This study adhered to the guidelines set forth by PRISMA31. Additionally, the protocol for this meta-analysis was registered in the PROSPERO database under the registration number CRD42022335594. The question posed in terms of PECO was: Is there an association between serum/plasma levels of IL-10 and the risk of CAD in studies with a case-control design?

An author of the study, M.S., carried out a comprehensive search in databases such as PubMed/MEDLINE, Web of Science, Scopus, and Cochrane Library up until March 25, 2022, without imposing any restrictions, to collect relevant articles. M.S. also reviewed the titles and abstracts of these articles. Subsequently, the full texts of the articles that met the selection criteria were obtained. The search strategy included keywords/title/abstract: ("coronary atherosclerotic heart disease" or "coronary heart disease" or "coronary artery disease" or "ischemic heart disease" or "myocardial infarction" or "acute coronary syndrome" or "angina pectoris") and ("interleukin-10" or "IL-10" or "IL10" or "interleukin 10") and ("plasma" or "serum" or "blood") and ("control" or "normal" or "healthy"). The bibliographies of the retrieved articles were scrutinized to ensure no significant studies were missed. Another author, R.H.M., verified the search and selection procedures. In the event of any discrepancies between the two authors, a third author, N.S., intervened in the resolution.

The inclusion criteria were as follows: 1) Any study that reported the levels of IL-10 in the serum or plasma of CAD patients and control subjects. 2) Studies that included more than 10 cases in both the case and control groups. 3) CAD was defined based on the criteria reported in Alshammary's study32, and Table 1 shows the criteria for each study. 4) CAD patients without any other systemic diseases and control subjects who were in good health. 5) CAD patients with or without medical treatment, such as statins. Conversely, review articles, meta-analyses, articles with missing data, studies conducted on animals, articles lacking a control group, commentary papers, conference papers, book chapters, duplicate studies, studies that included disease-afflicted controls, and studies involving cases under treatment were excluded.

Table 1

First author, publication year	CAD definition
Mazzone, 199933	Standard progressive changes in electrocardiography linked with a rise in CK values exceeding twice the upper normal limit and alterations in the ST-segment
Mizia-Stec, 200234	Coronangiography
Mizia-Stec, 200335	Coronangiography was performed if there was a constriction of the diameter by 75% or more in at least one of the three primary epicardial coronary arteries
Lee, 200636	Cardiac catheterization
Nilsson, 200637	Angiography
Szodoray, 200638	Angiography
Paulsson, 200839	Angiography
Cheng, 200940	Scanning with radioactive thallium or coronary angiogram
Jha, 200941	Angiography
Jha, 201042	Angiography
Khan, 201143	Angiography revealing a stenosis of more than 70% in at least one coronary vessel
Tapp, 201244	European Society of Cardiology definition
Karu, 201345	NR
Li, 201546	Clinical symptoms, ECG alterations, coronary angiography, and cardiac troponin tests
Mirhafez, 201547	Angiography
Cheng, 201648	Coronary stenosis with at least one main coronary vessel with 50% luminal narrowing
Liang, 201649	A narrowing of the lumen by 50% or more was observed in at least one primary coronary artery or its main branches
Bergström, 201725	Non-segment elevation myocardial infarction identified through characteristic ECG alterations and increased levels of troponins
Tajfard, 201750	An occlusion of 50% or more in at least one coronary artery
Xu, 201751	Stenosis exceeding 50% in at least one primary vessel
Boles, 201826	Angiography
Kharaeva, 201827	Angiography
Kumari, 201818	NR
Ansari, 201952	Angiography revealing stenosis exceeding 70% in a single coronary artery
Shipulin, 202053	Left ventricular ejection fraction ≤ 40%, stenosis left main or proximal part of the left descending artery or two or more epicedial vessels ≥ 75%
Nowrouzi-Sohrabi, 202254	Angiography confirmed stenosis of more than 50% in at least one coronary artery

Abbreviations: CAD: coronary artery disease, NR: not reported, ECG: electrocardiographic

The authors, M.S. and M.R., independently extracted data from the studies included in the meta-analysis. Extracted data encompassed authors' names, publication year, study country, case ethnicity, the number of coronary artery disease (CAD) patients and control subjects, sample size, average age, IL-10 levels in serum or plasma, and the quality score.

