Pulmonary arterial hypertension (PAH) is a relatively rare disease, with an estimated global incidence of 15–50 instances per million individuals [1]. However, it is more frequent in some groups, such as those with congenital heart disease (CHD). The increased blood flow and systemic pressure in the pulmonary circulation of patients with CHD put this group at higher risk of PAH [2]. According to the Indonesian registry, PAH develops in 66.9% of patients with CHD, and the most prevalent type of CHD is secundum atrial septal defect (ASD) [3]. Although it may occur in people of any age, PAH is typically identified in patients aged 30–60 years, and women are more likely than men to be diagnosed [1].

Patients and healthcare providers both suffer several difficulties because of the complicated nature of PAH. Through developments in medical care, the prognosis for PAH has improved recently; however, survival and quality of life are still significantly affected [4]. The 5-year survival rate of approximately 70% underscores the need for early and accurate detection and the development of effective biomarkers for monitoring and prognosis [5,6].

A particular type of endogenous noncoding RNAs known as circular RNAs (circRNAs) has now become possible biomarkers for several diseases owing to their stability and tissue-specific expression patterns [7]. CircRNAs have a closed-loop shape, giving them more strength and resistance to degradation than linear RNAs [8]. The identification of particular circRNAs dysregulated in PAH raises the potential of these molecules as biomarkers for early diagnosis and treatment [9–11].

CircRNAs might play a role in a wide range of biological processes [12]. CircRNAs serve an intriguing role in signaling networks, microRNA sponging, gene expression modulation, and cellular function regulation [13]. In PAH, circRNAs have been implicated in the pathogenesis of pulmonary vascular remodeling, a key feature of the disease. They regulate gene expression and protein translation through mechanisms such as microRNA sponging and binding with RNA-binding proteins [14]. For example, in chronic obstructive pulmonary disease-associated pulmonary hypertension (COPD-PH), specific circRNAs such as hsa_circ_0007608 and hsa_circ_0064656 are involved in cellular proliferation, migration, and endothelial-to-mesenchymal transition, which are critical processes in disease progression [15]. Gaining a thorough knowledge of the particular functions of circRNAs in the pathophysiology of PAH related to CHD can shed important light on the processes underlying the disease [16]. Furthermore, new diagnostic and prognostic approaches that can improve patient treatment, and outcomes might develop from investigating and analyzing circRNAs in patients with PAH related to CHD.

Despite these findings, the role of circRNAs in PAH, particularly in CHD, is still not comprehensively elucidated. Most studies have focused on idiopathic or other types of PAH, and PAH related to CHD received limited attention [17–19]. This review will fill this gap by explicitly focusing on the role and potential of circRNAs as biomarkers in PAH related to CHD. By synthesizing current research and highlighting novel circRNA targets, this review will provide a detailed overview of the current state of knowledge and identify future research directions. In summary, this review presents several potential circRNA targets as diagnostic biomarkers for PAH, discusses their biological functions, and addresses the challenges that need to be considered for their application in clinical settings. This review aimed to advance knowledge in this critical area and promote the development of effective diagnostic and prognostic strategies for PAH related to CHD in the context of circular RNA biomarkers.

A confluence of structural, hemodynamic, and genetic problems causes PAH related to CHD, a complex condition [2]. Often, this condition is caused by abnormalities in the left-to-right shunt that are not corrected. Poor functioning of the cells lining the blood vessels, anomalies in the usual signaling pathways inside the cells, and altered blood vessel structure are only a few of the complex interplay of factors that cause PAH related to CHD [20].

Genetic predisposition plays a significant role in PAH development in patients with CHD. Mutations in genes related to endothelium function, proliferation of smooth muscle cells, and fibroblast activity in the pulmonary vasculature have been linked to disease onset. Studies have identified numerous genetic variants linked to PAH susceptibility and development [21]. By modifying cyclooxygenase-2 (COX-2) expression levels, polymorphisms in COX-2 and associated miRNAs, such as rs20417, rs5275, miR-26b-5p, and miR-146a-5p, have been demonstrated to affect PAH development [22]. Furthermore, genetic studies have shown significant genetic heterogeneity in PAH, with disease development being linked to genes such as BMPR2, KCNK3, TBX4, and SOX17 [23,24]. These genetic mutations disrupt normal signaling pathways, leading to vascular remodeling and increased pulmonary vascular resistance (PVR), which are hallmarks of PAH.

