N6-Methyladenosine Modifications in Pulmonary Hypertension

Pulmonary hypertension (PH) is a progressive type of cardiovascular disease characterized by a persistent increase in pulmonary artery resistance and pulmonary artery pressure, which leads to increased right ventricular load and right ventricular hypertrophy, and ultimately to death from right heart failure [1]. The World Symposium on Pulmonary Hypertension categorizes PH into five disease types based on their etiology and causative mechanisms: (1) pulmonary arterial hypertension (PAH), which encompasses idiopathic, hereditary, drug and toxic, and disease-related forms of PH; (2) PH due to left heart disease; (3) PH due to lung disease and hypoxia; (4) PH due to obstructive pulmonary artery disease; (5) unspecified and multifactorial hypertension [2].

PAH is a rare disease, affects fifteen to fifty individuals per million in the USA and Europe. Idiopathic, hereditary, and anorexin-induced PAH account for 52.6% of all PAH cases [3]. Despite the availability of numerous targeted drugs, there is still an unmet clinical need. These drugs primarily focus on dilating the pulmonary vasculature rather than reversing pulmonary vascular remodeling (PVR), which is crucial for treating the underlying cause of PH [1]. PH remains a progressive disease, with a 5-year survival rate of approximately 57% for patients, and lung transplantation remains the sole treatment option for end-stage disease [4]. Since PVR is primarily responsible for PH progression in the later stages, discovering new PVR regulatory mechanisms and determining the interactions between key regulatory genes and pathway is important to find effective therapeutic targets and enrich the theoretical basis of PH, thus providing important references for developing new therapeutic strategies.

In general, N6-methyladenosine (m6A), an internal RNA modification, is ubiquitous, particularly for eukaryotic mRNA. There is growing evidence that m6A RNA methylation is involved in practically every aspect of RNA processing, thereby implying its key regulatory role in protein synthesis and determining cell fate [5]. Numerous investigations in recent years have established the role of m6A modifications in PH, but the molecular mechanisms of m6A regulation of PH have to be further examined. In this review, we summarized m6A RNA methylation modifications, their biological functions, and effects on RNA metabolism, and discussed the role of m6A methylation in PH. The findings of this review may provide some therapeutic targets for the treatment of PH in the future.

RNA methylation refers to its epitranscriptomic modification, wherein methyl groups are added to the adenosine base by methyltransferases. These RNA methylation reactions are involved in many biological processes like protein synthesis, splicing, RNA editing, RNA stability, control of mRNA lifetime, and regression [6]. In eukaryotes, tRNA and rRNA undergo a significant amount of posttranscriptional modification called m1A, while m7G methylation controls microRNA (miRNA) biosynthesis, mRNA transcription, biological activity, and tRNA stability [7]. However, m6A is seen to be the most common post-transcriptional RNA modification in eukaryotes, wherein the sixth N atom of adenine is methylated. It is responsible for about 97.4% of all known RNA modifications [8]. Various internal RNA modifications are extensively spread in various types of RNAs, while m6A is also widely present in noncoding RNAs (ncRNA), such as circular RNA (circRNA), miRNA, and long ncRNA [9]. The m6A modifications are regulated by different m6A-associated enzymes called “writers” and “erasers” (responsible for methylation and demethylation steps, respectively) and “readers” (recognition) [10], as described in Figure 1. A crucial catalytic enzyme is m6A methyltransferase, commonly called “Writers” [11]. Methyltransferases like 3/14, Vir like m6A methyltransferase associated, Wilms tumor 1-associated protein, and RNA-binding motif protein 15/15B are important components of m6A methyltransferases, which constitute a synergistic complex for catalytic action. The mRNA methyltransferase complex also includes the Cbl proto-oncogene like 1 and zinc finger CCCH-type containing 13. A group of RNA-binding protein known as m6A “readers” identifies the m6A modifications and supports the different biological activities of the target RNAs. Three major classes of readers have been widely examined. These include the heterogeneous nuclear ribonucleoprotein (HNRNP) family, the YT521-B homology (YTH) structural domain family of proteins, and the insulin-like growth factor 2 mRNA-binding proteins. The YTH structural domain family of proteins consists of 5 members, i.e., YTH structural domain 1 (YTHDC1), YTH structural domain 2 (YTHDC2), YTH structural domain family protein 1 (YTHDF1), YTH structural domain family protein 2 (YTHDF2), and YTH structural domain family protein 3 (YTHDF3).

