Network Pharmacology Approaches Used to Identify Therapeutic Molecules for Chronic Venous Disease Based on Potential miRNA Biomarkers.

1. Introduction

Chronic venous disease (CVD) is a widespread disease estimated to affect 83.6% of elderly individuals globally. This disease, which includes varicose veins and chronic venous insufficiency (CVI), is a prevalent medical condition among adults. CVD affects individuals worldwide, with the highest incidence in developed countries [ 1 ]. Reports describe venous leg ulcers as the most severe stage of chronic venous disease (CVD) [ 2 ]. CVD has significant consequences for individuals and society, affecting the quality of life through the experience of pain, heaviness, itching, and reduced mobility [ 3 ]. The most common cause of CVI is varicose veins in the lower extremities, with the most severe form resulting in venous ulceration. It is estimated that 30–40% of the adult population has varicose veins, and up to 6% of patients with varicose veins develop ulcers. In addition, 30% of varicose veins can develop into more severe forms of CVI.

The pathophysiology of CVD involves varicose veins, dysfunction of venous valves, and skin ulceration resulting from impaired cell proliferation, apoptosis, and angiogenesis [ 4 ]. Vascular cell functions, including cell differentiation, proliferation, migration, and apoptosis, are decisively regulated by microRNA (miRNA)-dependent gene expression modulation [ 5 , 6 ]. Many studies have revealed that miRNAs’ aberrant expression is associated with pathological conditions’ development [ 7 ]. In vascular disease, miRNAs have demonstrated modulatory functions in angiogenesis, endothelial cell dysfunction, and response to ischemic events [ 8 ]. Several miRNAs are regarded as potential biomarkers for cardiovascular diseases [ 8 ]. miRNAs and genes exhibit distinctive expression patterns in venous samples from patients with CVI compared to healthy patients [ 9 , 10 , 11 , 12 ]. Some works have separately proposed some biomarkers that could be used within CVD, and through functional analysis, seven genes (CDS2, HDAC5, PPP6R2, PRRC2B, TBC1D22A, WNK1, and PABPC3) selected as miRNA targets by in silico methods were recognized as potential biomarkers of CVD. However, their complete understanding remains elusive [ 13 ].

microRNAs (miRNAs) are small non-coding RNAs, typically 21–25 nucleotides in length, originating from both coding and non-coding transcription units found in genic (intronic or exonic) and intergenic regions [ 14 , 15 ]. They are pivotal in regulating essential processes such as tissue homeostasis and cell signaling, operating at the post-transcriptional gene expression level [ 16 ]. These molecules also have an extracellular role and can be released into microvesicles, modulating gene expression in several tissues [ 17 , 18 , 19 ]. Both intracellular and extracellular miRNAs can be detected in tissue samples and various biological fluids, including serum, plasma, urine, saliva, sweat, and tears. However, this approach remains unexplored, and is mainly used in personalized medicine as a diagnostic or therapeutic tool.

Network representations have proven essential in depicting interactions among entities of interest across various domains. This tool is precious for analyzing and visualizing complex biological processes [ 20 ]. Leveraging known interactions discovered from previous experiments creates biological networks. These intricate molecular interaction networks provide valuable insights into biological processes, enabling us to pinpoint critical nodes and other topological features associated with complex phenotypes in health and disease [ 21 ]. According to network medicine theory, disease-associated phenotypes are hypothesized to result from perturbations in gene networks rather than individual gene mutations acting in isolation [ 22 ].

Restoring or inhibiting specific miRNAs in diseased cells can be accomplished through various strategies, including (1) delivering miRNA mimics or inhibitors using modified nucleic acids, (2) modifying miRNAs using viral delivery vector systems, (3) delivering miRNA mimics or inhibitors using nonviral delivery vector systems, and (4) developing small-molecule drugs that target microRNAs [ 23 ]. A further strategy has been using drugs, natural products, or plant extracts, which have been shown to modulate the expression of miRNAs through multiple mechanisms in various pathologies [ 24 , 25 , 26 , 27 ].

Hesperidin, a drug widely used in the treatment of CVD [ 28 , 29 , 30 ], has been demonstrated to enhance endothelial function by increasing the availability of nitric oxide (NO) while simultaneously suppressing the production of reactive oxygen species (ROS) and plasma levels of pro-inflammatory markers [ 31 ]. This flavonoid has been involved in modulating miRNAs by downregulating miR-21-5p and upregulating miR-16-5p and miR-34a-5p [ 32 ], which have been involved in the physiopathology of several cardiovascular diseases, including CVD [ 4 , 33 , 34 , 35 ], and has been considered to be used as a reference compound in our study.

In this sense, because of the critical participation of miRNA in gene expression in the development of CVD, we focused on identifying potential miRNAs and drugs that could modulate the expression of those miRNAs and may antagonize their profile expression during CVD. To achieve this aim, we employed a network-based approach. The data were collected from both the literature and miRNA-related databases to identify, by network analysis, potential molecules that interact with miRNAs regulated by hesperidin and that modify the gene expression involved in CVD derived from miRNAs collected from several studies with potential miRNA biomarker candidates and as potential therapeutic agents [ 4 , 9 , 11 , 36 , 37 , 38 , 39 , 40 ].

2. Materials and Methods

2.1. Reviewing

Bibliographic Screening to Select MicroRNAs Involved in CVD

We searched the PubMed database from 2016 to 2024 using MESH terms such as microRNAs, miRNA, chronic venous disease, and chronic venous insufficiency. Two independent reviewers selected studies that met the eligibility criteria.

The bibliography considered in this work includes studies about miRNAs with significant expression profiles in CVD reported between 2016 and 2024, in vitro studies that included cases and control groups, and tests in human samples with validated CVD diagnoses. Studies not performed in English that do not include controls or duplicated data, studies conducted in animal models, or studies conducted in human-derived sources were excluded from this research.

2.2. Data Collection

We sought to analyze the genes influenced by the miRNAs identified through data extraction in both the reported bibliography (validated) and servers (predicted) from the Target Expression Analysis section of the miRDB-MicroRNA Target Prediction Database ( http://www.mirdb.org/mirdb/index.html , accessed on 1 May 2024), considering those targets with a prediction score > 95%. The network analysis used the mirNET database to search for small molecules responsible for modulating CVD-associated miRNAs ( https://www.mirnet.ca , accessed on 13 May 2024). The bar plot was generated using the ggplot2 package in R, leveraging information sourced from the literature and the Target Expression Analysis section of the miRDB-MicroRNA Target Prediction Database ( http://www.mirdb.org/mirdb/index.html , accessed on 28 June 2024).

2.3. Network Structural Analysis

We employed Cytoscape software (v.3.10.2) to construct networks aiming to identify the most connected miRNAs and relevant pathways within the context of CVD [ 41 ]. The Cytohubba plugin was used to identify the most interconnected genes within the network [ 42 ]. To elucidate the potential principal signaling pathways modified by the miRNAs, we employed the JEPETTO plugin within Cytoscape [ 43 ]. The pathways exhibiting the most significant enrichment ( p < 0.05) were adjusted using their XD scores based on the insights from the previous report [ 44 ].

2.4. Network Pharmacology Analysis

We utilized the Cytoscape software (v.3.10.2) to construct the structural networks [ 41 ] to analyze the information from the mirNET database and identify the chemical compounds responsible for modulating miRNAs involved in CVD ( https://www.mirnet.ca , accessed on 29 June 2024). The Cytohubba plugin was used to identify the most connected genes in the network. [ 42 ]. Furthermore, the highly interconnected small molecules were identified based on their scores, and an in-depth exploration of their respective functions was conducted in the literature.

To search for molecules with the same targeted miRNAs as hesperidin, (1) we generated a dataset of miRNAs targeted by hesperidin. These miRNAs were obtained from the SM2miR server ( http://www.jianglab.cn/SM2miR/ , accessed on 30 June 2024). This server allows us to identify whether those miRNAs targeted by hesperidin were up- or downregulated. (2) Subsequently, we obtained each sequence of those miRNAs upregulated or downregulated by hesperidin, searching the miRNA accession number in the miRbase server ( https://www.mirbase.org/ , accessed on 1 July 2024) [ 45 ]. (3) We analyzed each miRNA sequence and predicted other molecules that also regulate those same miRNAs in the PSRR (Prediction of SM-miRNA Regulation pairs by random forest) server ( https://rnadrug.shinyapps.io/PSRR/ , accessed on 2 July 2024). Using this random forest algorithm, we achieved the highest AUC values of 0.911 for the upregulation model and 0.896 for the downregulation model on the testing datasets, outperforming four other machine learning algorithms [ 46 ]. These molecules were employed to construct a structural network using the Cytoscape software (v.3.10.2) [ 41 ], and the results were analyzed with the Cytohubba plugin [ 42 ]. We considered those molecules with high degree centrality, which tends to highlight nodes (molecules) with higher degrees (more direct connections), and betweenness centrality, to identify those nodes (molecules) that lie on critical paths between others.

2.5. Using QED Index to Determine Drug-like Candidates

To determine the drug-likeness using the quantitative estimate of drug-likeness (QED), we used the DruLito software ( https://drulito.software.informer.com/ , accessed on 4 July 2024), as previously described [ 47 ]. This index has enabled us to identify compounds with favorable oral administration characteristics [ 48 ]. The DruLito software (version 1.0.0) evaluated SDF files from PubChem. Our analysis focused solely on molecules that were accessible via this server. The analytical data are reported in Supplementary Materials Tables S1–S10 .

