Biochemical and Molecular Study of Meteorin-Like Protein in Atherosclerosis Patients

Atherosclerosis is a chronic, multifocal inflammatory disease of the arterial vessels and represents the principal underlying cause of heart disease and stroke. It is estimated to account for approximately 50% of all deaths in Western societies [1]. The historical burden of this condition has been highlighted through paleopathological studies, where investigations of mummies dated between 3000 B.C. and 400 A.D. provided evidence of vascular atherosclerosis in ancient populations [2].

 

The pathological process of atherosclerosis often begins during adolescence and progresses throughout the lifespan. It is characterized by cholesterol deposition, macrophage infiltration, vascular smooth muscle cell proliferation, and accumulation of extracellular connective tissue within the intimal layer of the arterial wall [3]. The term "atherosclerosis" originates from the Greek words athero (porridge or paste) and sclerosis (hardening), with the suffix -osis denoting a diseased condition [2].

 

Atherosclerotic cardiovascular disease (ASCVD) develops through a complex interaction between genetic susceptibility and multiple modifiable and non-modifiable risk factors. Assessing the relative contributions of these risks to the overall disease burden is critical for devising effective prevention strategies [4]. In 2021, the global age-standardized rates (ASRs) per 100,000 population for ischemic heart disease (IHD) were reported as follows: 1856.5 disability-adjusted life years (DALYs), reflecting the overall disease burden; 365.7 for incidence; 3042.3 for prevalence; and 110.9 for mortality. Across all metrics, the burden of IHD was consistently higher among males [5].

 

Regionally, cardiovascular disease remains the leading cause of death in the United Arab Emirates (UAE), responsible for nearly 40% of all registered deaths [6]. Recent findings demonstrate that the UAE has the second-highest prevalence of established ASCVD (eASCVD) in the Middle East, with an overall rate of 29.7% comprising 35.6% among males and 20.5% among females. This is notably higher compared with the regional average of 20.9% (26.6% in males and 16.0% in females), encompassing countries such as Bahrain, Egypt, Jordan, Kuwait, Qatar, and South Africa [7].

 

Atherogenesis is a multifactorial process that involves several interconnected biological pathways, among which epigenetic modifications have recently gained considerable attention [8]

 

Emerging evidence suggests that pro-atherogenic factors, such as elevated levels of low-density lipoprotein (LDL) cholesterol and its oxidized derivatives, can induce persistent epigenomic reprogramming within innate immune cells. Such epigenetic alterations, although mitotically heritable, remain pharmacologically reversible, thereby highlighting their therapeutic potential.

 

In this context, a growing number of researches have been developed in recent years to selectively understand the role of Meteorin-like protein in the pathogenesis and progression of atherosclerosis. Emerging evidence suggests that Metrnl may serve not only as a promising biomarker for monitoring the onset and advancement of atherosclerotic disease but also as a potential therapeutic target. Such findings underscore its dual significance in both clinical diagnosis and the development of novel treatment strategies aimed at mitigating cardiovascular risk.

 

 

Table 1: METRNL and House Keeping gene Primers utilized in the RT-PCR process

 

CALCULATING OF GENE EXPRESSION FOLDING: (LEVAK CALCULATE)

In this research, folding of gene expression was calculated by using the double ΔCT method (ΔΔCT) based on the following equation: Gene Expression folding = 2 ΔΔCT[12].

 

2.6 DNA EXTRACTION

DNAwas extracted by applying the special protocol and instructions that was provided with TransgenBiotech, in which Proteinase K solution, Lysis buffer-2, Washing buffer 1, Washing buffer 2, Elution buffer as well as Collection tube and spin column were used then Biodrop spectrophotometer was used to determine both concentration and purity of the genomic DNA that was extracted from the samples

 