The quality assessment was conducted by one author, M.S., using the Newcastle-Ottawa Scale (NOS) tool to evaluate the quality and potential bias in case-control studies55. The highest possible score on the NOS is nine, with scores of seven or higher indicative of high quality.

The Review Manager 5.3 (RevMan 5.3, The Cochrane Collaboration, Oxford, UK) software was utilized to calculate effect sizes, providing the standardized mean difference (SMD) and a 95% confidence interval (CI) for IL-10 levels in the blood of CAD cases and controls. The Z-test determined the significance of the pooled SMD, considering a two-sided p-value of less than 0.05 significant. If heterogeneity was significant, indicated by a Pheterogeneity value of less than 0.1 and an I2 value greater than 50%, a random-effects model56 was used. Conversely, a fixed-effect model57 wad used in cases of insignificant heterogeneity.

Subgroup analysis, random meta-regression analysis, and sensitivity analysis ("one-study-removed" and "cumulative" analyses) were performed using the Comprehensive Meta-Analysis version 2.0 (CMA 2.0; Biostat Inc., Englewood, NJ, USA). Publication bias was assessed using Egger's58 and Begg’s tests59, with a 2-sided p < 0.10 indicating the presence of publication bias.

The NCSS 2021 version 21.0.2 (NCSS, Kaysville, UT, USA) software generated two plots (Radial and L'Abbé plots). The Radial, or Galbraith, plot displays the z-statistic60, and the L’Abbé plot illustrates event rates in cases compared to control groups61, 62, with a p-value less than 0.05 indicating statistically significant heterogeneity.

Trial Sequential Analysis (TSA) was conducted using TSA software (version 0.9.5.10 beta) (Copenhagen Trial Unit, Centre for Clinical Intervention Research, Rigshospitalet, Copenhagen, Denmark)63. The required information size (RIS) was calculated with an alpha risk of 5%, a beta risk of 20%, and a two-sided boundary type. The studies were considered to have included sufficient cases if the Z-curve intersected the RIS line, adhered to the boundary line, or entered the futility area. Otherwise, additional information and further studies were deemed necessary.

Functional interactions between examined genes were studied using the STRING software, a protein-protein interaction network tool accessed at https://string-db.org/ (accessed on 5 August 2023)64. Interaction settings were limited to "Homo sapiens" and required an interaction score threshold of more than 0.900. In the resulting networks, proteins are represented by nodes, and interactions are indicated by edges. STRING was used to identify potential interactions between differentially expressed genes (DEGs) in various tissues, with KEGG analysis obtained from the STRING software.

Figure 1

After removing duplicates and records that were not relevant, 63 out of the 1130 records that were initially retrieved from the databases met the criteria for inclusion as full-text articles (Figure 1). Subsequently, 37 full texts were removed for reasons. At last, 26 articles including 27 studies were entered into the meta-analysis.

The main features of 26 articles18, 25, 26, 27, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 incorporated in the meta-analysis are shown in Table 2. The articles were disseminated from 1999 to 2022. Seventeen articles25, 26, 27, 33, 34, 35, 37, 38, 39, 43, 44, 45, 47, 50, 51, 52, 53, 54 were reported in Caucasians and nine18, 36, 40, 41, 42, 46, 48, 49, 51 in Asians. Eleven articles25, 26, 33, 37, 41, 42, 44, 46, 51, 53, 54 were reported IL-10 level in plasma and fifteen18, 27, 34, 35, 36, 38, 39, 40, 43, 45, 47, 48, 49, 50, 52 in serum. Other variables and the quality score of each article are shown in Table 2 and Table 3, respectively. A few studies in some cases reported the CAD patients under statin therapy.

