Gene networks and canonical pathways associated with a favorable blood pressure response after CPAP treatment: the HIPARCO score post hoc analysis


 Background: Several studies report an association between microRNAs (miRNAs) and hypertension or cardiovascular disease. In a previous study, we observed a cluster of three miRNAs (miR-378a-3p, miR-100-5p, and miR-486-5p) functionally associated with the cardiovascular system that predicted a favorable blood pressure response to continuous positive airway pressure (CPAP) treatment in patients with resistant hypertension (RH) and obstructive sleep apnea (OSA) (HIPARCO score). However, little is known regarding the molecular mechanisms underlying this. Thus, the aim of the present study was to perform a post hoc analysis to investigate the genes, functions and pathways related to the previously found HIPARCO-score miRNAs. Methods: We performed an enrichment analysis using Ingenuity Pathway Analysis. The genes potentially associated with the miRNAs were filtered based on their confidence level. Particularly for cardiovascular disease, only the genes regulated by at least two of the miRNAs were studied. Results: We observed that the miRNAs studied regulate 200-249 molecules associated with several functions and diseases, including extracranial solid tumors and abdominal neoplasms, among others. The cardiac hypertrophy and NF-kB signaling pathways were identified as the cardiovascular pathways most influenced by these three miRNAs. Conclusions: Considering this, the mechanisms by which CPAP treatment decreases the blood pressure in OSA patients with RH could be related to these pathways. Further investigations will be necessary to confirm these findings, contributing to the elucidation of new therapeutic targets in patients that do not respond to CPAP treatment.

Gene networks and canonical pathways associated with a favorable blood pressure response after CPAP treatment: the HIPARCO score post hoc analysis in the middle-aged population (9,29). This disorder is caused by the collapse of the upper airway during sleep, which promotes intermittent hypoxemia and hypercapnia. These events are usually accompanied by cortical arousals during the night, leading to daytime somnolence and poor quality of life. The repetitive cycles of upper airway collapse during sleep induce the activation of various pathways related to endothelial dysfunction, oxidative stress, sympathetic activation, in ammation, hypercoagulability, and metabolic dysregulation (13,22). This explains, at least in part, the relationship between OSA and atherosclerosis. Basic research and epidemiological and clinical data support the concept that OSA has a role in the initiation or progression of several cardiovascular diseases (14,27).
Continuous positive airway pressure (CPAP) is the rst-choice treatment for sleep apnea. Although CPAP treatment does not demonstrate effectiveness for secondary cardiovascular prevention (17), observational studies show a lower incidence of cardiovascular disease (CVD) in patients treated with CPAP (16). Additionally, treatment with CPAP showed effectiveness in controlling blood pressure in patients with resistant hypertension (RH), which is of extreme importance, considering that increased blood pressure is the main factor for CVD development (4,8). However, despite good CPAP compliance, substantial variability exists in its effects. This increases the interest and necessity to develop tools to identify patients who could bene t from this treatment in terms of blood pressure reduction.
MicroRNAs (miRNAs) are small RNAs that inhibit protein-coding messenger RNAs (mRNAs) through translational repression and degradation. These molecules regulate a wide range of biological processes, including development, differentiation, proliferation, metabolism and apoptosis (5). Accordingly, miRNAs are linked with the pathogenesis of human diseases, such as cancer, viral infections, metabolic disorders and cardiovascular diseases. Considering this, miRNAs are interesting diagnostic and prognostic biomarkers (7,15).
Our research group described the existence of a singular cluster of circulating miRNAs that predicts blood pressure responses to CPAP treatment in patients with RH and OSA (29). This speci c expression pro le gives a quantitative score parameter, the HIPARCO-score, which identi es those patients with RH and OSA who are more likely to exhibit a favorable blood pressure response to CPAP. This approach constitutes the rst validated precision medicine tool for RH management in OSA patients. However, the biological mechanisms that underlie this outcome are still unknown. The identi cation of genes, functions, and pathways related to this molecular signature will improve the understanding of the role of OSA in the pathogenesis of cardiovascular diseases, ultimately bene ting OSA patients with RH who do not present a favorable blood pressure response after CPAP treatment. Considering this, the aim of the present study was to perform a post hoc analysis to investigate the genes, functions and pathways related to the previously found HIPARCO-score miRNAs.