2.7 DNA SEQUENCE ANALYSIS

In this study, the sequences of the nitrogenous bases of the METRNL gene were determined to make sure the accuracy of the designed primer used in the PCR technique also to identify the presence of the genetic variation in the target genes that may influence the effect on the gene effectiveness as well as its rule in the development of the disease. The products of the PCR reaction for the Metrnl genes were sent with the primers. Then the sequence was read at the Psomagen Center in the United States of America. After that, the obtained gene sequences were matched with the reference gene sequences documented at the National Center for Biotechnology Information (NCBI). Finally, the results were analyzed by using the bioinformatics Basic Local Alignment Search Tool (BLAST) program. The sequences of primers used are shown in table (2)

 

PCR reaction was conducted, using the DNA, For. Primer, Rev. primer, Master mix of PCR and Distilled water. While the qPCR program involve an 1 cycle initial pre-denaturation step at 94°C for 5 minutes, succeeded by 35 cycle denaturation at 94°C for 45 min,  as well as 1 cycle of annealing at 57OC  for 1 minute as well as 1 cycle of extension at 72°C for 1 minutes followed by 1 cycle of final extension at 72 o C for about 1 minute and finally   1 cycle of stop reaction  was used at 4o C for 4 minutes.

 

Table 2: displays primer sequence used in the DNA sequenc

 

2.8 STATISTICAL ANALYSIS

Analysis of the results was done by using the statistical program SPSS 25, An independent sample t-test is used to compare the means of healthy and patient groups to determine if there is a statistically significant difference between them while the receiver operating characteristic (ROC) curve analysis was utilized to assess the diagnostic performance of Meteorin-Like Protein and area under the curve (AUC). [13].

A total 240 blood sample was collected from individuals, including patients diagnosed with atherosclerosis and healthy controls ,180 of them for biochemical study and 60 blood sample were collected for molecular biology  study.

 

3.1 THE LEVEL OF METEORIN-LIKE PROTEIN IN SERUM OF CONTROL AND PATIENTS WITH ATHEROSCLEROSIS

Fig. 1:  a. Comparison of Meteorin-Like Protein levels between the healthy control group and patients at P ≤0.001                                                                  b.Diagnostic accuracy of serum Metrnl level in the predication atherosclerosis evaluated by ROC curve

.

Table 3: Analysis of ROC Curve of Meteorin-Like Protein in Atherosclerosis Predicting.

 

MEASURING THE LEVEL OF GENE EXPRESSION OF THE METRNL AND ANALYSIS OF DNA SEQUENCE RESULTS.

 

Table (4): gene expression level of metrnl and the housekeeping gene in control group and atherosclerosis patients in the case study

Fig.2: melting curve  for q PCR and the level of atherosclerosis Metrnl gene measured in this study.

 

 Fig.4:the matching results of nucleotide sequence of the Metrnl gene in the studied blood sample with the nucleotide sequence of the main gene at national center of biotechnology information (NCBI)

Arteries are essential conduits of the circulatory system, along with veins and the heart. They are tube-like structures responsible for transporting fluids—namely blood in the circulatory system and lymph in the lymphatic system—to and from every organ in the body. Fundamentally, arteries transport oxygenated blood and nutrients to body tissues[14] Atherosclerosis is a multifocal inflammatory disease of the arterial vessels and represents the principal underlying cause of heart disease and stroke [15]. The condition often originates in adolescence and gradually advances with age. It is marked by lipid accumulation, inflammatory cell infiltration, smooth muscle proliferation, and extracellular matrix deposition within the intimal layer [16]. Atherosclerotic cardiovascular disease (ASCVD) develops through the interaction of genetic predisposition with multiple modifiable along with non-modifiable risk factors.

 

Recent research highlights the involvement of epigenetic regulation in disease progression. Meteorin-like protein (Metrnl) has recently attracted attention for its potential role in vascular pathology. This study was designed to assess its involvement in atherosclerosis.