Table 2

First author, publication year	Country	Ethnicity	Sample	Groups	Number	Mean age, years	IL-10 level (Mean ± SD)	Detection assay
Mazzone, 199933	Italy	Caucasian	Plasma	CAD	42	61	10.8 ± 1.8	ELISA
				CO	39	55	1.9 ± 1.1
Mizia-Stec, 200234	Poland	Caucasian	Serum	CAD	100	58.0	52.37 ± 106.15	ELISA
				CO	20	55.3	14.3 ± 28.5
Mizia-Stec, 200335	Poland	Caucasian	Serum	CAD	33	60.9	68.0 ± 152.5	ELISA
				CO	20	55.4	14.3 ± 28.5
Lee, 200636	Taiwan	Asian	Serum	CAD	30	65.5	1.56 ± 1.37	ELISA
				CO	73	63	1.22 ± 0.85
Nilsson, 200637	Sweden	Caucasian	Plasma	CAD	65	56.9	2.58 ± 2.04	ELISA
				CO	28	52.5	2.4 ± 2.5
Szodoray, 200638	Hungary	Caucasian	Serum	CAD	62	64.2	32.3 ± 95.3	ELISA
				CO	58	72.3	6.95 ± 15.38
Paulsson, 200839	Sweden	Caucasian	Serum	CAD	19	61	0.73 ± 0.67	Immunoassay
				CO	19	60	0.55 ± 0.49
Cheng, 200940	Taiwan	Asian	Serum	CAD	138	65.5	2.1 ± 0.2	ELISA
				CO	74	63	1.2 ± 0.1
Jha, 200941	India	Asian	Plasma	CAD	192	-	1.83 ± 0.16	ELISA
				CO	192	-	1.95 ± 0.19
Jha, 201042	India	Asian	Plasma	CAD (male)	148	-	1.83 ± 0.15	ELISA
				CAD (female)	42	-	1.82 ± 0.16
				CO (male)	142	-	1.94 ± 0.17
				CO (female)	50	-	1.94 ± 0.18
Khan, 201143	Pakistan	Caucasian	Serum	CAD	98	40	2.07 ± 1.70	ELISA
				CO	74	35	1.7 ± 1.85
Tapp, 201244	UK	Caucasian	Plasma	CAD	40	60.4	0.55 ± 0.93	Flow cytometry
				CO	40	59.5	0.88 ± 1.16
Karu, 201345	Estonia	Caucasian	Serum	CAD	39	64	0.53 ± 0.19	High-sensitivity array
				CO	39	62	0.65 ± 0.21
Li, 201546	China	Asian	Plasma	CAD	29	66.9	18.74 ± 20.84	ELISA
				CO	11	62.3	20.92 ± 14.69
Mirhafez, 201547	Iran	Caucasian	Serum	CAD	289	59.1	0.75 ± 0.29	Sandwich Chemiluminescence
				CO	89	58.7	0.84 ± 0.39
Cheng, 201648	China	Asian	Serum	CAD	52	60	7.42 ± 3.81	ELISA
				CO	50	59	17.46 ± 5.01
Liang, 201649	China	Asian	Serum	CAD	128	65.3	134.43 ± 38.24	ELISA
				CO	106	64.7	164.38 ± 36.45
Bergström, 201725	Sweden	Caucasian	Plasma	CAD	57	66	0.32 ± 0.12	ELISA
				CO	41	67	1.03 ± 0.13
Tajfard, 201750	Iran	Caucasian	Serum	CAD	231	59.5	0.76 ± 0.29	Biochip array
				CO	120	53.3	0.83 ± 0.34
Xu, 201751	China	Asian	Plasma	CAD	264	59	98.65 ± 34.79	ELISA
				CO	186	58	32.18 ± 12.15
Boles, 201826	Sweden	Caucasian	Plasma	CAD	69	64.5	0.29 ± 0.21	ELISA
				CO	140	58.6	0.25 ± 0.15
Kharaeva, 201827	Russia	Caucasian	Serum	CAD	27	57	53.6 ± 3.2	ELISA
				CO	20	55	10.0 ± 3.0
Kumari, 201818	India	Asian	Serum	CAD	290	51.6	5.33 ± 3.34	ELISA
				CO	290	51.7	5.83 ± 2.63
Ansari, 201952	Pakistan	Caucasian	Serum	CAD	340	42	0.83 ± 0.53	ELISA
				CO	310	39	0.87 ± 0.36
Shipulin, 202053	Russia	Caucasian	Plasma	CAD	26	59.2	25.16 ± 4.07	ELISA
				CO	14	58.6	20.5 ± 4.44
Nowrouzi-Sohrabi, 202254	Iran	Caucasian	Plasma	CAD	15	58.9	1.98 ± 0.73	ELISA
				CO	15	56.2	1.87 ± 0.92

Abbreviations: CAD: coronary artery disease; SD: standard deviation; CO: control; IL-10: interleukin-10, ELISA: Enzyme-linked immunosorbent assay