Study population and miRNA analysis
The selection of miRNAs was based on a study by Sánchez-de-la-Torre et al (29). Brie y, the study was conducted with patients diagnosed with RH and OSA from the HIPARCO study (NCT00616265), which was carried out in 24 hospitals in Spain. Patients with RH and OSA who used CPAP for more than 4 hours per night were selected to determine the responder patients in relation to blood pressure (BP) levels and therefore establish whether there was a miRNA pro le associated with a positive response to CPAP treatment.
To identify this pro le, patients were divided into training and validation sets with two different groups regarding their response to CPAP treatment: responder (BP change > 4.5 mmHg) or nonresponder (BP change < 4.5 mmHg) groups. Human cardiovascular system-speci c miRNA arrays (84 miRNAs; Qiagen) were used with the blood plasma of the patients to identify differentially expressed miRNAs. Seven differentially expressed miRNAs were found, of which three miRNAs (miR-378-3p, miR-438-5p, and miR-100-5p) were included in a logistic regression model for the training set. These three miRNAs had the highest statistical associations and were linked to an average favorable BP response to CPAP treatment. We simpli ed the model's use by producing a simple numeric score (the HIPARCO-score, ranging from 0 to 6) that predicts the probability of a favorable BP response to adherent CPAP use. The model showed high discrimination, with an area under the curve (AUC) of 0.88. This model was validated in a validation set with an AUC of 0.92. The miRNAs miR-378-3p, miR-438-5p, and miR-100-5p were chosen for in silico analysis to investigate the genes, functions and pathways related to the HIPARCO-score miRNAs.
This study was conducted with permission to use the patients' records and the con dentiality was maintained (CEIC number 48/2008. Consorci Hospital Universitari General de València).

Data analysis
The overall design and data analysis work ow are presented in Fig. 1. We performed enrichment analysis using Ingenuity Pathway Analysis (IPA) (Ingenuity Systems, Redwood City, CA) to link the miRNAs with their mRNA target genes. This platform provides a literature-curated analysis of networks, biological processes, canonical pathways, diseases and functions associated with miRNAs and target genes. The IPA miRNA Target Filter tool was used to identify the genes regulated by each miRNA. This software identi es a list of potentially regulated genes based on four databases (TargetScan, miRecords, Ingenuity Knowledge Base and TarBase). The genes potentially associated with the miRNAs were ltered based on their con dence level, and only experimentally observed and highly predicted target genes were used in the analysis (Fig. 4).
The associations between the predicted miRNA target genes and networks, canonical pathways, diseases and functions were analyzed. We rst established the mRNAs regulated by these miRNAs, and then, from the list of mRNAs/genes, the canonical pathways were examined. Different diseases and associated pathways were also explored, especially cardiovascular-related ones (for this, only the genes regulated by at least two of the miRNAs were studied).

Canonical pathway analysis
The HIPARCO-score molecular signature comprises three miRNAs (miR-378a-3p, miR-100-5p, and miR-486-5p). The diseases and functions most signi cantly associated with these miRNAs are summarized in Table 1. The studied miRNAs regulate 200-249 molecules in each disease or function, including digestive organ tumors, extracranial solid tumors, abdominal neoplasms, nonhematologic cancers, solid tumors, and carcinomas. Regarding the canonical pathways (Table 1), we observed that these miRNAs target 8-11 genes in each of the processes in which they are involved. In axonal guidance signaling, for example, 11 genes are targeted by these miRNAs, while 10 genes are targeted in glucocorticoid receptor signaling and molecular mechanisms of cancer.