 

Results shows that the level of Meteorin-Like Protein is Serum of atherosclerosis patients is significantly decreased (mean = 1.322ng/ml) as compared to the healthy control group (mean = 3.3132 ng/ml) as shown in Figure1: (a). This notable reduction suggests that there is an inverse relationship between Meteorin-Like Protein levels and atherosclerosis disease. ROC curve analysis Figure1: (b) & Table (3) establish a very good diagnostic accuracy, which reveal area under the curve (AUC) of 0.871 for serum Meteorin-Like Protein level. The best cut-off value of serum its level that predicted atherosclerosis was ≤2.2585 ng/ml, and the sensitivity and specificity were 0.949 and0.643 respectively These results indicate that serum meteorin-like protein has reasonable characteristic power and could serve as a biomarker for prediction of atherosclerosis.

 

The qPCR amplification curves demonstrated clear differences in Metrnl gene expression between patients with atherosclerosis and healthy controls. As shown in table (4) and the amplification plot figure (2), patient samples exhibited delay threshold cycle (Ct) values compared to controls, indicating a reduced abundance of Metrnl transcripts. This observation was further confirmed by the relative quantification analysis, where the normalized expression level of Metrnl in the patient group (0.45) was markedly lower than that of the control group (1.0). These results indicate a 55% decrease in Metrnl expression among those with atherosclerosis.

 

These findings suggest that Metrnl expression is significantly downregulated in individuals with atherosclerosis. The observed reduction may reflect its involvement in vascular homeostasis and inflammation, processes that are dysregulated during the development of atherosclerotic lesions. Given its consistent decrease in patients, Metrnl could potentially serve as a biomarker for disease progression and may represent a novel therapeutic target. Nevertheless, further studies with larger cohorts and mechanistic investigations are required to clarify its precise role in the pathophysiology of atherosclerosis

 

The downregulation of Metrnl observed in atherosclerosis patients aligns with previous studies suggesting a disease suppressive role. For example, research conducted by Huang& Hou in 2023 found that lower Metrnl levels were linked with a higher risk of both first-time and recurrent ACS. Even after adjusting for the usual risk factors (like high cholesterol, diabetes, hypertension, smoking) so Metrnl could be a protective factor for the heart[17].  Another research found that in a mouse model of doxorubicin-induced cardiotoxicity, METRNL activates the cAMP/PKA/SIRT1 signaling pathway, conferring resistance to myocardial oxidative stress, apoptosis, and myocardial injury[18] . Future studies should seek to investigate the mechanistic contributions of Metrnl to atherosclerosis progression, consider its potential rule as a biomarker for early detection of atherosclerosis, and to assess its therapeutic applications.

 

Analyses Of Sequence Results

From the results of the DNA sequence analysis and when the results of nucleotide sequence of the Metrnl gene with in the studied blood sample matched with the nucleotide sequence of the main gene at national center of biotechnology information (NCBI) as shown in figure(3 and 4), this BLAST result show that the Metrnl DNA  sequence  which successfully deposited in the GenBank (Accession number: LC874112 (  is exactly identical (100% match) to Metrnl sequence in the database nucleotide sequence of the main gene at national center of biotechnology information (NCBI), this matching reflect that there is no mutation within the gene sequence and the disease progression may result from unhealthy lifestyle, dietary habits , environmental effects and obesity.

Metrnl rule as atherosclerosis disease progression suppressor, indicated by its significant downregulation in atherosclerosis patients. This notable reduction in expression could influence the severity or progression of the disease. disease progression may result from unhealthy lifestyle, dietary habits , environmental effects and obesity These findings suggest that Metrnl holds potential as a biomarker for diagnosing of atherosclerosis and its complications and may serve as a treatment target.

 

SOURCE OF FUNDING: no source of funding

 

ETHICAL CLEARANCE: ethics committee of Nineveh health directorate (department of health, Mosul, Iraq) approved the study under official documentNo.59239, date11/12/2024.

 

CONFLICT OF INTEREST. No conflict

 

ACKNOWLEDGMENTS: we thank the physician at Al Salam Hospital -Cardiac Surgery Center- Mosul, for their priceless support and assistance during the period of study. We also extend our deepest appreciation to all patients and healthy volunteers who participated in this research, for their cooperation and trust, which made this work possibl.  

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