3. Results

After reviewing the literature and applying the filters mentioned in the methods for bibliographical screening, we created different networks using the selected studies. Scheme 1 , according to the PRISMA statement, outlines the article selection criteria for subsequent analysis. A carefully selected database of differentially expressed miRNAs in cardiovascular disease was acquired to determine the most pertinent miRNAs. A structural network analysis was built to find the common miRNAs between the countries where these studies were performed. The data for this network analysis were collected from studies where the detection methods, tissues, and expression of each miRNA were found ( Figure 1 ). The three most outstanding sources were the proximal part of the significant saphenous vein tissue, vein tissues, and peripheral blood mononuclear cells (PBMCs). This network analysis found that Poland researched CVD-associated miRNAs most, followed by China, the UK, and the USA. The most effective methods for diagnosing CVD were microarray and RT-PCR. The analysis identified several interconnected miRNAs within the network, including miR-34a, miR-34c, miR-202-3, miR-1202, and miR-130a. The analysis detected miR-34a in wound-edge epidermal keratinocytes and the proximal part of the significant saphenous vein tissue using microarray, RT-PCR, and RT-PCR validation methods.

To identify the miRNAs that regulate the most significant number of genes and to find critical miRNAs that could regulate numerous genes, we utilized our complete dataset of miRNAs ( Table S1 ) from the miRBD database ( http://www.mirdb.org/mirdb/index.html , accessed on 28 June 2024) to identify genes and pathways that interact with them. We only considered target genes with a score above 95% ( Figure 2 ). Figure 2 A depicts the genes regulated by each miRNA involved in CVD found in the miRBD database, including miR-593-3p (135 genes), miR-19a-3p (127 genes), miR-92a-3p (116 genes), miR-301a-3p (92 genes), and mir-181a-2-3p (87 genes). Figure 2 B depicts the number of nodes (genes) interacting with the whole miRNA dataset and shows the most regulated targets indicated by the miRBD database. Using this new set, we created another network with the genes altered by these miRNAs.

We further analyze the structural network in Figure 2 , which shows the most connected nodes. The most connected genes were WNK-1, IL1RN, PPP6R2, PRRC2B, and ADIPOQ, and the most connected miRNAs were hsa-miR-106b-3p, has-miR-92a-3p, hsa-miR-454-3p, hsa-miR-548ac, and hsa-miR-128-3p ( Figure 3 ).

After defining these most connected and relevant genes involved during CVD, they were used to perform an enrichment analysis to better understand the pathophysiology of CVD. The targeted genes of the miRNAs involved in CVD were analyzed to correlate CVD with other pathways or processes. Table 1 lists the most relevant pathways or processes associated with CVI. Lately, a pharmacology network analysis was conducted considering the most relevant miRNAs from the dataset and the compounds present in the mirNET database ( https://www.mirnet.ca/upload/MirUploadView.xhtml , accessed on 29 June 2024) ( Figure 4 and Table 2 ). This analysis was performed to find small molecules of different natures that could interact with our most relevant miRNAs. Interestingly, the interactions inside the network showed that drugs and toxicants could modulate such miRNAs. This network showed that the reported phlebotonic drug hesperidin shares several miRNAs with the natural compound curcumin. Table 2 displays the most relevant features of each compound. Furthermore, the interactions within the network suggest that drugs and toxicants have the potential to modulate these miRNAs. As a result, it is crucial to consider their influence on toxicological analysis and in conducting further ecological and epidemiological studies ( Table 3 ).

To further analyze the interactions between the miRNAs targeted by hesperidin, determining if this molecule influences their regulation, and thus determine new candidates that can also share and similarly regulate these miRNAs targeted by hesperidin, we generated a network structural analysis to determine those molecules that were most connected and shared more intermediates within this network ( Figure 5 ). For more information on how this network was built, see the Materials and Methods. Table 4 shows the degree and betweenness values of the most relevant molecules in this network (20 molecules).

Finally, we determined their drug-likeness through the QED index to analyze whether these most relevant molecules could be suitable candidates for oral administration. At the end of the analysis of the eighteen molecules obtained from the study of the structural networks that share miRNAs with hesperidin, only six met this QED index criteria of a value greater than 0.5, identifying desoximetasone, curcumin, flurandrenolide, fludrocortisone, diflorasone, and reversine as the most suitable candidates to be administered orally in this study ( Table 4 ).

4. Discussion

CVD is a multifactorial condition characterized by complex pathophysiological mechanisms [ 40 ], among which the non-coding RNAs, such as miRNAs, regulate multiple functions in biological and pathological processes, positively or negatively, promoting or not the action of the corresponding mRNA product. Numerous miRNAs can be biomarkers for several diseases and conditions, including cardiovascular diseases, cancer, and sepsis [ 92 ]. MicroRNAs (miRNAs) can be released into the bloodstream, where they exist as stable circulating free miRNAs or remain safeguarded from endogenous RNAse enzyme activity through encapsulation in exosomes or proteins [ 93 ]. Numerous reports have described the significant role of miRNAs in processes closely associated with vascular pathology, such as angiogenesis, endothelial dysfunction, and vascular inflammation. Therefore, dysregulation of the miRNA expression profiles could be considered a promising biomarker of CVD [ 94 ].

Although few studies have been carried out, most of these studies come from the USA, Poland, China, and the UK. However, there were no studies in Latin America, so many aspects of CVD and miRNAs are still unknown in this population. Thus, urgent research must be translated into clinical practice.

One of the most connected miRNAs is miR-34a. This miRNA has been extensively studied in cancer biology in in vitro conditions due to its role as a tumor suppressor. During cancer development, this miRNA has been demonstrated to impact several crucial processes, including cell cycle progression, self-renewal, senescence, apoptosis, epithelial-to-mesenchymal transition, differentiation, migration, and metastasis. Evidence indicates that miR-34a is a crucial regulator of age-related tissue modifications and a promoter of cellular senescence. Levels of miR-34a have shown an increase with age. This increase is found in several organs, including the muscle, heart, and aorta, and it promotes detrimental organ remodeling and functional decline. In the cardiovascular system, miR-34a promotes the senescence of endothelial cells (ECs) in the vascular wall and vascular smooth muscle cells (VSMCs), principally by direct negative regulation of its best-known target, the longevity-associated gene sirtuin 1 (SIRT1) [ 95 ]. This pathway promotes arterial inflammation and the development of age-related VD.

Furthermore, miR-34a expression could be influenced by other risk factors promoting vascular changes similar to aging [ 95 ]. These features make this miRNA a suitable target for treating VD. It has been described as a target of flavonoids inhibiting miR-34a synthesis and upregulation of SIRT1 expression, protecting endothelial cells from senescence [ 96 ]. This finding correlates with the literature because most drugs to treat VD come from a flavonoid nature [ 30 ].

Furthermore, miR-34a targets XIAP [ 97 ], which has been implicated in resistance to apoptosis, a phenomenon possibly related to the downregulation of apoptosis in the medial layer of varicose veins [ 98 ]. This resistance to apoptosis has also been found in SASP (senescence-associated secretory phenotype), where varicose veins lead to vascular endothelium dysfunction, causing a pro-inflammatory environment [ 99 ]. Expression of miR-34a has shown significant upregulation in both the plasma and atherosclerotic plaques of patients diagnosed with CAD (coronary artery disease). This finding suggests that overexpression of miR-34a in our study is common in both arterial and venous pathology. Also, this result agrees with the research performed by Zalewski et al. [ 4 ], where this miRNA was found to be upregulated in patients with CVD. By integrating both experimental and predictive data analysis in our review, mir-34a is an outstanding miRNA and may be used as a potential biomarker for the early detection of CVD.

miR-34c has been associated with several cellular processes in the cardiovascular system [ 100 ], playing a protective role in hypoxia by activating autophagy through repressing BCL2. Significant cellular stress is produced during hypoxia that originates diverse pathological consequences, such as cardiovascular diseases and cancers [ 101 ].

miR-202-3p is downregulated during CVD. This downregulation is involved in metalloproteinase-1 (MMP-1) overexpression. MMP1 promotes trans-endothelial migration by degrading inter-endothelial junctions and disrupting endothelial integrity [ 102 ]. Thus, this loss in miR-202-3p expression may increase the activity of the MMP-1, which plays a role in CDV pathogenesis [ 103 ]. miR-1202 plays regulatory roles in MMP9 function during CVD development. However, it is required to evaluate their potential diagnostic and therapeutic utility [ 37 ]. This finding agrees with the results obtained by Zalewski et al., where miR-1202 is downregulated in patients with varicose veins. Thus, our findings also propose this miRNA as a valuable potential biomarker [ 94 ]. miR-130a was the most interconnected miRNA in this network. miR-130a has been reported in skin biopsies from venous ulcers, compared to standard skin specimens [ 94 ]. An association with the overexpression of these miRNAs has been established because they may inhibit wound healing by targeting LepR [ 4 , 94 ].

Interestingly, the other most connected miRNAs, miR-106b-3p, miR-92a-3p, miR-454-3p, miR-548ac, and miR-128-3p, were obtained by next-generation sequencing (NGS) of PBMCs of patients with varicose veins [ 94 ]. These findings allow us to know the relevance of NGS and PBMCs in diagnosing VD.

