).

Discussion

To our knowledge, this is the first study to collect and analyze aortic tissue samples from patients in whom sleep studies were also performed. This unique approach allowed us to directly correlate molecular markers of inflammation—such as S100A8 and S100A9—with sleep disordered breathing. By combining immunohistochemical data from the aorta with detailed parameters from sleep study in the same individuals, we were able to gain novel insights into the relationship between vascular inflammation and overnight physiological processes.

Repetitive episodes of intermittent hypoxia and reoxygenation, which are a hallmark of OSA, promote oxidative stress and evoke systemic inflammation [17]. Inflammatory stimulation leads to a rapid release of S100A8 and S100A9 from neutrophils, monocytes, and macrophages at sites of inflammation and activates multiple inflammatory signaling pathways. We specifically chose these proteins as their role in cardiovascular diseases has gained interest. They were shown to mediate the inflammation, recruit and activate leucocytes at the site of vascular injury, and promote plaque formation and its destabilization [18, 19]. One of the leading pathways of S100A8 and S100A9 is mediated by TLR4 and RAGE [4, 20–22]. Activation of these receptors may lead to plaque destabilization and rupture [7]. In our previous study, we demonstrated an association between the levels of TLR4 and RAGE in human carotid plaques and the severity of OSA in patients undergoing carotid endarterectomy [9]. Our current study is a continuation of exploring this atherogenic pathway, but for this study, we specifically focused on levels of S100A8 and S100A9.

Our data demonstrate the relationship between the severity of OSA and the expression of S100A8 and S100A9 in the aortic walls of patients undergoing coronary artery bypass grafting procedures. We clearly showed that both proteins were overexpressed in patients with moderate-to-severe disease, with a statistically significant increase for S100A9. The S100A8 didn’t reach statistical significance, which may result from a small sample size or protein instability.

S100A8 and S100A9 were confirmed in atherosclerotic plaques in human macrophages, foam cells, and neovessels [13]. Moreover, S100A9 was shown to be strongly associated with rupture-prone atherosclerotic lesions [23].

Interestingly, increased levels of plasma and plaque S100A8/S100A9 were related to an increased risk of adverse cardiovascular events after a carotid endarterectomy during a 3-year follow-up, independent of traditional cardiovascular risk factors [24]. Serum levels of S100A8 and S100A9 were analyzed in several studies showing their potential role as a reliable biomarker of cardiovascular risk severity in these patients [14, 18, 21].

These proteins were also evaluated in patients with OSA [15, 20, 25]. A recent study of 173 patients by Tian et al. showed a significantly positive dose-response relationship between serum levels of S100A9 and OSA severity [16]. Cholidou et al. showed that CPAP treatment significantly decreased S100A8 and S100A9 levels in patients with moderate-to-severe OSA (p < 0,0001) [14]. Two other studies demonstrated increased S100A8 and S100A9 levels in serum in children with OSA in a dose-dependent manner [26, 27].

Remarkably, specimens from our previous studies [9] only included atherosclerotic plaque from the carotid arteries, not the whole arterial wall. In our current study, we managed to obtain small pieces of the whole aortic walls, with all layers from the CABG surgery. This allowed us to quantitively measure S100A8 and S100A9 in all the layers of the major vessel. Therefore, we were able to examine inflammatory markers at the site of atherosclerosis formation, previously for both TLR and RAGE and now for S100A8 and S100A9 proteins. Of note, the only studies to date in patients with OSA have demonstrated S100A8 and S100A9 levels from plasma only, likely a representation of the systemic effects of their local release.

Finally, our study revealed a high prevalence of moderate-and-severe OSA in a group of patients undergoing CABG, as compared to general population of Polish adults (52,2% vs. 7,5%) [28]. This group should be perceived as a high-risk group, and OSA screening should be performed in patients undergoing this common cardiovascular procedure. More importantly, OSA is an independent risk factor for developing atherosclerosis [29].

Limitations of the study

Only small samples of the aortic wall are taken during CABG (diameter of 3 mm). Therefore, it was not possible to histologically assess the morphology of the vessel wall or how advanced atherosclerosis is in this patient population. We used Immunohistochemistry (IHC) as a powerful laboratory technique that is widely used in a variety of settings, including clinical medicine. The use of other methods such as Western blotting or RT-PCR was not possible due to the limited amount of material tested from patients. Another limitation of the study is the use of a home sleep apnea testing (HSAT) device. While WatchPAT™ demonstrates high sensitivity and specificity for detecting obstructive sleep apnea, it is not the full PSG, which is a gold standard for OSA diagnosis.

Conclusions

This study showed that S100A8 and S100A9 were overexpressed in patients with moderate-and-severe OSA, with statistically significant increases of S100A9. Both S100A8 and A9 are potent activators of TLR4 and RAGE - mediated atherogenesis and could play a pivotal role in cardiovascular plaque formation.

Author contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Ewa Migacz, Wojciech Kuwa, Wioletta Olejarz, Andrzej Łoś, Katarzyna Bednarek-Rajewska, David F. Smith and Andrzej Kluk. The first draft of the manuscript was written by Ewa Migacz and Wojciech Kukwa and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Data availability

Data supporting reported results can be found at Department of Otorhinolaryngology, Faculty of Medicine and Dentistry, Medical University of Warsaw, Warsaw, Poland.

Declarations

Compliance with ethical standards

This study was approved by the Bioethical Committee of the Medical University of Warsaw. Written informed consent was obtained from each patient.

Conflict of interest

Author Wojciech Kukwa is a shareholder of Clebre Company. All other authors declare that they have no conflicts of interest. The authors did not receive any financial support for preparation of this paper.