Cardiovascular-targeted canonical pathway analysis
An in-depth study was carried out to investigate the cardiovascular-related functions and canonical pathways signi cantly altered by at least two of the miRNAs studied ( Table 2). The major cardiovascular functions associated with these miRNAs are related to the development of blood vessels and cardiovascular tissues. We observed 30, 26 and 18 molecules altered in angiogenesis, vasculogenesis and cardiogenesis, respectively (p-value < 0.05). To focus on the most representative canonical pathways related to cardiovascular disease, we studied the cardiac hypertrophy and nuclear factor kappa beta (NF-kB) signaling canonical pathways. In the cardiac hypertrophy signaling canonical pathway, miR-100 regulates the expression of mammalian target of rapamycin (mTOR), and together with miR-378a, it regulates insulin-like growth factor 1 (IGF1R). miR-378a-3p also affects the expression of the growth factor receptor-bound protein 2 (GRB2) and interleukin 6 receptor (IL-6R). Moreover, miR-486 interferes with the regulation of the expression of eukaryotic translation initiation factor 4E (eIF4E), p70S6 kinase (p70S6K) and the complex TAK1/TAB1 (Fig. 1). The three miRNAs also participate in the regulation of nuclear factor of activated T-cells (NFAT). miR-100-5p and miR-378a-3p jointly regulate the expression of leukemia inhibitory factor (LIF) and IGF1R, while miR-486 regulates transforming growth factor-β-activated kinase 1 (TAK1). In addition, miR-378a-3p alters the expression of the glycoprotein 130 (GP130) dimer and SHC-GRB2-SOS complex (Fig. 2).
Regarding the NF-kB signaling canonical pathway, miR-486-5p and miR-378a-3p regulate the expression of TAK1 and tumor necrosis factor receptor-associated factor 6 (TRAF6), respectively, leading to an alteration in the activation of the NF-kB pathway (Fig. 3). This ultimately leads to an alteration in the mechanisms of cell survival and cell proliferation.

Discussion
In the present study, we performed an enrichment analysis using IPA to observe the pathways regulated by the HIPARCO-score miRNAs (miR-378a-3p, miR-100-5p and miR-486-5p), a set of miRNAs that identify patients more likely to exhibit a favorable BP response to CPAP. Our main objective was to investigate the main biological functions regulated by these three miRNAs that are involved in the favorable response to CPAP treatment in patients with OSA and RH.
We observed the contribution of miR-378a-3p, miR-100-5p and miR-486-5p in the regulation of a variety of genes and pathways, especially in those related to cardiovascular function and cancer. Speci cally, we observed a role for these miRNAs in heart hypertrophy, heart failure, apoptosis, and heart tissue injury. This nding is corroborated by a variety of studies in the literature demonstrating an association between miRNAs and cardiovascular disease and hypertension (18,25). Similarly, studies have shown a strong relation between OSA and these events. Therefore, it has been reported that OSA is associated with hypertension and with hypertension-associated end-stage organ diseases such as stroke, coronary heart disease, and arrhythmia (33). Moreover, previous studies suggest that OSA has a role in the initiation and progression of several cardiovascular diseases (28). Considering this, the effect of CPAP in RH patients could be mediated by these miRNAs through their actions on cardiovascular-associated genes and functions. In fact, we observed that the studied miRNAs are associated with the NF-kB signaling pathway, which is known to upregulate the transcription of genes involved in endothelin signaling (23). Accordingly, several NF-kB binding sites were identi ed in the endothelin gene promoter region (10). Thus, the mechanism by which CPAP treatment decreases BP may be associated with the involvement of these miRNAs with the NF-kB signaling pathway, ultimately affecting the endothelin signaling. Further studies will be necessary to con rm this.
Cardiac failure is often associated with prolonged and maladaptive cardiac hypertrophy (11,21). Considering that cardiomyocytes lose their ability to divide soon after birth, cardiac hypertrophy is an important adaptive response to maintain or increase the cardiac output of the organism (19). We observed that miR-378a-3p, miR-100-5p and miR-486-5p are involved in the regulation of several molecules related to activation pathways that lead to a hypertrophic response at the cellular level. For instance, the miR-378a-3p-mediated induction of the cytokine receptor GP130 activates the MAPK, PI3K and signal transducer and activator of transcription 3 (STAT3) pathways, which leads to the activation of genes involved in hypertrophy and survival pathways (32). Consequently, the translation of various signals through the GRB2-SOS complex and Akt activation that targets mTOR also occurs. mTOR speeds up the process of protein synthesis by activating its downstream targets p70S6K and eIF4E, resulting in protein synthesis, cell growth and cell proliferation. In turn, these kinases mediate cellular responses to stress, such as DNA damage and nutrient deprivation (26). Interestingly, Wen et al demonstrated that intermittent hypoxia, which is one of the main features of OSA, led to the inhibition of mTOR phosphorylation and activation of AMPK phosphorylation, inducing cellular apoptosis (31). On the other hand, a variety of studies reported an association between cardiac hypertrophy and the severity of OSA (20). Considering these ndings, it is possible to observe the role of the studied miRNAs in the cardiovascular-related pathways that are also affected by OSA. In addition, it is important to address that RH may lead to cardiac hypertrophy (6). Thus, the studied miRNAs associated with a favorable response to CPAP treatment could be involved in the regulation of such adverse outcome in the long-term. This highlights the importance of these miRNAs not only as biomarkers but also as possible therapeutic targets. This relationship should be further explored in future studies.
miR-378a-3p, miR-100-5p and miR-486-5p also seem to regulate the effect of proin ammatory cytokines in beta cells via NF-kB (2,24). miR-378a regulates TAK1, a kinase that mediates the signal transduction induced by TGF-beta and bone morphogenetic protein (BMP), and controls a variety of cell functions, including transcription regulation and apoptosis. Additionally, in response to interleukin-1 (IL-1), TAK1 forms a kinase complex that includes TRAF6 (also regulated by miR-486), MAP3K7P1/TAB1 and MAP3K7P2/TAB2; this complex is required for the activation of NF-kB(1). Therefore, these miRNAs modify NF-kB activity, which is pivotal for activating or preventing the overstimulation of the toll-like receptor (TLR) pathway (2). On the other hand, rapid reoxygenation at the end of apneas/hypopneas leads to the production of free radicals, inducing oxidative stress and the upregulation of NF-kB. In addition, there is evidence indicating that CPAP treatment reduces the levels of in ammatory mediators, such as interleukin-6, tumor necrosis factor-α, and C-reactive protein (3). Thus, there is considerable compatibility between the molecules and pathways affected by the studied miRNAs and those affected by OSA or CPAP treatment regarding in ammatory mechanisms.
A substantial number of studies have reported the oncogenic and tumor suppressing roles of miRNAs. Additionally, several miRNAs are markers for the early diagnosis of cancer (12,30). In fact, the present study suggests a role for the studied miRNAs in a variety of cancer-related pathways. This leads to a possibility for which there is still no evidence that the phenotype of good CPAP responders in terms of BP might be associated with other characteristics, such as different prognosis in tumor-related diseases.
It is important to note that there are still considerable limitations in miRNA studies, and more investigations are needed to con rm the role of miR-378a-3p, miR-100-5p, and miR-486-5p in cardiovascular diseases. Considering that these miRNAs were measured in plasma, no distinction was made between miRNAs derived from exosomes and those derived from other events, such as cell death.
Thus, the results discussed herein should be interpreted with caution. Regardless, the observed signi cant change in the circulating pro le of miRNAs is su cient as a biomarker to be interpreted by itself.