According to structural network analysis, the most connected genes were WNK-1, IL1NR, PPP6R2, PRRC2B, and ADIPOQ WNK1, which have been associated with downregulation of miR-181a-2-3p and miR-106b-3p in previous reports [ 4 ]. WNK1 plays a role in endothelial cell proliferation, chemotaxis, and invasion [ 104 ]. In mouse model studies, the lack of the WNK1 gene leads to embryonic death due to angiogenic and cardiovascular defects [ 104 ]. The hypothesis postulates that the upregulation of WNK1 is associated with the downregulation of miR-181a-2-3p and miR-106b-3p, thereby leading to the induction of inflammation and aging in response to oxidative stress in CVD [ 4 ]. Interleukin-1 receptor antagonist (IL1RN) has a biological role in the pro-inflammatory cytokine interleukin-1. This downregulation may be implicated in the chronic inflammation of the CVD [ 105 ]. The PPP6R2 gene is upregulated during CVD. This gene encodes proteins that act as a significant T-loop phosphatase for Aurora A, a crucial mitotic kinase. Aurora A activation is very complex and occurs at specific subcellular locations due to signaling events, including phosphorylation and oxidation [ 106 ]. Aurora A controls key oncogenic pathways associated with drug resistance and poor patient outcomes [ 106 ]. According to the findings of this study, several miRNAs are present in both CVD and some types of cancer. PPP6R2 may be a potential early biomarker for CVD and cancer.

PRRC2B has been reported as possibly upregulated by miR-92a-3p [ 4 ]. This gene-encoding protein is involved in brain development. As noted by Zalewski et al., specific neurodevelopmental processes might be implicated in the pathogenesis of CVD, highlighting potential associations between vascular and neurodegenerative disorders [ 4 ]. However, the network analysis showed another potential biomarker in this study, the ADIPOQ gene. ADIPOQ gene polymorphisms are related to adiponectin levels, insulin resistance, and metabolic diseases such as diabetes mellitus type II (DMT2) [ 107 ]. This finding also connects with WNK, which is involved in DMT2 [ 108 , 109 ] and vascular disorders [ 110 ]. In this context, evidence has been proposed that the incidence of diabetes mellitus is much higher in patients with CVI. More intense therapy directed against CVI in diabetic patients could potentiate the prevention of complications such as leg edema, deep vein thrombosis, progression of CVI, and post-thrombotic syndrome and improve the quality of life [ 111 ].

For the first time, small molecules (toxins and drugs) that regulate the expression of several miRNAs are involved in a CVD study through structural network analysis. Certain identified compounds include standard air or water contaminants and drugs that possess the potential to regulate miRNAs associated with CVD and its progression. Formaldehyde (FA), a prevalent air pollutant, is identified among xenobiotics and is widely encountered in nature, industrial processes, and various consumer products [ 67 , 112 ]. Its long-term exposure may provoke skin disorders, cancer, and cardiovascular disease [ 113 ]. This small molecule alters vascular function and oxidative stress, influencing cardiovascular health [ 113 ]. Exposure to FA from various sources can lead to heart diseases, including arrhythmia, myocardial infarction (MI), heart failure (HF), and atherosclerosis (AS) [ 112 ]. Acute FA exposure has influenced adult female vascular function in the arms and legs, decreasing conduit vessel function without altering microvascular function [ 113 ]. Also, FA causes an increase in blood pressure by activating the ACE/AT1R axis [ 114 ], so its exposure may be involved in the development of hypertension, which has been proposed as a potential cause of the early development of CVD [ 114 , 115 , 116 , 117 , 118 ]. Additional research is imperative to ascertain the diverse effects of varying concentrations and durations of FA exposure in both genders, older adults, and individuals who are particularly susceptible to cardiovascular disease, including CVD. Ethanol has been shown to induce vascular toxicity, linking patients with disorders associated with excessive ethanol consumption and the development of CVD. This process has been described through the modulation of the AT1 receptor with vascular hyper-contractility, promoting mitochondrial dysfunction, mtROS production, and reduced bioavailability of NO and H 2 O 2 [ 119 ].

17beta-estradiol (E2) and diethylstilbestrol are estrogens [ 68 , 73 ]. Estrogens are critical regulators of vascular homeostasis, primarily functioning through ERα and ERβ, which serve as ligand-gated transcription factors. Research findings have revealed a substantial upregulation in the expression of ERα, ERβ, and GPER in the tissue of individuals with CVD. This upregulation has been associated with disease severity and has shown a correlation with the clinical stage of VD [ 120 ]. In addition, a large group of heterogeneous drugs with anticancer properties showed significant interactions with the miRNAs proposed by our network analysis. These results could be because most of the miRNAs in this study are involved in the development of, or are potential targets of, some cancers [ 102 , 121 , 122 , 123 , 124 , 125 , 126 , 127 , 128 , 129 ]. Our data agree with the evidence that shows that CVD may have a relationship with the development of cancer [ 130 , 131 ]. These results encourage more extensive and in-depth studies to be carried out to elucidate the pathogenesis mechanisms shared by both CVD and the development of some types of cancer.

Among the compounds that share those miRNAs that are targeted by the reference compound, hesperidin, we find a group of heterogeneous molecules of different types, such as corticosteroids, diarylheptanoids, antivirals, nucleoside analogs, oncology drugs, purine derivatives, estrogen receptor modulators, histone deacetylase inhibitors, and HMG-CoA reductase inhibitors. Interestingly, many of these small molecules are oncology drugs. This result correlates with that last obtained with the network analysis because of the close relationship that may occur between the miRNAs involved in CVD and cancer. In this context, hesperidin has been established to have potential use in cancer therapy [ 132 ]. Furthermore, this is the first study proposing candidates for the treatment of CVD using miRNA-based and network pharmacology approaches, compared to previous studies using virtual screening and network pharmacology approaches [ 133 ]. This approach would open new possibilities for finding new candidates to treat CVD.

The employing of corticosteroids in cardiovascular diseases has been controversial because it has been associated with adverse cardiac effects such as cardiac hypertrophy, cardiac fibrosis, and cardiac remodeling [ 134 ]. However, Ospina-Quintero et al. [ 135 ] found that a low dose of vitamin D and dexamethasone can protect against heart disease when administered subcutaneously and repeatedly. This is because it induces the production of IL-10 by a network of lymphoid and myeloid immune cells. Also, Wang et al. [ 136 ] indicated that administering low doses of dexamethasone yielded cardioprotective effects in myocardial ischemia/reperfusion (MIR) mice. These effects were manifested through the enhancement of cardiac function, attenuation of reactive oxygen species (ROS), suppression of inflammatory responses, and stimulation of anti-oxidant responses. Additionally, the study by Wang et al. revealed that the protective mechanisms of dexamethasone on myocardial tissues were mediated through the Keap1/Nrf2/HO-1 pathway. [ 136 ]. Therefore, dexamethasone could have a protective effect on cardiovascular diseases, including CVD. However, more studies are necessary to support these hypotheses. These analyses may also be carried out at a clinical level, along with the other corticosteroids in this study.

Curcumin and hesperidin share many miRNAs. Hesperidin, a flavonone predominantly found in citrus fruits, is proposed to have the potential to serve as a therapeutic agent for the modulation of several cardiovascular diseases [ 58 ]. Curcumin demonstrates substantial efficacy in mitigating cardiomyocyte injury after ischemia and hypoxia, as well as in inhibiting myocardial hypertrophy and fibrosis, ameliorating ventricular remodeling, and attenuating drug-induced myocardial injury. Furthermore, it shows promise in improving diabetic cardiomyopathy (DCM), alleviating vascular endothelial dysfunction, impeding foam cell formation, and reducing the proliferation of vascular smooth muscle cells (VSMCs). Clinical studies have substantiated the protective impact of curcumin on blood vessels, while toxicological investigations have confirmed its safety profile. Nonetheless, it is imperative to acknowledge that elevated doses of curcumin may induce adverse effects, including hepatic impairment and perturbations in embryonic heart development [ 137 ]. These characteristics could propose curcumin as a drug with therapeutic potential for CVD.

Reversine, alternatively known as 2-(4-morpholinoanilino)-6-cyclohexylaminopurine, is a derivative of 2,6-disubstituted purine. This diminutive molecule exhibits encouraging antitumor characteristics by impeding diverse kinases that govern the cell cycle and cytokinesis. Through the application of network pharmacology prediction, molecular docking, and systematic review, it has been determined that MEK1 could potentially serve as a promising target for the action of reversine [ 138 ]. MEK1-ERK1/2 pathways are part of the MAPK cascade and have been implicated in regulating myocyte survival following ischemia–reperfusion injury, oxidative stress, and anthracycline exposure. These pathways have been associated with cardioprotective effects by directly antagonizing myocyte apoptosis [ 139 ] and play a crucial role in the signaling and transcriptional pathways that contribute to the pathogenesis of heart and vascular disease via the MEK-ERK-Egr-1 axis [ 140 ]. These data open a new line of study on miRNAs as potential biomarkers of CVD, genes, and contaminants or drugs that could be involved in developing or treating CVD.