The m6A modification was first identified in cancer cells in the early 1970s. Earlier studies have reported that the m6A sites are significantly conserved, enriched in the shared motif, i.e., RRACH (R = G or A and H = A, C or U), and are easily detected in the 3′-untranslated region of mRNA, closer to the stop codon and in the inner ministerial exon. Several studies have confirmed the involvement of its regulators in different human diseases. For example, it was found that the overexpression of METTL3 promoted tumor angiogenesis and glycolysis in gastric cancer, which highlighted the role of METTL3 expression as a potential prognostic biomarker molecule and a therapeutic target [12]. Furthermore, Huang et al. [13] revealed the expression and potential role of the m6A RNA methylation regulators in chronic obstructive pulmonary disease. They also observed that the METTL3, FTO, and YTHDC2 expression levels were significantly related to the signaling pathways that promote the development of chronic obstructive pulmonary disease. Vascular endothelial cell dysfunction is a key factor in atherosclerosis. Reduced METTL14 expression was found to inhibit endothelial inflammation and atherosclerosis development, and thus the METTL14-dependent m6A modifications can be used as a potential target for treating atherosclerosis [14]. Under hypoxia-induced PH, the METTL3/YTHDF2/PTEN axis is involved in the hypoxia-induced pulmonary artery smooth muscle proliferation [15]. Many researchers, who investigated the m6A levels and methylation-related enzyme expression changes in the PH rats, stated that FTO and YTHDF1 may play a key role [16]. Yao et al. [17] revealed that myeloid ecotropic viral integration site 1 (MEIS1) overexpression inhibited the proliferation and migration of pulmonary artery smooth muscle cells (PASMCs) afforded by hypoxia. MEIS1-mediated hypoxia induced the proliferation and migration of PASMCs via the METTL14/MEIS1/p21 signaling pathway. In another study, m6A-modified GRAP mRNA was upregulated in PAH lung samples, cHx/Su-induced mouse models, and hypoxia-stimulated HPASMCs, while METTL14 and m6A-binding proteins like YTHDF2, were significantly increased in PAH. Furthermore, GRAP expression was consistently and negatively correlated with METTL14 and YTHDF2 [18]. All the above studies have strived to understand and highlight the importance of RNA epigenetics in PAH.

Many researchers have reported that m6A plays a key role in various processes related to RNA metabolism and processing, such as mRNA expression, precursor mRNA processing in the nucleus, and mRNA translation and degradation in the cytoplasm [19]. With regard to lipid metabolism, it has been shown that overexpression of METTL3 exacerbates the high-fat diet-induced hepatic metabolic disorders and insulin resistance, such that prolonging the half-life of the Lpin1 (which is a key regulator of the lipid metabolism process) by specific knockdown of METTL3, significantly alleviates insulin resistance and high-fat diet-induced metabolic disorders [20]. In another study, the researchers noted that the expression of the fatty acid synthase enzyme (FASN) and the intracellular lipid content were significantly reduced after the FTO gene knockdown. However, the FTO knockdown increased the m6A levels in the FASN mRNA, which further decreased the FASN mRNA expression levels through the m6A-mediated RNA decay [21]. In another study, the researchers noted that YTHDC1 promotes the biogenesis of the mature miR-30d, which inhibits the aerobic glycolysis pathway and suppresses pancreatic tumorigenesis by regulating GLUT1 and HK1 expression [22]. In a different study, the investigators confirmed that IMP2/insulin-like growth factor 2 mRNA-binding proteins acts as a key regulator of pancreatic β-cell proliferation and function, its deletion leads to reduced compensatory β-cell proliferation and function, and its post-transcriptional gene expression promotes susceptibility to type-2 diabetes [23].

As a dynamic and reversible process regulated by m6A regulators, there is now much evidence that m6A modification plays a crucial role in innate immunity, inflammation, and antitumor effects through interactions with different m6A regulators [24]. Therefore, an increasing number of studies are being conducted on the expression and significance of m6A modifications in the lungs and pulmonary vasculature. These studies have identified valuable biomarkers for diagnostic and prognostic assessments, while also suggesting novel therapeutic targets.