Table 3

First author, publication year	Selection#	Comparability&	Exposure$	Total score
Mazzone, 199933	***	*	**	6
Mizia-Stec, 200234	****	**	**	8
Mizia-Stec, 200335	****	**	**	8
Lee, 200636	****	**	**	8
Nilsson, 200637	****	**	**	8
Szodoray, 200638	****	**	**	8
Paulsson, 200839	****	**	**	8
Cheng, 200940	****	**	**	8
Jha, 200941	****	**	**	8
Jha, 201042	****	**	**	8
Khan, 201143	***	**	**	7
Tapp, 201244	****	**	**	8
Karu, 201345	****	**	**	8
Li, 201546	****	**	**	8
Mirhafez, 201547	****	**	**	8
Cheng, 201648	****	**	**	8
Liang, 201649	****	**	**	8
Bergström, 201725	****	**	**	8
Tajfard, 201750	****	**	**	8
Xu, 201751	****	**	**	8
Boles, 201826	***	**	**	7
Kharaeva, 201827	****	**	**	8
Kumari, 201818	****	**	**	8
Ansari, 201952	***	**	**	7
Shipulin, 202053	****	**	**	8
Nowrouzi-Sohrabi, 202254	***	**	**	7

^#Maximum 4 stars, ^&Maximum 2 stars, ^$Maximum 3 stars

The quality scores for case-control studies incorporated in the meta-analysis are shown in Table 3. Most studies involved a high quality (total score ≥ 7).

As Figure 2 shows, the pooled SMD for 85 studies reporting CAD patients was 0.33 (95%CI: ˗ 0.12, 0.78; p = 0.15; I² = 98%). Based on the result, the two groups (patients with CAD and controls) did not exhibit a significant difference in blood IL-10 levels.

Figure 2

The subgroup analysis derived from ethnicity, blood sample, and sample size is shown in Table 4. The findings reported that sample size could be an effective factor in the pooled result.

Table 4

Variable	Number of studies	SMD	95%CI	p-value	Heterogeneity, %
Ethnicity
Caucasian	18	0.30	˗ 0.16, 0.76	0.20	96
Asian	9	˗ 0.29	˗ 1.07, 0.49	0.46	99
Sample
Serum	15	0.42	˗ 0.08, 0.93	0.10	97
Plasma	12	0.12	˗ 0.77, 1.01	0.78	99
Sample size
≥200	11	0.93	0.24, 1.63	0.008	99
<200	16	˗ 0.08	˗ 0.72, 0.56	0.81	96

SMD: Std. Mean difference. CI: Confidence interval. Bold data donates statistical significance (p < 0.05).

The results of "one-study-removed" and "cumulative" analyses indicated that the combined analysis was stable.

Figure 3 indicates funnel plots of blood IL-10 levels in controls compared to CAD patients. With regards to blood IL-10 levels, the p-values of tests were Egger’s: 0.274 and Begg’s: 0.128 for the CAD patients compared to controls. Publication bias was not observed.

Figure 3

Figure 4 presents the outcome of the TSA for blood IL-10 levels in CAD patients versus controls. The data indicates that there are ample cases for comparing blood IL-10 levels in CAD patients and controls.

Figure 4

The results of both radial and L'Abbé plots for blood IL-10 levels in the CAD patients versus the controls are identified in Figure 5 and Figure 6, respectively. The radial plot indicated that one possible cause of heterogeneity in the initial analysis could be outliers. Also, the L'Abbé plot shows evidence of high heterogeneity (p < 0.001).

Figure 5

Figure 6

Table 5 represents the random-effects meta-regression of IL-10 levels for CAD patients compared to controls. The results indicated that the publication year and blood sample were moderator factors in the pooled initial analyses (p < 0.01).

Table 5

Variable		Data
Publication year	Point Estimate	˗ 0.00065
	SE	0.00020
	Lower Limit	˗ 0.00103
	Upper Limit	˗ 0.00026
	Z	˗ 3.30123
		0.00096
Sample Size	Point Estimate	0.00023
	SE	0.00016
	Lower Limit	˗ 0.00008
	Upper Limit	0.00053
	Z	1.45301
		0.14622
Blood sample	Point Estimate	˗ 0.26738
	SE	0.06390
	Lower Limit	˗ 0.39263
	Upper Limit	˗ 0.14214
	Z	˗ 4.18444
		0.00003

