Evaluation of miRNA-133a-3p and miRNA-124-3p expression in atherosclerosis using real-time PCR and immunohistochemical methods

Methods

A total of 25 patients who underwent coronary artery bypass graft surgery using internal mammary artery grafts (IMA) between April 2021 and February 2023 at the department of Cardiovascular Surgery, Faculty of Medicine, Mersin University, were included in the study. The study was approved by the Clinical Research Ethics Committee of Mersin University Faculty of Medicine, and written informed consent was obtained from all patients with atherosclerosis prior to their inclusion in the study.

Results

The study included 25 patients with atherosclerosis who underwent coronary artery bypass graft surgery using IMA. Most of the patients were male, comprising 80% of the study population. Approximately half of the patients were current or former smokers (44%), and 56% had type 2 diabetes mellitus (Table I).

The mean expression level of miRNA-133a-3p was significantly decreased by 0.35-fold, while miRNA-124-3p decreased by 0.25-fold in the patient group compared to control group ( Figure).

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Figure.

Fold change in tissue miRNA expression between in two groups.

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The medians of miRNA expression values were compared between the atherosclerosis group and the IMA control group. As shown in Table II, the atherosclerosis group showed a significant downregulation of two miRNAs compared to the control group, with a P value of 0 for each.

When the relationship between miRNAs and demographic parameters in atherosclerosis was examined, no significant correlation was found between miRNA-133a-3p, miRNA-124-3p, and demographic risk factors (P> 0.05, data not shown).

Histopathological examination revealed distinct morphological differences between IMA tissues (control group) and atherosclerotic aortic tissues (case group). In IMA tissues, the Tunica (T.) intima exhibited a thin structure and was lined with a single layer of uniformly arranged endothelial cells. The T. media consisted of regularly organized smooth muscle cells and intact elastic laminae, indicating preserved structural integrity. The T. adventitia presented a histologically normal appearance, characterised by a loose connective tissue structure, vasa vasorum, and nerve fibres. No significant inflammatory cell infiltration was detected. In atherosclerotic aortic tissue samples, the boundaries of the vessel wall layers were disrupted and prominent pathological changes were observed. The T. intima was markedly thickened, with lipid accumulation, infiltration of foam cells, proliferation of fibrous tissue, and occasional necrotic lipid cores observed in this area. In the T. media, a pronounced loss of structural integrity, irregularities, and segmental fragmentation of the elastic laminae were observed. These alterations are consistent with elastic fibre degeneration and matrix structural changes associated with the atherosclerotic process. In addition, a disorganised architecture characterized by an irregular arrangement of smooth muscle cells was observed. This indicates a transition of smooth muscle cells from a contractile phenotype to a synthetic phenotype and a remodelling process in the vascular structure. The T. adventitia was notable for chronic inflammatory cell infiltration, predominantly lymphocytes and macrophages, concentrated especially around the vasa vasorum. These findings suggest that the inflammatory process is not limited to the intima but also extends into the deeper layers of the vessel wall (Supplementary Fig. 1).

In the immunohistochemical analysis using the S100A4 protein, IMA tissues exhibited limited and physiological levels of immunoreactivity. The distribution of the staining pattern across the different layers of the vessel wall was evaluated as follows: In the intima layer, endothelial integrity was preserved, and the cellular composition remained within normal limits. No significant immunoreactivity for S100A4 protein was detected in this region. S100A4 positivity was observed at a level suggesting low infiltration of dendritic or other immune cells. The media layer was typically composed of smooth muscle cells, which exhibited a mild level of S100A4 immunoreactivity. In the T. media, no inflammatory cells were detected, and the structural integrity was observed to be preserved. S100A4 positivity was observed at a mild to moderate level. In contrast, positive immunoreactivity for the S100A4 protein was detected in the adventitia layer. These findings, however, support the existence of neuronal elements that are physiologically located in the T. adventitia. Moreover, S100A4 positivity appeared in the connective tissue components as well as in the perivascular area.

In the immunohistochemical examination of aortic tissues from regions associated with atherosclerotic lesions (case group), variable levels of immunoreactivity were observed in different vascular layers following staining with the S100A4 protein. S100A4-positive cells were identified within the cellular components accompanying the atheromatous plaque in the intima layer. Marked S100A4 immunoreactivity was observed especially in the group of cells located around the necrotic core and morphologically resembling dendritic cells. This finding suggests that antigen-presenting cells, probably dendritic cells, are present in this region as part of the intimal inflammatory response. Smooth muscle cells in the media layer showed positive immunoreactivity for S100A4. In some cases, due to pathological conditions, there is a transition among VSMCs from a physiological contractile state to a synthetic and proinflammatory phenotype. A large number of S100A4 positive cells were found in the medial layer, and it was thought that these cells may be associated with an inflammatory response. In the adventitia layer, strong S100A4 protein staining was observed, consistent with the presence of nerve sheath structures. Additionally, S100A4 positivity was detected in cells located in the connective tissue of the adventitia and around small-caliber vessels, which, based on their morphological features, suggest dendritic cells. This finding indicates that the adventitia may also play an active role in inflammatory and immunological processes (Supplementary Fig. 2 and Table III).