For example, it was found that the expression level of methyltransferase METTL3 was significantly increased in lung adenocarcinoma patients and lung cancer cells, and METTL3 could promote proliferation in different lung adenocarcinoma cell models [25]. The demethylase ALKBH1 was significantly upregulated in lung cancer cells and mouse lung cancer tissues after overexpression. Overexpression and silencing of ALKBH1 in cells played opposite roles in regulating m6A levels in RNA [26]. Furthermore, the binding protein YTHDF2 showed higher expression levels in lung adenocarcinoma, and its knockdown promoted migration and invasion of lung adenocarcinoma cells [27]. Hu et al. [28] discovered that the binding protein YTHDF1 enhances MAGED1 translation, promoting proliferation of PASMCs and the development of PH. This study identifies m6A RNA modification as a novel mediator of pathological changes in PASMCs and PH. Additionally, it has been found that YTHDF1 can alleviate pulmonary vascular changes, reduce right ventricular systolic pressure, and inhibit hypoxic PASMC proliferation by regulating Foxm6 translation [29].

PVR is regarded as a key factor that increases pulmonary vascular resistance and causes right heart failure in PH. The main cellular mechanism responsible for vascular remodeling in PH involves the excessive proliferation of vascular cells. Although there have been many studies on endothelial cell dysfunction, apoptosis, and proliferation of the smooth muscle cells during disease progression, the main drugs that are currently used for PH treatment are vasodilators, which are unable to prevent or reverse the thickening, stiffness, and excessive constriction of the developed vessel wall [30]. Hence, proper knowledge and understanding of the different intracellular mechanisms that drive cellular dysfunction have to be acquired. In recent years, numerous experimental studies have established a new epigenetic mechanism, called RNA methylation, which acts as a major factor that leads to cell proliferation and vascular remodeling in PH [31].

The results of in vitro: m6A is the most popular inner modification of mRNA. The methyltransferases involved in m6A modification like METTL3 and METTL14 and regulatory proteins such as the binding proteins YTHDF1 and YTHDF2 and the demethylase FTO have been studied to demonstrate their involvement in and regulation of pulmonary vascular cell hyperproliferation [25]. The m6A and YTHDF1 proteins were overexpressed in a few PH cell samples, derived from rodents and humans, called the hypoxic-treated PASMCs. However, YTHDF1 knockdown inhibited PASMCs proliferation, phenotypic transformation, and PH symptoms. MAGED1 was identified as an immediate target of m6A managing PH pathogenesis, and YTHDF1 recognized and promoted MAGED1 translation in a m6A-dependent manner. When the MAGED1 gene was silenced, it inhibited the hypoxia-induced proliferation of PASMCs by downregulating the proliferating cell nuclear antigens. Interestingly, YTHDF1 does not display any regulatory effect on MAGED1 protein expression in the METTL3-deficient PASMCs. This showed that METTL3 helps in regulating PH pathogenesis by YTHDF1 [28]. Studies have demonstrated that METTL3-driven m6A-modified PTEN mRNA promotes the development of hypoxic PH through the m6A-binding protein YTHDF2. Specifically, YTHDF2 recognizes m6A-modified PTEN mRNA and facilitates its degradation. This leads to reduced PTEN expression, which in turn promotes proliferation of PASMCs by activating the PI3K/Akt signaling pathway. These findings unveil a novel epigenetic regulatory mechanism of hypoxic PH, and targeting the METTL3/YTHDF2/PTEN axis offers a promising therapeutic approach for hypoxic PH [15]. In a separate study by Zhou et al. [32], it was discovered that knockdown of SETD2, a specific protein involved in H3K36me3 modification, in PASMCs resulted in the inactivation of hypoxia-induced PAH. Conversely, the expression of METTL14, a RNA modification protein, was elevated in PASMCs of mice with hypoxia-induced PAH. Furthermore, the expression of RNA modification decreased after SETD2-specific knockdown in PASMCs. Additionally, the absence of SETD2 in PASMCs was found to reduce METTL14 expression levels and total RNA methylation levels. In conclusion, SETD2-mediated H3K36me3 modification is implicated in the onset and development of hypoxia-induced PAH through METTL14-mediated m6A RNA modifications.