5. Conclusions

Despite efforts to compile the available information on miRNAs and CVD, this review has limitations. Among the challenges encountered were the following: the studies were heterogeneous and there was an absence of pertinent clinical data, as well as a lack of a comprehensive survey on individual lifestyles, ethnicities, sample collection, methodologies, heterogeneity in protocols, miRNA isolation, comorbidities, and servers used for miRNA analysis. Additional epidemiological research is urgently required to comprehend the potential of miRNAs as a treatment for CVD or as a prospective risk factor for its development. Furthermore, a notable absence of Latin American representation in cardiovascular disease (CVD) profiles has been observed, underscoring the necessity of incorporating this demographic in forthcoming research endeavors. Notwithstanding these constraints, our study meticulously consolidated the foremost findings, delineating miRNA profiles prevalent in CVD. Our in silico strategies suggest possible associations between CVD and xenobiotic agents, including 5-Aza-CdR, arsenic, trichostatin A, and endogenous hormones.

Furthermore, the network analysis allowed us to identify potential drug-like compounds that share targeted miRNAs by hesperidin: desoximetasone, curcumin, flurandrenolide, fludrocortisone, diflorasone, and reversine. These compounds may be investigated in future preclinical or clinical studies to treat CVD. It is crucial to investigate these drugs deeper. Furthermore, the clinical use of miRNAs as biomarkers for the diagnosis and prognosis of CVD should be supported.

Acknowledgments

UNAM: School of Medicine assisted with implementing project 008-CIC-2022. Oscar Salvador Barrera-Vázquez is grateful to the Dirección General de Asuntos del Personal Académico (DGAPA), Universidad Nacional Autónoma de México, for the Subprograma de Incorporación de Jóvenes Académicos de Carrera (SIJA).

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/jox14040083/s1 , Figure S1: Network analysis of CVD-associated miRNAs with their expression, sources, countries of origin, and detection methods; Figure S2: Network analysis of miRNAs associated with CVD and their predicted targets; Figure S3: The structural network represents the ten most connected nodes, including miRNAs and targets. Figure S4: The structural network of small molecules, phlebotonic, and miRNAs. Figure S5: Structural network of hesperidin-specific miRNAs that are also altered by small molecules. Table S1: CVD bibliography dataset; Table S2: Sources, countries, and methods; Table S3: miRNAs, targeted genes and their regulation; Table S4: Most connected nodes of genes and miRNAs; Table S5: Enrichment analysis of the most outstanding genes; Table S6: miRNAs and targeted small molecules; Table S7: Analysis of the most connected small molecules; Table S8: Results of network analysis of miRNAs and small molecules; Table S9: The most connected nodes between hesperidin, small molecules and miRNAs; Table S10: Druglikeness determined by QED index of the small molecules that shared the miRNA expression profile with hesperidin.

Author Contributions

Conceptualization, G.A.M.-G. and O.S.B.-V.; methodology, O.S.B.-V.; software, O.S.B.-V.; validation, G.A.M.-G. and O.S.B.-V.; formal analysis, G.A.M.-G. and O.S.B.-V.; investigation, O.S.B.-V.; resources, G.A.M.-G.; data curation, O.S.B.-V.; writing—original draft preparation, G.A.M.-G. and O.S.B.-V.; writing—review and editing, G.A.M.-G., J.L.E.-R., and O.S.B.-V.; visualization, G.A.M.-G. and J.L.E.-R.; supervision, G.A.M.-G.; project administration, G.A.M.-G.; funding acquisition, G.A.M.-G. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available in this article and Supplementary Materials .

Conflicts of Interest

The authors declare no conflicts of interest.

References

1. Sinikumpu S.P. Keränen M.H. Jokelainen J. Keinänen-Kiukaanniemi S. Huilaja L. The association between chronic venous disease and measures of physical performance in older people: A population-based study BMC Geriatr. 2021 21 556 10.1186/s12877-021-02528-9 34649528 PMC8518156

2. Nicolaides A.N. The Most Severe Stage of Chronic Venous Disease: An Update on the Management of Patients with Venous Leg Ulcers Adv. Ther. 2020 37 19 24 10.1007/s12325-020-01219-y 31970660 PMC7004438

3. Duque M.I. Yosipovitch G. Chan Y.H. Smith R. Levy P. Itch, pain, and burning sensation are common symptoms in mild to moderate chronic venous insufficiency with an impact on quality of life Am. Acad. Dermatol. 2005 53 504 508 10.1016/j.jaad.2005.04.079 16112363

4. Zalewski D.P. Ruszel K.P. Stępniewski A. Gałkowski D. Bogucki J. Komsta Ł. Kołodziej P. Chmiel P. Zubilewicz T. Feldo M. Dysregulations of MicroRNA and Gene Expression in Chronic Venous Disease J. Clin. Med. 2020 9 1251 10.3390/jcm9051251 32344947 PMC7287878

5. Fernández-Hernando C. Suárez Y. MicroRNAs in endothelial cell homeostasis and vascular disease Curr. Opin. Hematol. 2018 25 227 236 10.1097/MOH.0000000000000424 29547400 PMC6175704

6. Qin S. Zhang C. MicroRNAs in vascular disease J. Cardiovasc. Pharmacol. 2011 57 8 12 10.1097/FJC.0b013e318203759b 21052012 PMC4517184

7. Pahlavan Y. Mohammadi Nasr M. Dalir Abdolahinia E. Pirdel Z. Razi Soofiyani S. Siahpoush S. Nejati K. Prominent roles of microRNA-142 in cancer Pathol. Res. Pract. 2020 216 153220 10.1016/j.prp.2020.153220 33007646

8. Schulte C. Karakas M. Zeller T. microRNAs in cardiovascular disease—Clinical application Clin. Chem. Lab. Med. 2017 55 687 704 10.1515/cclm-2016-0576 27914211

9. Cui C. Liu G. Huang Y. Lu X. Lu M. Huang X. Li W. Jiang M. MicroRNA profiling in great saphenous vein tissues of patients with chronic venous insufficiency Tohoku J. Exp. Med. 2012 228 341 350 10.1620/tjem.228.341 23132275

10. Markovic J.N. Shortell C.K. Genomics of varicose veins and chronic venous insufficiency Semin. Vasc. Surg. 2013 26 2 13 10.1053/j.semvascsurg.2013.04.003 23932556

11. Pastar I. Khan A.A. Stojadinovic O. Lebrun E.A. Medina M.C. Brem H. Kirsner R.S. Jimenez J.J. Leslie C. Tomic-Canic M. Induction of specific microRNAs inhibits cutaneous wound healing J. Biol. Chem. 2012 287 29324 29335 10.1074/jbc.M112.382135 22773832 PMC3436197

12. Jin Y. Xu G. Huang J. Zhou D. Huang X. Shen L. Analysis of the association between an insertion/deletion polymorphism within the 3′ untranslated region of COL1A2 and chronic venous insufficiency Ann. Vasc. Surg. 2013 27 959 963 10.1016/j.avsg.2013.04.001 23849651

13. Serra R. Ssempijja L. Provenzano M. Andreucci M. Genetic biomarkers in chronic venous disease Biomark. Med. 2020 14 75 80 10.2217/bmm-2019-0408 32053001

14. Chen R. Jiang N. Jiang Q. Sun X. Wang Y. Zhang H. Hu Z. Exploring microRNA-like small RNAs in the filamentous fungus Fusarium oxysporum PLoS ONE 2014 9 e104956 10.1371/journal.pone.0104956 25141304 PMC4139310

15. Wahid F. Shehzad A. Khan T. Kim Y.Y. MicroRNAs: Synthesis, mechanism, function, and recent clinical trials Biochim. Biophys. Acta 2010 1803 1231 1243 10.1016/j.bbamcr.2010.06.013 20619301

16. Galvão-Lima L.J. Morais A.H.F. Valentim R.A.M. Barreto E. miRNAs as biomarkers for early cancer detection and their application in the development of new diagnostic tools Biomed. Eng. Online 2021 20 21 10.1186/s12938-021-00857-9 33593374 PMC7885381

17. Backes C. Meese E. Keller A. Specific miRNA Disease Biomarkers in Blood, Serum and Plasma: Challenges and Prospects Mol. Diagn. Ther. 2016 20 509 518 10.1007/s40291-016-0221-4 27378479

18. Izzotti A. Carozzo S. Pulliero A. Zhabayeva D. Ravetti J.L. Bersimbaev R. Extracellular MicroRNA in liquid biopsy: Applicability in cancer diagnosis and prevention Am. J. Cancer Res. 2016 6 1461 27508091 PMC4969398

19. Shigeyasu K. Toden S. Zumwalt T.J. Okugawa Y. Goel A. Emerging role of microRNAs as liquid biopsy biomarkers in gastrointestinal cancers Clin. Cancer Res. 2017 23 2391 2399 10.1158/1078-0432.CCR-16-1676 28143873 PMC5433899

20. Yu D. Kim M. Xiao G. Hwang T.H. Review of biological network data and its applications Genom. Inform. 2013 11 200 210 10.5808/GI.2013.11.4.200 PMC3897847 24465231

21. Charitou T. Bryan K. Lynn D.J. Using biological networks to integrate, visualize and analyze genomics data Genet. Sel. Evol. 2016 48 27 10.1186/s12711-016-0205-1 27036106 PMC4818439