Bold data donates statistical significance (p < 0.05). SE: Standard Error

Figure 7 shows the IL-10/STAT3 pathway and its role in suppressing inflammation. IL-10 activates STAT3, and the IL-10/STAT3 axis can have a powerful anti-inflammatory property65 and its role can be crucial in limiting undesirable immune reactions. It is possible that this axis could play a role in regulating inflammation in the context of CAD17. SOCS3 plays a role in the mechanism by which IL-10 modulates inflammation and acts as a feedback inhibitor of the JAK/STAT pathway. Further research would be needed to determine the exact role of the IL-10/STAT3/SOCS3 axis in CAD. The STRING PPI interaction contains 5 nodes with 10 edges with the highest confidence (0.900). The average node degree was 4 and the PPI enrichment p-value was 6.73e^-5. In addition, the KEGG suggested a network analysis for the JAK-STAT signaling pathway based on 5 nodes entered into STRING (Figure 8).

Figure 7

Figure 8

Coronary artery disease (CAD), a prevalent cardiovascular condition, is characterized by the buildup of plaque within the coronary artery walls, leading to reduced blood flow to the heart muscle66. Our main findings showed no difference in the blood levels of IL-10 between the CAD patients and the controls. The analyses reported that factors such as ethnicity, blood sample type (serum or plasma), sample size, and publication year could affect the pooled results.

Limited knowledge exists regarding the function of anti-inflammatory cytokines in CAD. IL-10, an anti-inflammatory cytokine that effectively inhibits immune responses, is produced by T and B cells, monocytes, and macrophages67. It suppresses pro-inflammatory cytokines and has a broad range of anti-inflammatory capabilities, including the inhibition of early pro-inflammatory transcription factors, which consequently reduces cytokine production68.

In vitro, IL-10 plays a crucial role in diminishing lesion growth and preventing the advancement of atherosclerosis69, 70, as well as in modulating the immune response to atherosclerosis, a major contributor to CAD71. However, in clinical settings, the role of IL-10 in patients with CAD has yielded inconsistent results25, 26, 33, despite numerous studies investigating the relationship between IL-10 and CAD25, 27, 40.

One study suggested that CAD patients of both sexes who smoked and consumed alcohol had lower levels of IL-1042, while another demonstrated that CAD patients with low serum iron had significantly higher levels of IL-10 compared to normal controls or CAD patients with normal/high serum iron36. The current meta-analysis suggested that factors such as ethnicity, blood sample, sample size, and publication year could significantly influence the pooled results. Therefore, CAD progression and development may be influenced by several factors, and future studies should examine the effect of each factor individually to identify the most significant influences. According to one study, there was no significant correlation between serum IL-10 levels in CAD patients and factors such as smoking, hypertension, diabetes, obesity, and dyslipidemia72. The findings suggest that certain risk factors for CAD, such as dietary habits (e.g., low intake of fruits and vegetables, high intake of saturated fats), smoking, physical inactivity, and obesity, may reduce IL-10 levels and increase inflammation in the body73, 74, 75, 76, thus contributing to the disease's development. Understanding the underlying mechanisms of these associations may assist in identifying new preventive and therapeutic strategies for CAD.

IL-10 has been shown to suppress the generation of pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α) and IL-1β, which are key contributors to the formation of atherosclerotic plaques68, 77. Additionally, IL-10 has been found to promote the survival of endothelial cells that line the inner surfaces of blood vessels and prevent their apoptosis, thereby reducing the risk of plaque formation78, 79.

Various studies have indicated that increased IL-10 levels in the bloodstream could signal ongoing systemic inflammation in CAD patients27, 33, 40, while other studies noted that in human coronary disease, IL-10 is present and associated with diminished signs of inflammation71, 80. The available evidence suggests that IL-10 plays a protective role against CAD by modulating the inflammatory response and promoting endothelial cell survival. Nonetheless, additional studies are needed to fully comprehend the mechanisms underlying the relationship between IL-10 and CAD.