Through several pathways, structural defects in CHD result in PAH. In CHD with left-to-right shunting, increased pulmonary blood flow results in endothelial dysfunction and vascular remodeling, which increases PVR and sets off a series of proliferative and inflammatory reactions in the pulmonary vasculature [2,25]. Large systemic-to-pulmonary shunts, such as those seen in untreated ventricular septal defects (VSDs) and ASDs aggravate PAH by contributing to the steady increase in pulmonary arterial pressure (PAP) caused by this aberrant vascular homeostasis [25]. Vascular resistance and PAP increase with age in specific CHD lesions, including VSDs, atrioventricular septal defects, and transposition of the great arteries. If these defects are not repaired early, the persistent high flow and pressure can lead to irreversible pulmonary vascular disease [2]. Unlike the tetralogy of Fallot with significant aortopulmonary collateral arteries, postoperative PAH related to CHD can be caused by thrombosis, hypoplasia, or pulmonary agenesis, underscoring the multiplicity of anatomical and hemodynamic characteristics that contribute to PAH [26].

The complex interaction of genetic predisposition changes in hemodynamics and vascular remodeling highlights the complicated pathophysiology of PAH in CHD [25,27]. Hemodynamic indices, including mean PAP (mPAP), PVR, right atrial pressure, and cardiac output index, are significantly associated with clinical outcomes in patients with PAH. Changes in these indices, particularly mPAP and PVR, have high explanatory power for predicting adverse clinical events and mortality, underscoring their importance in risk stratification and treatment monitoring, to consider more contributing elements. These can influence PAH onset and course and include oxidative stress, metabolic abnormalities, and epigenetic changes [21]. In addition, noncoding RNAs, histone acetylation, and DNA methylation are some epigenetic changes that can affect gene expression patterns and exacerbate pathways linked to PVR and vascular remodeling, indicating the involvement of both genetic and epigenetic mechanisms [28].

Currently, numerous biomarkers that have shown promise in diagnosing and monitoring PAH related to CHD have been identified [29]. These biomarkers include endothelin-1 [30], cardiac troponins [31], natriuretic peptides [32], and microRNAs [33]. These biomarkers can give important information for risk stratification, prognosis, and therapy response in patients with PAH related to CHD and are linked to the disease severity [34].

The N-terminal pro-B-type natriuretic peptide (NT-proBNP) has attracted interest as one of the essential indicators in this area [32]. This biomarker can give important information regarding the prognosis of patients with CHD and has shown the potential to assist in evaluating PAH severity. Jin et al identified Serum S-adenosyl methionine, guanine, and NT-proBNP as among the panel of serum biomarkers promising to differentiate children with PAH related to CHD from those with CHD alone. The combined use of these indicators showed good predictive accuracy, making them potentially helpful instruments for screening and diagnosis of patients with PAH related to CHD [35].

Another possible biomarker for PAH related to CHD is endothelin-1. Endothelin-1 levels in patients with PAH are high and are linked to both disease severity and higher PVR [30,36]. Other promising biomarkers in PAH related to CHD research are microRNAs, small noncoding RNAs involved in the control of gene expression, and cardiac troponins, markers of myocardial damage [33]. Studies have demonstrated that the development and progression of PAH are caused by the deregulation of specific pathways, notably the BMPR2 signaling pathway [37]. Biomarkers linked to these pathways might shed important light on the etiology of diseases and possible targets for treatments.

Biomarkers are still being used in clinical practice for PAH related to CHD, and more studies are required to confirm their efficacy and create common standards for their application. In this context, early diagnosis, prognosis prediction, and construction of customized treatment plans depend on discovering new biomarkers [38].

CircRNAs are an unusual type of endogenous noncoding RNAs. These molecules are formed without 3′ poly-(A) tails or 5′ cap structures by covalently closed-loop-like structures produced by back-splicing during pre-mRNA processing [13]. After their synthesis in the nucleus via back splicing, circRNAs are carried across the nuclear membrane. RNAs can circularize in both the nucleus and cytoplasm, with minor spliceosome perhaps mediating cytoplasmic biogenesis [39].