22. Barabási A.-L. Gulbahce N. Loscalzo J. Network medicine: A network-based approach to human disease Nat. Rev. Genet. 2011 12 56 68 10.1038/nrg2918 21164525 PMC3140052

23. Lee T.J. Yuan X. Kerr K. Yoo J.Y. Kim D.H. Kaur B. Eltzschig H.K. Strategies to Modulate MicroRNA Functions for the Treatment of Cancer or Organ Injury Pharmacol. Rev. 2020 72 639 667 10.1124/pr.119.019026 32554488 PMC7300323

24. Ahmed F. Ijaz B. Ahmad Z. Farooq N. Sarwar M.B. Husnain T. Modification of miRNA Expression through plant extracts and compounds against breast cancer: Mechanism and translational significance Phytomedicine 2020 68 153168 10.1016/j.phymed.2020.153168 31982837

25. Lindholm E.M. Ragle Aure M. Haugen M.H. Kleivi Sahlberg K. Kristensen V.N. Nebdal D. Børresen-Dale A.L. Lingjaerde O.C. Engebraaten O. miRNA expression changes during the course of neoadjuvant bevacizumab and chemotherapy treatment in breast cancer Mol. Oncol. 2019 13 2278 2296 10.1002/1878-0261.12561 31402562 PMC6763780

26. Arora A. MicroRNA targets: Potential candidates for indirect regulation by drugs Pharmacogenet. Genom. 2015 25 107 125 10.1097/FPC.0000000000000111 25590610

27. Cervena K. Novosadova V. Pardini B. Naccarati A. Opattova A. Horak J. Vodenkova S. Buchler T. Skrobanek P. Levy M. Analysis of MicroRNA Expression Changes During the Course of Therapy In Rectal Cancer Patients Front. Oncol. 2021 11 702258 10.3389/fonc.2021.702258 34540669 PMC8444897

28. Santiago F. Quality of Life in Chronic Venous Disease: Bridging the Gap Between Patients and Physicians Clin. Drug Investig. 2023 43 3 8 10.1007/s40261-023-01264-9 PMC10219877 37162624

29. Rabe E. Guex J.-J. Morrison N. Ramelet A.-A. Schuller-Petrovic S. Scuderi A. Staelens I. Pannier F. Treatment of chronic venous disease with flavonoids: Recommendations for treatment and further studies Phlebology 2013 28 308 319 10.1177/0268355512471929 23395842

30. Martinez-Zapata M.J. Vernooij R.W. Simancas-Racines D. Uriona Tuma S.M. Stein A.T. Moreno Carriles R.M.M. Vargas E. Bonfill Cosp X. Phlebotonics for venous insufficiency Cochrane Database Syst. Rev. 2020 11 Cd003229 10.1002/14651858.CD003229.pub4 33141449 PMC8094625

31. Imperatrice M. Cuijpers I. Troost F.J. Sthijns M.M.J.P.E. Hesperidin Functions as an Ergogenic Aid by Increasing Endothelial Function and Decreasing Exercise-Induced Oxidative Stress and Inflammation, Thereby Contributing to Improved Exercise Performance Nutrients 2022 14 2955 10.3390/nu14142955 35889917 PMC9316530

32. Magura J. Moodley R. Mackraj I. The effect of hesperidin and luteolin isolated from Eriocephalus africanus on apoptosis, cell cycle and miRNA expression in MCF-7 J. Biomol. Struct. Dyn. 2022 40 1791 1800 10.1080/07391102.2020.1833757 33050842

33. Wu R. Zhou S. Liu M. An H. Wang Z. Liu T. Clinical Significance of miR-21-5p in Predicting Occurrence and Progression of Uremic Vascular Calcification in Patients with End-Stage Renal Disease Yonsei Med. J. 2022 63 252 258 10.3349/ymj.2022.63.3.252 35184427 PMC8860934

34. Toro R. Pérez-Serra A. Mangas A. Campuzano O. Sarquella-Brugada G. Quezada-Feijoo M. Ramos M. Alcalá M. Carrera E. García-Padilla C. miR-16-5p Suppression Protects Human Cardiomyocytes against Endoplasmic Reticulum and Oxidative Stress-Induced Injury Int. J. Mol. Sci. 2022 23 1036 10.3390/ijms23031036 35162959 PMC8834785

35. Hua C.C. Liu X.M. Liang L.R. Wang L.F. Zhong J.C. Targeting the microRNA-34a as a Novel Therapeutic Strategy for Cardiovascular Diseases Front. Cardiovasc. Med. 2021 8 784044 10.3389/fcvm.2021.784044 35155600 PMC8828972

36. Wu J. Li X. Li D. Ren X. Li Y. Herter E.K. Qian M. Toma M.A. Wintler A.M. Sérézal I.G. MicroRNA-34 Family Enhances Wound Inflammation by Targeting LGR4 J. Investig. Dermatol. 2020 140 465 476.e411 10.1016/j.jid.2019.07.694 31376385

37. Biranvand A.S. Khosravi M. Esfandiari G. Poursaleh A. Hosseini-Fard S.R. Amirfarhangi A. Najafi M. Associations between miR-661, miR-1202, lncRNA-HOTAIR, lncRNA-GAS5 and MMP9 in differentiated M2-macrophages of patients with varicose veins Int. Angiol. 2018 37 451 456 10.23736/S0392-9590.18.04022-1 30558403

38. Huang X. Liu Z. Shen L. Jin Y. Xu G. Zhang Z. Fang C. Guan W. Liu C. Augmentation of miR-202 in varicose veins modulates phenotypic transition of vascular smooth muscle cells by targeting proliferator-activated receptor-γ coactivator-1α J. Cell. Biochem. 2019 120 10031 10042 10.1002/jcb.28287 30556158

39. Anwar M.A. Adesina-Georgiadis K.N. Spagou K. Vorkas P.A. Li J.V. Shalhoub J. Holmes E. Davies A.H. A comprehensive characterisation of the metabolic profile of varicose veins; implications in elaborating plausible cellular pathways for disease pathogenesis Sci. Rep. 2017 7 2989 10.1038/s41598-017-02529-y 28592827 PMC5462754

40. Ortega M.A. Fraile-Martínez O. García-Montero C. Álvarez-Mon M.A. Chaowen C. Ruiz-Grande F. Pekarek L. Monserrat J. Asúnsolo A. García-Honduvilla N. Understanding Chronic Venous Disease: A Critical Overview of Its Pathophysiology and Medical Management J. Clin. Med. 2021 10 3239 10.3390/jcm10153239 34362022 PMC8348673

41. Shannon P. Markiel A. Ozier O. Baliga N.S. Wang J.T. Ramage D. Amin N. Schwikowski B. Ideker T. Cytoscape: A software environment for integrated models of biomolecular interaction networks Genome Res. 2003 13 2498 2504 10.1101/gr.1239303 14597658 PMC403769

42. Chin C.H. Chen S.H. Wu H.H. Ho C.W. Ko M.T. Lin C.Y. cytoHubba: Identifying hub objects and sub-networks from complex interactome BMC Syst. Biol. 2014 8 (Suppl. 4) S11 10.1186/1752-0509-8-S4-S11 25521941 PMC4290687

43. Winterhalter C. Widera P. Krasnogor N. JEPETTO: A Cytoscape plugin for gene set enrichment and topological analysis based on interaction networks Bioinformatics 2014 30 1029 1030 10.1093/bioinformatics/btt732 24363376 PMC3967109

44. Astore C. Zhou H. Ilkowski B. Forness J. Skolnick J. LeMeDISCO is a computational method for large-scale prediction & molecular interpretation of disease comorbidity Commun. Biol. 2022 5 870 10.1038/s42003-022-03816-9 36008469 PMC9411158

45. Griffiths-Jones S. miRBase: The microRNA sequence database Methods Mol. Biol. 2006 342 129 138 10.1385/1-59745-123-1:129 16957372

46. Yu F. Li B. Sun J. Qi J. De Wilde R.L. Torres-de la Roche L.A. Li C. Ahmad S. Shi W. Li X. PSRR: A Web Server for Predicting the Regulation of miRNAs Expression by Small Molecules Front. Mol. Biosci. 2022 9 817294 10.3389/fmolb.2022.817294 35386297 PMC8979021

47. Bickerton G.R. Paolini G.V. Besnard J. Muresan S. Hopkins A.L. Quantifying the chemical beauty of drugs Nat. Chem. 2012 4 90 98 10.1038/nchem.1243 22270643 PMC3524573

48. Barrera-Vázquez O.S. Montenegro-Herrera S.A. Martínez-Enríquez M.E. Escobar-Ramírez J.L. Magos-Guerrero G.A. Selection of Mexican Medicinal Plants by Identification of Potential Phytochemicals with Anti-Aging, Anti-Inflammatory, and Anti-oxidant Properties through Network Analysis and Chemoinformatic Screening Biomolecules 2023 13 1673 10.3390/biom13111673 38002355 PMC10669844

49. Rathod N.B. Elabed N. Punia S. Ozogul F. Kim S.K. Rocha J.M. Recent Developments in Polyphenol Applications on Human Health: A Review with Current Knowledge Plants 2023 12 1217 10.3390/plants12061217 36986905 PMC10053535

50. Hewlings S.J. Kalman D.S. Curcumin: A Review of Its Effects on Human Health Foods 2017 6 92 10.3390/foods6100092 29065496 PMC5664031