Research has established that CAD is associated with a continuous inflammatory response81. STAT3 has been identified as a crucial molecule in IL-10's operation, with its activation necessary for the cytokine's anti-inflammatory effects82, 83. Furthermore, evidence suggests that SOCS3 plays a role in how IL-10 modulates inflammation17, 84. Acting as a feedback inhibitor of the JAK/STAT pathway, SOCS3 is crucial in preventing STAT3 activation, cytokine signaling, and the expression of inflammatory genes in immune cells such as macrophages and microglia85. One study found that IL-10 increased SOCS3 expression in cultured cardiomyocytes17. With regards to protein-protein interactions between IL-10, STAT3, and SOCS3, evidence supports that both STAT3 and SOCS3 are involved in IL-10's regulation of inflammation. Additionally, the KEGG analysis reported several biomarkers.

The effects of statins on IL-10 levels in CAD patients versus controls have varied across studies39. One trial reported that statin therapy did not affect IL-10 mRNA expression in patients with CAD86, while another study confirmed this for serum IL-10 levels87. Our meta-analysis included a few studies that involved cases with statin therapy; thus, we were unable to analyze data related to the effect of statins on IL-10 levels comprehensively. Future research should investigate the impact of statins on IL-10 levels in CAD patients. The meta-regression identified the publication year as a confounding factor in the pooled result, indicating possible significant differences in the therapeutic approach to CAD patients over time.

The significant limitations of this meta-analysis include heterogeneity among the studies, limited case count in some analyses, and the lack of patient-level data; diverse criteria for study inclusion, such as varying definitions of CAD, which could introduce bias; different patient populations; and varying methodologies. Additionally, the study mentions factors such as statin therapy that could influence IL-10 levels in CAD patients, but these confounding variables were not fully explored in the analysis. The quality assessment of the included studies was conducted by a single author, which may introduce subjective bias. A more systematic and independent quality assessment process could enhance the reliability of the results. While no evidence of publication bias was found, it is possible that studies with significant results may have been more likely to be published, leading to an over representation of certain findings in the meta-analysis. However, the strengths of the meta-analysis include the absence of publication bias, the high quality of most included studies, and the consistency of the combined results.

According to this systematic review and meta-analysis, no significant difference was observed in blood IL-10 levels between CAD patients and controls, suggesting that IL-10 may not serve as a reliable biomarker for CAD. The analysis included a sufficient number of cases for robust comparison, highlighting the complex and multifactorial nature of CAD. Further research is needed to better understand the role of inflammation and specific inflammatory markers in the development of CAD. Future studies should include larger sample sizes and explore the interaction of IL-10 with other proteins to enhance our understanding of CAD's pathogenesis and identify potential therapeutic targets. This study underscores the importance of continued research efforts to improve our comprehension of the mechanisms and risk factors involved in CAD.

This meta-analysis is clinically significant as it informs clinicians and researchers that IL-10 levels may not be useful for diagnosing or predicting CAD. It highlights the complex and multifactorial nature of CAD, indicating that many factors, not just IL-10, contribute to the disease's development. This insight is vital for clinicians when diagnosing and treating CAD.

CAD - Coronary Artery Disease, CI - Confidence Interval, CK - Creatine Kinase, CO - Control, DEGs - Differentially Expressed Genes, ECG - Electrocardiographic/Electrocardiography, HDs - Heart Diseases, IL-10 - Interleukin-10, JAK - Janus Kinase, KEGG - Kyoto Encyclopedia of Genes and Genomes, NR - Not Reported, NOS - Newcastle-Ottawa Scale, PECO - Population, Exposure, Comparator, Outcome, PPI - Protein-Protein Interactions, PRISMA - Preferred Reporting Items for Systematic Reviews and Meta-Analyses, PROSPERO - International Prospective Register of Systematic Reviews, RIS - Required Information Size, SD - Standard Deviation, SE - Standard Error, SMD - Standardized Mean Difference, SOCS3 - Suppressor of Cytokine Signaling 3, STAT3 - Signal Transducer and Activator of Transcription 3, TNF-α - Tumor Necrosis Factor-Alpha, TSA - Trial Sequential Analysis

None.

Conceptualization, R.H.M.; methodology, M.S.; software, M.S.; validation, R.H.M., and N.S.; formal analysis, M.S.; investigation, M.R. and M.S.; resources, M.S.; data curation, M.S.; writing—original draft preparation, N.S.; writing—review and editing, M.R. and M.S.; visualization, R.H.M.; supervision, N.S.; project administration, R.H.M.; funding acquisition, N.S. All authors have read and agreed to the published version of the manuscript.

None.

Data and materials used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Not applicable.

Not applicable.

The authors declare that they have no competing interests.