CircRNAs are involved in many biological processes, and diseases, including PAH related to CHD, have been linked to their aberrant control. These noncoding RNAs have been connected to controlling gene expression, serving as microRNA sponges, modifying protein functions, and affecting several cellular functions [40] (Fig. 1).

Among the most engaging roles of circRNAs is that of miRNA sponges [41]. MicroRNAs, small noncoding RNA molecules are essential to the post-transcriptional control of gene expression. MicroRNAs can be competitively bound by circRNAs, blocking their interactions with target mRNAs [42]. CircRNAs control the expression of miRNA target genes, affecting several biological pathways by sequestering miRNAs [43].

CircRNAs have also been reported to control transcription, affect alternative splicing, and interact with RNA-binding proteins to change cellular processes. CircRNAs have numerous modes of action, so they are significant participants in the complex networks of regulation seen in cells [39,44]. Because of their special stability and abundance, circRNAs in body fluids are compelling candidates for noninvasive diagnostic and prognostic tools [45,46]. Significantly, circRNAs demonstrate higher tissue specificity than mRNAs of their host genes. This indicates that circRNAs could provide superior analytical validity, encompassing better analytical specificity, robustness, reproducibility, and repeatability when utilized as biomarker molecules [47,48]. A recent study examining circRNA expression in 20 clinically relevant tissue samples highlighted the potential of circRNAs as disease biomarkers, reinforcing the findings that circRNA expression is highly tissue-specific [49]. These findings underscore the significant potential of circRNAs as biomarkers in various diseases, including cancers, neurodegenerative disorders, and cardiovascular diseases, highlighting their importance in advancing diagnostic and therapeutic strategies in clinical research.

Knowledge of the precise functions of circRNAs in endothelial dysfunction, vascular remodeling, and aberrant cellular signaling pathways will provide crucial new information on the pathophysiology of PAH related to CHD [45]. In recent research, numerous potential circRNAs demonstrated different expression in the PAH group than in the control group (Table 1). These circRNAs, such as has_circ_0003416 and has_circ_0068481, may be candidate biomarkers for early diagnosis and prognostic evaluation of PAH in CHD patients.

CircRNAs are detected and quantified using various methods, each with strengths and limitations. RNA sequencing (RNA-seq) is a fundamental technique for detecting circRNAs, employing computational tools to identify their distinctive back-splice junctions (BSJs). However, RNA-seq may yield false positives because of sequencing artifacts, requiring meticulous algorithmic management to reduce these errors [53]. In contrast, microarray technology improves circRNA detection and profiling by specifically targeting their unique BSJs. A major advantage of microarray technology is its ability to simultaneously examine the expression profiles of thousands of circRNAs, offering a comprehensive view of their differential expression across various conditions. A significant drawback is its reliance on predesigned probes targeting known back-splice sites, which restricts its ability to discover novel circRNAs. This limitation contrasts with RNA-seq, which can identify new circRNAs even without prior knowledge of their sequences [54].

A benchmarking study of 16 circRNA detection tools revealed high precision but variable sensitivities, highlighting the importance of tool selection and the potential benefit of combining multiple tools to enhance detection sensitivity while minimizing false discoveries [55]. For validation, RT-qPCR is commonly used; however, linear RNA contaminants can confound it. To address this, Rnase R treatment is employed to degrade linear RNAs; however, the protocol must be optimized to avoid partial degradation of circRNAs [56].

An advanced method, RT-hyperbranched rolling circle amplification (RT-HRCA), and its droplet digital variant (ddRT-HRCA), offer sensitive and quantitative detection of circRNAs, achieving low detection limits and high selectivity without the need for Rnase R enrichment [57]. Despite these advancements, traditional RT-qPCR remains limited by potential strand substitution during RT, which can hinder accurate quantification. In addition, the tissue-specific expression and evolutionary conservation of circRNAs necessitate precise and sensitive detection platforms to fully understand their biological roles and potential as disease biomarkers [58]. Overall, while RNA-seq and RT-qPCR are foundational techniques in circRNA research, emerging methods such as ddRT-HRCA provide promising alternatives for more accurate and sensitive circRNA quantification, addressing some of the limitations inherent in conventional approaches [53,56–58].