The experimental animal in vivo studies: Zeng et al. [16] conducted a study using MCT-PAH rats and discovered that changes in signaling pathways and metabolic molecules may be closely linked to methylation alterations in the coding region during the pathogenesis of PAH. M6A modifications may contribute to the development of PAH by regulating mRNA to influence protein translation or transcription of key enzymes involved in glycolysis, as well as through the TGF-β pathway and the regulation of inflammatory processes. Moreover, alterations in the expression of FTO and YTHDF1 were found to promote PAH, potentially by upregulating m6A methylation and enhancing mRNA translation associated with various molecular functions, biological processes, or KEGG pathways related to PAH. Furthermore, studies have utilized MeRIP-seq to analyze specific m6A methylation changes following exposure to hypoxia. Among these findings, the m6A methyltransferases METTL3 and METTL14 were found to be reduced, as were the demethylases FTO and ALKBH5 after postnatal hypoxia. METTL3 expression consistently showed lower levels in the hypoxic group compared to the control group. Additionally, varying numbers of hypermethylated and hypomethylation peaks were observed in 2 rats exposed to hypoxia and in adult rats subjected to postnatal hypoxia. These differentially associated genes are implicated in numerous respiratory-related physiological processes, such as the Notch signaling pathway and endothelial cell activation, suggesting that m6A is likely involved in the pathogenesis of PH. Moreover, mRNA methylation of the trichorhinophalangeal syndrome 1 (Trps1) gene was analyzed and found to regulate PH following postnatal hypoxia by affecting epithelial-mesenchymal transition in neonatal rats. This effect persists into adulthood, and further studies will determine the specific role of epithelial-mesenchymal transition in the development of hypoxia-induced PH [33].

Additionally, ncRNA has become an important regulator in PH development in recent years. It is the transcriptional product of genes that cannot be translated into proteins. CircRNAs are involved in many physiological and biological processes, while m6A modifications play a vital role in regulating circRNA mechanisms. According to one study, the enrichment of m6A in the circRNAs was remarkably reduced under hypoxic conditions, during the in vitro experiments. During hypoxia, m6A affects the circRNA-miRNA-mRNA network. For instance, in hypoxia-induced PH, the circXpo6 and circTmtc3 levels were downregulated [34], as summarized in Table 1. The abovementioned 2 m6A-circRNAs associated with hypoxia-mediated PH have been investigated in this study, where the pathological mechanisms and PH treatment strategies were also determined.

PH is a complex disease. Numerous studies have confirmed that development of PH is inextricably linked to genetic and environmental factors. RNA methylation, as one of the major epigenetic regulatory mechanisms, has made great progress in the study of its impact on PH. Targeting key regulators of m6A RNA methylation could facilitate future treatment of PH. Based on the majority of current studies, it can be concluded that targeting the inhibition of m6A methylation holds greater promise for exploring potential therapies for PH. However, many unknown RNA gene methylation processes can affect PH, as well as the mechanisms by which the key regulators of RNA methylation occur, or what clinical epigenetic therapies show effective reversibility or cure for PH. All these issues indicate the limitations of our present notion of PH and would be studied in the future. By studying the methylation patterns in PH patients and the RNA-related genes, and exploring the m6A methylation processes, some novel PH therapeutic targets can be determined and a few potential molecular markers can be identified in humans.

We declare that we have no financial and personal relationships with other people organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service, or company that could be construed as influencing the position presented in the review of the manuscript entitled “N6-methyladenosine (m6A) Modifications in Pulmonary Hypertension.”

This work was supported by the General Project of Jiangsu Provincial Health Commission (M2022055), the sixth “521 Project” of Lianyungang City (LYG06521202122), and National Natural Science Foundation of China (31871155).

Yu Xia, Yanyan Zhang, Jie Huang, Bing Chen, and Yanjiao Jiang performed experiments; Yun Liu and Zengxian Sun designed experiments, analyzed data, and supported the preparation of the manuscript; Yun Liu and Yu Xia led the investigation and wrote the manuscript.

Yu Xia and Yanyan Zhang contributed equally to this work.