51. Safa A. Bahroudi Z. Shoorei H. Majidpoor J. Abak A. Taheri M. Ghafouri-Fard S. miR-1: A comprehensive review of its role in normal development and diverse disorders Biomed. Pharmacother. 2020 132 110903 10.1016/j.biopha.2020.110903 33096351

52. Yan B. Guo Q. Fu F.J. Wang Z. Yin Z. Wei Y.B. Yang J.R. The role of miR-29b in cancer: Regulation, function, and signaling OncoTargets Ther. 2015 8 539 548 10.2147/ott.S75899 PMC4354468 25767398

53. Katoh M. Cardio-miRNAs and onco-miRNAs: Circulating miRNA-based diagnostics for non-cancerous and cancerous diseases Front. Cell Dev. Biol. 2014 2 61 10.3389/fcell.2014.00061 25364765 PMC4207049

54. Gross C. Yao X. Engel T. Tiwari D. Xing L. Rowley S. Danielson S.W. Thomas K.T. Jimenez-Mateos E.M. Schroeder L.M. MicroRNA-Mediated Downregulation of the Potassium Channel Kv4.2 Contributes to Seizure Onset Cell Rep. 2016 17 37 45 10.1016/j.celrep.2016.08.074 27681419 PMC5061042

55. Smalheiser N.R. Lugli G. Rizavi H.S. Torvik V.I. Turecki G. Dwivedi Y. MicroRNA expression is down-regulated and reorganized in prefrontal cortex of depressed suicide subjects PLoS ONE 2012 7 e33201 10.1371/journal.pone.0033201 22427989 PMC3302855

56. Wang K. Zhang D.L. Long B. An T. Zhang J. Zhou L.Y. Liu C.Y. Li P.F. NFAT4-dependent miR-324-5p regulates mitochondrial morphology and cardiomyocyte cell death by targeting Mtfr1 Cell Death Dis. 2015 6 e2007 10.1038/cddis.2015.348 26633713 PMC4720883

57. Hervé M. Ibrahim E.C. MicroRNA screening identifies a link between NOVA1 expression and a low level of IKAP in familial dysautonomia Dis. Model. Mech. 2016 9 899 909 10.1242/dmm.025841 27483351 PMC5007982

58. Mas-Capdevila A. Teichenne J. Domenech-Coca C. Caimari A. Del Bas J.M. Escoté X. Crescenti A. Effect of Hesperidin on Cardiovascular Disease Risk Factors: The Role of Intestinal Microbiota on Hesperidin Bioavailability Nutrients 2020 12 1488 10.3390/nu12051488 32443766 PMC7284956

59. Bian H. Zhou Y. Zhou D. Zhang Y. Shang D. Qi J. The latest progress on miR-374 and its functional implications in physiological and pathological processes J. Cell. Mol. Med. 2019 23 3063 3076 10.1111/jcmm.14219 30772950 PMC6484333

60. Di Mauro G. Frontini F. Torreggiani E. Iaquinta M.R. Caselli A. Mazziotta C. Esposito V. Mazzoni E. Libener R. Grosso F. Epigenetic investigation into circulating microRNA 197-3p in sera from patients affected by malignant pleural mesothelioma and workers ex-exposed to asbestos Sci. Rep. 2023 13 6501 10.1038/s41598-023-33116-z 37081052 PMC10119131

61. Ahn J. Lee H. Jung C.H. Ha S.Y. Seo H.D. Kim Y.I. Ha T. 6-Gingerol Ameliorates Hepatic Steatosis via HNF4α/miR-467b-3p/GPAT1 Cascade Cell. Mol. Gastroenterol. Hepatol. 2021 12 1201 1213 10.1016/j.jcmgh.2021.06.007 34139323 PMC8445893

62. Lu L. Wang X. Zhao H. Jiang F. Li Y. Yao Y. Shi C. Yang Y. MiR-291a/b-5p inhibits autophagy by targeting Atg5 and Becn1 during mouse preimplantation embryo development RSC Adv. 2019 9 9331 9341 10.1039/C9RA00017H 35517663 PMC9062091

63. Rider B.J. 5 Fluorouracil xPharm: The Comprehensive Pharmacology Reference Enna S.J. Bylund D.B. Elsevier New York, NY, USA 2007 1 5

64. Hantzidiamantis P.J. Awosika A.O. Lappin S.L. Physiology, Glucose StatPearls [Internet] StatPearls Publishing Treasure Island, FL, USA 2024 Available online: https://www.ncbi.nlm.nih.gov/books/NBK545201/ (accessed on 22 July 2024) 31424785

65. Manohar S. Leung N. Cisplatin nephrotoxicity: A review of the literature J. Nephrol. 2018 31 15 25 10.1007/s40620-017-0392-z 28382507

66. Liu Y. Yu S. Xing X. Qiao J. Yin Y. Wang J. Liu M. Zhang W. Ginsenoside Rh2 stimulates the production of mitochondrial reactive oxygen species and induces apoptosis of cervical cancer cells by inhibiting mitochondrial electron transfer chain complex Mol. Med. Rep. 2021 24 873 10.3892/mmr.2021.12513 34713297 PMC8569524

67. Bernardini L. Barbosa E. Charão M.F. Brucker N. Formaldehyde toxicity reports from in vitro and in vivo studies: A review and updated data Drug Chem. Toxicol. 2022 45 972 984 10.1080/01480545.2020.1795190 32686516

68. Reed C.E. Fenton S.E. Exposure to diethylstilbestrol during sensitive life stages: A legacy of heritable health effects Birth Defects Res. Part C Embryo Today Rev. 2013 99 134 146 10.1002/bdrc.21035 PMC3817964 23897597

69. Jałbrzykowska K. Chrzanowska A. Roszkowski P. Struga M. The New Face of a Well-Known Antibiotic: A Review of the Anticancer Activity of Enoxacin and Its Derivatives Cancers 2022 14 3056 10.3390/cancers14133056 35804828 PMC9264829

70. Zhou H. Xie Y. Baloch Z. Shi Q. Huo Q. Ma T. The effect of atorvastatin, 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor (HMG-CoA), on the prevention of osteoporosis in ovariectomized rabbits J. Bone Miner. Metab. 2017 35 245 254 10.1007/s00774-016-0750-2 27119523

71. Jiang Y. Shen X. Zhi F. Wen Z. Gao Y. Xu J. Yang B. Bai Y. An overview of arsenic trioxide-involved combined treatment algorithms for leukemia: Basic concepts and clinical implications Cell Death Discov. 2023 9 266 10.1038/s41420-023-01558-z 37500645 PMC10374529

72. Le V.K.H. Pham T.P.D. Truong D.H. Delivery systems for vorinostat in cancer treatment: An updated review J. Drug Deliv. Technol. 2021 61 102334 10.1016/j.jddst.2021.102334

73. Yaşar P. Ayaz G. User S.D. Güpür G. Muyan M. Molecular mechanism of estrogen-estrogen receptor signaling Reprod. Med. Biol. 2017 16 4 20 10.1002/rmb2.12006 29259445 PMC5715874

74. Ping Y.H. Lee H.C. Lee J.Y. Wu P.H. Ho L.K. Chi C.W. Lu M.F. Wang J.J. Anticancer effects of low-dose 10-hydroxycamptothecin in human colon cancer Oncol. Rep. 2006 15 1273 1279 10.3892/or.15.5.1273 16596197

75. Sritharan S. Sivalingam N. A comprehensive review on time-tested anticancer drug doxorubicin Life Sci. 2021 278 119527 10.1016/j.lfs.2021.119527 33887349

76. Sharifi-Rad J. Herrera-Bravo J. Kamiloglu S. Petroni K. Mishra A.P. Monserrat-Mesquida M. Sureda A. Martorell M. Aidarbekovna D.S. Yessimsiitova Z. Recent advances in the therapeutic potential of emodin for human health Biomed. Pharmacother. 2022 154 113555 10.1016/j.biopha.2022.113555 36027610

77. Abdulfattah A.Y. Tajuddin S. Akkari N. Elsayed O.I. Graham-Hill S. A Rare Case of Bicalutamide-Induced Severe Congestive Heart Failure in a Patient With Advanced Prostate Cancer Cureus 2024 16 e60298 10.7759/cureus.60298 38872672 PMC11175563

78. Bouyahya A. El Omari N. Bakha M. Aanniz T. El Menyiy N. El Hachlafi N. El Baaboua A. El-Shazly M. Alshahrani M.M. Al Awadh A.A. Pharmacological Properties of Trichostatin A, Focusing on the Anticancer Potential: A Comprehensive Review Pharmaceuticals 2022 15 1235 10.3390/ph15101235 36297347 PMC9612318

79. Combs G.F. Chapter 9—Vitamin C The Vitamins 4th ed. Combs G.F. Academic Press San Diego, CA, USA 2012 233 259

80. Felippe M.J.B. Chapter 66—Immunotherapy Equine Infectious Diseases 2nd ed. Sellon D.C. Long M.T. W.B. Saunders St. Louis, MO, USA 2014 584 597.e585

81. Liang C. Qiao G. Liu Y. Tian L. Hui N. Li J. Ma Y. Li H. Zhao Q. Cao W. Overview of all-trans-retinoic acid (ATRA) and its analogues: Structures, activities, and mechanisms in acute promyelocytic leukaemia Eur. J. Med. Chem. 2021 220 113451 10.1016/j.ejmech.2021.113451 33895500