Furthermore, circRNAs are known for their stability owing to their closed-loop structures, which makes them resistant to exonuclease-mediated degradation. This stability makes circRNAs promising candidates for clinical biomarkers [46]. However, sample collection and processing can still affect such stability. For example, circRNAs remain stable during sample processing delays, showing minimal expression changes up to 48 h at 4°C, whereas linear mRNAs and lncRNAs degrade significantly after 12 h [59]. The half-life of circRNAs in blood samples is approximately 24.56  ±  5.2 h, longer than mRNAs (16.4 h) and other types of noncoding RNAs such as miRNAs (16.42 h) and lncRNAs (17.46 h) [60]. Although the half-life of circRNAs is better than that of linear RNAs, appropriate and professional methods must be employed for preserving blood samples to ensure the accuracy and reliability of test results. This is important in clinical trials where obtaining high-quality experimental samples is essential. Therefore, the effects of specimen analysis and processing must be carefully considered [61].

The feasibility of translating circRNA findings into clinical practice is supported by advanced techniques such as qRT-PCR and bioinformatics, which allow for precise detection and functional analysis of circRNAs. Moreover, the circRNA-miRNA-mRNA regulatory networks provide a comprehensive understanding of disease mechanisms, offering new avenues for targeted therapies [62]. Overall, the use of circRNAs as biomarkers in PAH related to CHD presents several advantages over existing biomarkers, including higher stability, specificity, and ability to reflect complex regulatory networks, making them highly feasible for clinical application and potentially improving current diagnostic and therapeutic strategies.

Prior research has demonstrated that circRNAs undergo substantial dysregulation and may serve as potential biomarkers in CHD [63,64], various types of PAH [11,18,19], and PAH related to CHD [9,10]. However, the identification of circRNAs in PAH related to CHD remains limited, highlighting a significant gap in the current understanding and diagnostic capabilities for this condition. Thus, a multifaceted approach is necessary to identify and validate a broader spectrum of circRNAs comprehensively. This could include high-throughput technologies, such as RNA-seq [65] or microarray [54], followed by rigorous validations using RT-qPCR and functional assays [66]. Integrating these findings with other omics data, such as transcriptomics, proteomics, and metabolomics, could provide a more holistic view of the molecular landscape of PAH related to CHD. In addition, leveraging advanced bioinformatics tools to analyze circRNA-miRNA-mRNA interaction networks could elucidate the regulatory mechanisms underlying PAH related to CHD, offering new therapeutic targets [39,53,67]. Encouraging collaborative research efforts and longitudinal studies is crucial in establishing the diagnostic accuracy and clinical relevance of these biomarkers.

In addition, future studies should not only look at the molecular underpinnings of circRNAs but also at developing noninvasive techniques for identifying and tracking circRNA biomarkers in PAH related to CHD. This might include investigating circRNA signatures in biofluids such as serum or blood, providing a less intrusive method of determining the disease state and course [68].

Reproducibility factors for each circRNA target must be further validated to ensure their reliability across different studies and clinical settings. To the best of our knowledge, in PAH, only has_circ_0003416 and has_circ_0068481 have been reported based on findings from two studies, although conducted from the same population. Moreover, validating these biomarkers in large, well-characterized cohorts of patients with CHD, both with and without PAH, would enhance their clinical relevance and utility [69]. A multi-omics approach could uncover complex regulatory networks and potential crosstalk across various molecular pathways, identifying new targets for therapeutic intervention and personalized treatment plans [70].

To overcome sample size constraints, establishing a sample storage system in conjunction with registries such as COHARD PH [3] is essential for advancing future research and understanding this rare, life-limiting disease. Consequently, each center must develop such registries and sample banks to manage and utilize valuable data and samples effectively. In addition, confounding factors that may influence circRNA expression must be considered. Although previous studies [9,11] have not found significant differences in sex, age, and body mass index between cases and controls, information regarding other comorbidities, such as diabetes or other diseases, was not provided. This gap underscores the need to apply strict criteria to minimize the effects of confounding factors.

The multifaceted roles of circRNAs in biological processes underscore their significance in molecular biology and their potential for therapeutic applications. The potential of circRNAs as diagnostic and therapeutic targets in the PAH related to CHD is a promising area for future exploration. It may pave the way for advancements in managing this complex disease. Although the AUC values for circRNAs as a diagnostic biomarker are high, confirmation and validation through longitudinal multicenter studies involving large, well-characterized patient cohorts and unbiased molecular phenotyping are still required.