The correlation analysis results obtained in this study revealed strong associations between S100A4 immunopositivity scores and the expression levels of miRNA-133a-3p and miRNA-124-3p. Specifically, the analyses demonstrated a very strong negative correlation between S100A4 and miR-124-3p (r = –0.84, P<0.001), and a strong negative correlation between S100A4 and miR-133a-3p (r = –0.77, P<0.001). These findings suggest that the downregulation of miRNA-133a-3p and miRNA-124-3p, along with the upregulation of S100A4, may contribute to the phenotypic switch of VSMCs from a contractile to a synthetic/proinflammatory state (Supplementary Fig. 3).

Discussion

MicroRNA expression profiles involved in atherosclerotic processes such as endothelial dysfunction, plaque progression, instability, and rupture provide valuable insights into the underlying biological mechanisms and hold potential as biomarkers for prognosis, diagnosis, and treatment.^24,25 In this study, we found no association between miRNA-133a-3p, miRNA-124-3p, and demographic risk factors. However, we determined that miR-133a-3p and miR-124-3p were downregulated, while S100A4 protein expression was upregulated in human atherosclerotic tissues. Therefore, our findings support the hypothesis that miRNAs dysregulation actively contributes to inflammatory remodelling in atherosclerosis.

A study by Carmona-Maurici et al²⁶ reported an association between miR-133a-3p and BMI, while Escate et al²⁷ found a correlation between this miRNA and cholesterol levels. Both findings are in contrast with our results. On the other hand, Andiappan et al⁹ did not observe any association between miR-133a-3p and triglyceride, HDL, LDL, or glucose levels, which is consistent with our findings. In contrast to our results, de Ronde et al¹⁵ reported that high miR-124-3p expression was associated with an increased risk of subclinical atherosclerosis in smokers.

The relationship between miRNA-133a-3p and Coronary Artery Disease (CAD), examined in this study, has also been investigated in previous studies to identify biomarkers for early diagnosis and treatment.^8,9,26,27 While a plasma-based study reported a 28-fold increase in miR-133a levels in CAD patients⁹, Shi et al⁸ observed decreased miR-133a-3p expression in blood and vascular plaques; consistent with this, our tissue-based study also found reduced miR-133a-3p levels in atherosclerotic samples.We interpreted the downregulation of miR-133a-3p in vascular tissue as a potential indicator of increased atherosclerotic risk, in contrast to plasma studies^26,27 showing the opposite. The roles of miR-124-3p, another miRNA examined in our study, in atherosclerosis have been investigated in previous studies.^14,16,28 Decreased miR-124 levels, consistent with our findings, have been reported in CAD patient serum, atherosclerotic tissues of high-fat diet-fed ApoE⁻/⁻ mice, and RAW264.7 macrophages.¹⁶ miR-124-3p also downregulates CETP, PCSK9, MTTP, and APOB genes, reducing cell viability and suggesting therapeutic potential.²⁸ Conversely, it inhibits P4HA1 in vascular smooth muscle cells (VSMCs), decreasing collagen synthesis and plaque stability.¹⁴ Our findings demonstrate that S100A4 expression is markedly increased in atherosclerotic aortic tissue compared to IMA, while both miR-124-3p and miR-133a-3p levels are significantly reduced. This inverse relationship supports the concept of VSMC phenotypic switching, in which the loss of contractile markers (e.g., α-SMA, MYH11) is paralleled by the upregulation of S100A4, promoting migration, proliferation, and fibrotic remodelling.²⁹ Importantly, miR-124-3p has been shown to directly target S100A4, thereby limiting VSMC proliferation and neointimal formation¹⁹, while miR-133a-3p plays a central role in preserving the contractile phenotype and its downregulation facilitates pathological remodelling.²⁰ Taken together, these data suggest that the miR-124/133–S100A4 axis represents a critical regulatory pathway in atherosclerosis.

This study offers novel insights into the regulatory axis involving miR-133a-3p and miR-124-3p, and S100A4 protein in the context of atherosclerosis. On the other hand, there were several limitations. First, our study was not designed to directly assess clinical outcomes (such as the degree of atherosclerosis, major cardiovascular events, or survival). Therefore, larger-scale, prospective clinical studies are needed to demonstrate the clinical prognostic value of our findings. Second, the sample size is limited, which particularly limits the power of subgroup analyses. Furthermore, our study focused solely on miR-133a-3p and miR-124-3p and did not include an analysis of gene regulatory pathways involved in the pathogenesis of atherosclerosis. In conclusion, this study demonstrates the downregulation of miR-133a-3p and miR-124-3p expression levels alongside increased S100 protein expression in human atherosclerotic tissues. Our findings support the hypothesis of miRNA-S100 interplay in vascular inflammation. This relationship may offer a novel perspective for the treatment of atherosclerotic cardiovascular diseases and stimulate continued scientific exploration in this field. However, further large-scale studies are required to confirm the potential diagnostic implications.