82. Majeed M. Hakeem K.R. Rehman R.U. Mistletoe lectins: From interconnecting proteins to potential tumour inhibiting agents Phytomed. Plus 2021 1 100039 10.1016/j.phyplu.2021.100039

83. Fan G. Wei X. Xu X. Is the era of sorafenib over? A review of the literature Ther. Adv. Med. Oncol. 2020 12 1758835920927602 10.1177/1758835920927602 32518599 PMC7252361

84. Saleem K. Franz J. Klem M.L. Yabes J.G. Boyiadzis M. Jones J.R. Shaikh N. Lontos K. Second primary malignancies in patients with haematological cancers treated with lenalidomide: A systematic review and meta-analysis Lancet Haematol. 2022 9 e906 e918 10.1016/S2352-3026(22)00289-7 36354020

85. Kirstine W.V. Galbally I.E. Ethanol in the Environment: A Critical Review of Its Roles as a Natural Product, a Biofuel, and a Potential Environmental Pollutant Crit. Rev. Environ. Sci. Technol. 2012 42 1735 1779 10.1080/10643389.2011.569874

86. Lyseng-Williamson K.A. Fenton C. Docetaxel: A review of its use in metastatic breast cancer Drugs 2005 65 2513 2531 10.2165/00003495-200565170-00007 16296875

87. Pourmadadi M. Aslani A. Hassan D. Sani A. Rahdar A. Medina D.I. Abdouss M. Romanholo Ferreira L.F. Recent advancements in the targeted delivery of Gemcitabine: Harnessing nanomedicine for enhanced cancer therapy OpenNano 2023 13 100177 10.1016/j.onano.2023.100177

88. Vogel C.L. Cobleigh M.A. Tripathy D. Gutheil J.C. Harris L.N. Fehrenbacher L. Slamon D.J. Murphy M. Novotny W.F. Burchmore M. Efficacy and safety of trastuzumab as a single agent in first-line treatment of HER2-overexpressing metastatic breast cancer J. Clin. Oncol. 2002 20 719 726 10.1200/JCO.2002.20.3.719 11821453

89. Zanwar A.A. Badole S.L. Shende P.S. Hegde M.V. Bodhankar S.L. Chapter 21—Anti-oxidant Role of Catechin in Health and Disease Polyphenols in Human Health and Disease Watson R.R. Preedy V.R. Zibadi S. Academic Press San Diego, CA, USA 2014 267 271

90. Jeong S.P. Sharma N. An S.S.A. Role of Calcitriol and Vitamin D Receptor (VDR) Gene Polymorphisms in Alzheimer’s Disease Int. J. Mol. Sci. 2024 25 4806 10.3390/ijms25094806 38732025 PMC11084202

91. Kolatorova L. Vitku J. Suchopar J. Hill M. Parizek A. Progesterone: A Steroid with Wide Range of Effects in Physiology as Well as Human Medicine Int. J. Mol. Sci. 2022 23 7989 10.3390/ijms23147989 35887338 PMC9322133

92. Condrat C.E. Thompson D.C. Barbu M.G. Bugnar O.L. Boboc A. Cretoiu D. Suciu N. Cretoiu S.M. Voinea S.C. miRNAs as Biomarkers in Disease: Latest Findings Regarding Their Role in Diagnosis and Prognosis Cells 2020 9 276 10.3390/cells9020276 31979244 PMC7072450

93. Chakrabortty A. Patton D.J. Smith B.F. Agarwal P. miRNAs: Potential as Biomarkers and Therapeutic Targets for Cancer Genes 2023 14 1375 10.3390/genes14071375 37510280 PMC10378777

94. Zalewski D. Chmiel P. MicroRNA expression biomarkers of chronic venous disease Curr. Issues Pharm. Med. Sci. 2022 35 21 26 10.2478/cipms-2022-0005

95. Raucci A. Macrì F. Castiglione S. Badi I. Vinci M.C. Zuccolo E. MicroRNA-34a: The bad guy in age-related vascular diseases Cell. Mol. Life Sci. 2021 78 7355 7378 10.1007/s00018-021-03979-4 34698884 PMC8629897

96. Guo Y. Xing L. Chen N. Gao C. Ding Z. Jin B. Total flavonoids from the Carya cathayensis Sarg. leaves inhibit HUVEC senescence through the miR-34a/SIRT1 pathway J. Cell. Biochem. 2019 120 17240 17249 10.1002/jcb.28986 31106472

97. Niederer F. Trenkmann M. Ospelt C. Karouzakis E. Neidhart M. Stanczyk J. Kolling C. Gay R.E. Detmar M. Gay S. Down-regulation of microRNA-34a* in rheumatoid arthritis synovial fibroblasts promotes apoptosis resistance Arthritis Rheum. 2012 64 1771 1779 10.1002/art.34334 22161761

98. Ducasse E. Giannakakis K. Speziale F. Midy D. Sbarigia E. Baste J.C. Faraggiana T. Association of primary varicose veins with dysregulated vein wall apoptosis Eur. J. Vasc. Endovasc. Surg. 2008 35 224 229 10.1016/j.ejvs.2007.08.015 17936650

99. Mikuła-Pietrasik J. Uruski P. Aniukiewicz K. Sosińska P. Krasiński Z. Tykarski A. Książek K. Serum from Varicose Patients Induces Senescence-Related Dysfunction of Vascular Endothelium Generating Local and Systemic Proinflammatory Conditions Oxid. Med. Cell. Longev. 2016 2016 2069290 10.1155/2016/2069290 27994710 PMC5141312

100. Chen L.B. An Z. Zheng H.K. Wang X.P. Shan R.T. Mao C.Y. Zhang W.Q. MicroRNA-34c suppresses proliferation of vascular smooth muscle cell via modulating high mobility group box protein 1 J. Clin. Lab. Anal. 2020 34 e23293 10.1002/jcla.23293 32157741 PMC7370740

101. Kim S. Han J. Ahn Y.H. Ha C.H. Hwang J.J. Lee S.E. Kim J.J. Kim N. Protective Role of miR-34c in Hypoxia by Activating Autophagy through BCL2 Repression Mol. Cells 2022 45 403 412 10.14348/molcells.2022.2010 35611688 PMC9200661

102. Harati R. Hafezi S. Mabondzo A. Tlili A. Silencing miR-202-3p increases MMP-1 and promotes a brain invasive phenotype in metastatic breast cancer cells PLoS ONE 2020 15 e0239292 10.1371/journal.pone.0239292 33002044 PMC7529272

103. Papazafiropoulou A. Tentolouris N. Matrix metalloproteinases and cardiovascular diseases Hippokratia 2009 13 76 82 19561775 PMC2683462

104. Dbouk H.A. Weil L.M. Perera G.K. Dellinger M.T. Pearson G. Brekken R.A. Cobb M.H. Actions of the protein kinase WNK1 on endothelial cells are differentially mediated by its substrate kinases OSR1 and SPAK Proc. Natl. Acad. Sci. USA 2014 111 15999 16004 10.1073/pnas.1419057111 25362046 PMC4234555

105. Shiiba M. Saito K. Yamagami H. Nakashima D. Higo M. Kasamatsu A. Sakamoto Y. Ogawara K. Uzawa K. Takiguchi Y. Interleukin-1 receptor antagonist (IL1RN) is associated with suppression of early carcinogenic events in human oral malignancies Int. J. Oncol. 2015 46 1978 1984 10.3892/ijo.2015.2917 25738940

106. Tavernier N. Sicheri F. Pintard L. Aurora A kinase activation: Different means to different ends J. Cell Biol. 2021 220 e202106128 10.1083/jcb.202106128 34287649 PMC8298103

107. Al-Nbaheen M.S. Effect of Genetic Variations in the ADIPOQ Gene on Susceptibility to Type 2 Diabetes Mellitus Diabetes Metab. Syndr. Obes. 2022 15 2753 2761 10.2147/DMSO.S377057 36101664 PMC9464438

108. Kim J.H. Kim H. Hwang K.H. Chang J.S. Park K.S. Cha S.K. Kong I.D. WNK1 kinase is essential for insulin-stimulated GLUT4 trafficking in skeletal muscle FEBS Open Bio 2018 8 1866 1874 10.1002/2211-5463.12528 PMC6212645 30410865

109. Henriques A.F.A. Matos P. Carvalho A.S. Azkargorta M. Elortza F. Matthiesen R. Jordan P. WNK1 phosphorylation sites in TBC1D1 and TBC1D4 modulate cell surface expression of GLUT1 Arch. Biochem. Biophys. 2020 679 108223 10.1016/j.abb.2019.108223 31816312

110. Jaykumar A.B. Plumber S. Barry D.M. Binns D. Wichaidit C. Grzemska M. Earnest S. Goldsmith E.J. Cleaver O. Cobb M.H. WNK1 collaborates with TGF-β in endothelial cell junction turnover and angiogenesis Proc. Natl. Acad. Sci. USA 2022 119 e2203743119 10.1073/pnas.2203743119 35867836 PMC9335306

111. Fejfarová V. Roztočil K. Svědínková A. Čermáková H. Dubský M. Bém R. Jirkovská A. Wosková V. Němcová A. Skibová J. The relationship between chronic venous insufficiency and diabetes mellitus Int. Angiol. 2017 36 90 91 10.23736/S0392-9590.16.03736-6 28124879