Conclusions
We observed the contribution of miR-378a-3p, miR-100-5p and miR-486-5p in the regulation of a variety of genes and pathways, especially in those related to cardiovascular function and cancer. The cardiac hypertrophy and NF-kB signaling pathways were identi ed as the cardiovascular pathways most in uenced by these three miRNAs. Considering this, the mechanisms by which CPAP treatment decreases the blood pressure in OSA patients with RH could be related to these pathways. Further investigations in different populations, as well as an evaluation of their functional impacts on putative target genes and pathways will be necessary to con rm these ndings. Improved knowledge of the pathways altered by these 3 miRNAs will ultimately lead to a better understanding of the molecular mechanisms underlying lowered BP levels after CPAP treatment. In addition, this will contribute to the elucidation of new therapeutic targets in patients that do not respond to CPAP treatment.   miRNA target genes in the cardiac hypertrophy signaling canonical pathway: miR-100-5p directly regulates mTOR. Additionally, miR-100-5p and miR-378a-3p directly modulate the IGF1 membrane receptor. miR-378a-3p also regulates the membrane receptor of IL-6 and GRB2 at the cytoplasmic level. miR-486-5p has an indirect effect on eIF4E, p70S6K and on the TAK1/TAB1 complex. miRNA target genes in the NFAT pathway: miR-100-5p and miR-378a-3p directly regulate the IGF1 membrane receptor and LIF. miR-378a-3p acts on the formation of the SCH-GRB2-SOS complex at the cytoplasmic level. In addition, this miRNA is directly involved in the activity of the GP130 dimer. miR-486-5p is indirectly implicated in the regulation of TAK1.

Figure 4
Flowchart of the study