112. Zhang Y. Yang Y. He X. Yang P. Zong T. Sun P. Sun R.C. Yu T. Jiang Z. The cellular function and molecular mechanism of formaldehyde in cardiovascular disease and heart development J. Cell. Mol. Med. 2021 25 5358 5371 10.1111/jcmm.16602 33973354 PMC8184665

113. Augenreich M. Stickford J. Stute N. Koontz L. Cope J. Bennett C. Ratchford S.M. Vascular dysfunction and oxidative stress caused by acute formaldehyde exposure in female adults Am. J. Physiol. Heart Circ. Physiol. 2020 319 H1369 H1379 10.1152/ajpheart.00605.2020 33064555

114. Wang S. Han Q. Wei Z. Wang Y. Deng L. Chen M. Formaldehyde causes an increase in blood pressure by activating ACE/AT1R axis Toxicology 2023 486 153442 10.1016/j.tox.2023.153442 36706861

115. Bergan J.J. Pascarella L. Schmid-Schönbein G.W. Pathogenesis of primary chronic venous disease: Insights from animal models of venous hypertension J. Vasc. Surg. 2008 47 183 192 10.1016/j.jvs.2007.09.028 18178472

116. Raju S. Knight A. Lamanilao L. Pace N. Jones T. Peripheral venous hypertension in chronic venous disease J. Vasc. Surg. Venous Lymphat. Disord. 2019 7 706 714 10.1016/j.jvsv.2019.03.006 31196767

117. Perrin M. Ramelet A.A. Pharmacological Treatment of Primary Chronic Venous Disease: Rationale, Results and Unanswered Questions Eur. J. Vasc. Endovasc. Surg. 2011 41 117 125 10.1016/j.ejvs.2010.09.025 21126890

118. Santillán-Aguayo E. Rivera Sánchez J.d.J. Carbajal-Robles V. Duarte-Acuña J. Piña-Avilés F.A. Ramírez-Berumen M.V. Chronic venous insufficiency symptoms and its potential causes: Are we doing it right? Rev. Mex. Angiol. 2021 49 57 66 10.24875/RMA.20000046

119. Awata W.M.C. Alves J.V. Costa R.M. Bruder-Nascimento A. Singh S. Barbosa G.S. Tirapelli C.R. Bruder-Nascimento T. Vascular injury associated with ethanol intake is driven by AT1 receptor and mitochondrial dysfunction Biomed. Pharmacother. 2023 169 115845 10.1016/j.biopha.2023.115845 37951022

120. Serra R. Gallelli L. Perri P. De Francesco E.M. Rigiracciolo D.C. Mastroroberto P. Maggiolini M. de Franciscis S. Estrogen Receptors and Chronic Venous Disease Eur. J. Vasc. Endovasc. Surg. 2016 52 114 118 10.1016/j.ejvs.2016.04.020 27220899

121. Kalfert D. Ludvikova M. Pesta M. Ludvik J. Dostalova L. Kholová I. Multifunctional Roles of miR-34a in Cancer: A Review with the Emphasis on Head and Neck Squamous Cell Carcinoma and Thyroid Cancer with Clinical Implications Diagnostics 2020 10 563 10.3390/diagnostics10080563 32764498 PMC7459507

122. Fu J. Imani S. Wu M.Y. Wu R.C. MicroRNA-34 Family in Cancers: Role, Mechanism, and Therapeutic Potential Cancers 2023 15 4723 10.3390/cancers15194723 37835417 PMC10571940

123. Takashima Y. Kawaguchi A. Iwadate Y. Hondoh H. Fukai J. Kajiwara K. Hayano A. Yamanaka R. miR-101, miR-548b, miR-554, and miR-1202 are reliable prognosis predictors of the miRNAs associated with cancer immunity in primary central nervous system lymphoma PLoS ONE 2020 15 e0229577 10.1371/journal.pone.0229577 32101576 PMC7043771

124. Lohcharoenkal W. Li C. Das Mahapatra K. Lapins J. Homey B. Sonkoly E. Pivarcsi A. MiR-130a Acts as a Tumor Suppressor MicroRNA in Cutaneous Squamous Cell Carcinoma and Regulates the Activity of the BMP/SMAD Pathway by Suppressing ACVR1 J. Investig. Dermatol. 2021 141 1922 1931 10.1016/j.jid.2021.01.028 33766507

125. Sagar S.K. miR-106b as an emerging therapeutic target in cancer Genes. Dis. 2022 9 889 899 10.1016/j.gendis.2021.02.002 35685464 PMC9170583

126. Larrue R. Fellah S. Boukrout N. De Sousa C. Lemaire J. Leboeuf C. Goujon M. Perrais M. Mari B. Cauffiez C. miR-92a-3p regulates cisplatin-induced cancer cell death Cell Death Dis. 2023 14 603 10.1038/s41419-023-06125-z 37704611 PMC10499794

127. Shao N. Xue L. Wang R. Luo K. Zhi F. Lan Q. miR-454-3p Is an Exosomal Biomarker and Functions as a Tumor Suppressor in Glioma Mol. Cancer Ther. 2019 18 459 469 10.1158/1535-7163.MCT-18-0725 30413650

128. Song F. Yang Y. Liu J. MicroRNA-548ac induces apoptosis in laryngeal squamous cell carcinoma cells by targeting transmembrane protein 158 Oncol. Lett. 2020 20 69 10.3892/ol.2020.11930 32863902 PMC7436114

129. Du X. Li Y. Lian B. Yin X. microRNA-128-3p inhibits proliferation and accelerates apoptosis of gastric cancer cells via inhibition of TUFT1 World J. Surg. Oncol. 2023 21 47 10.1186/s12957-023-02906-0 36797791 PMC9936645

130. Sala A.C.A. Crespo A.C.F. Stelini R.F. Leite O. Moraes A.M. Velho P. Souza E.M. Cintra M.L. Chronic Venous Insufficiency as a Predisposing Factor for Basal Cell Carcinoma on Legs Ann. Vasc. Surg. 2020 68 185 191 10.1016/j.avsg.2020.04.053 32422291

131. Serra R. Buffone G. Miglietta A.M. Abonante S. Giordano V. Renne M. Lugarà M. de Franciscis S. Breast cancer and venous disease: A retrospective cohort study Ann. Vasc. Surg. 2013 27 762 766 10.1016/j.avsg.2012.10.020 23809843

132. Rahmani A.H. Babiker A.Y. Anwar S. Hesperidin, a Bioflavonoid in Cancer Therapy: A Review for a Mechanism of Action through the Modulation of Cell Signaling Pathways Molecules 2023 28 5152 10.3390/molecules28135152 37446814 PMC10343639

133. Barrera-Vázquez O.S. Escobar-Ramírez J.L. Santiago-Mejía J. Carrasco-Ortega O.F. Magos-Guerrero G.A. Discovering Potential Compounds for Venous Disease Treatment through Virtual Screening and Network Pharmacology Approach Molecules 2023 28 7937 10.3390/molecules28247937 38138427 PMC10745828

134. Han Q.F. Liu J.H. Mo D.G. The Effects of Dexamethasone on Cardiovascular Disease: Friend or Foe? Drugs Ther. 2023 37 847 848 10.1007/s10557-022-07375-3 36001199

135. Ospina-Quintero L. Jaramillo J.C. Tabares-Guevara J.H. Ramírez-Pineda J.R. Reformulating Small Molecules for Cardiovascular Disease Immune Intervention: Low-Dose Combined Vitamin D/Dexamethasone Promotes IL-10 Production and Atheroprotection in Dyslipidemic Mice Front. Immunol. 2020 11 743 10.3389/fimmu.2020.00743 32395119 PMC7197409

136. Wang H. Yao X. Huang K. Zhang J. Xiao J. Guo J. Wei D. Xiang B. Low-dose dexamethasone in combination with luteolin improves myocardial infarction recovery by activating the antioxidative response Biomed. Pharmacother. 2022 151 113121 10.1016/j.biopha.2022.113121 35605295

137. Yang C. Zhu Q. Chen Y. Ji K. Li S. Wu Q. Pan Q. Li J. Review of the Protective Mechanism of Curcumin on Cardiovascular Disease Drug Des. Dev. Ther. 2024 18 165 192 10.2147/DDDT.S445555 38312990 PMC10838105

138. Chen X. Zhong Y. Wang S. Xu S. Chen J. Cheng X. Yang X. Reversine inhibits proliferation and induces apoptosis of human osteosarcoma cells through targeting MEK1 J. Bone Oncol. 2024 46 100601 10.1016/j.jbo.2024.100601 38706714 PMC11063522

139. Lips D.J. Bueno O.F. Wilkins B.J. Purcell N.H. Kaiser R.A. Lorenz J.N. Voisin L. Saba-El-Leil M.K. Meloche S. Pouysségur J. MEK1-ERK2 signaling pathway protects myocardium from ischemic injury in vivo Circulation 2004 109 1938 1941 10.1161/01.CIR.0000127126.73759.23 15096454

140. Khachigian L.M. The MEK-ERK-Egr-1 axis and its regulation in cardiovascular disease Vasc. Pharmacol. 2023 153 107232 10.1016/j.vph.2023.107232 37734428

Figures, Scheme and Tables