Role and predictive value of microRNAs 204 and 210 in the diagnosis of pulmonary arterial hypertension and the distinction between idiopathic, systemic sclerosis, and schistosomiasis-associated pulmonary arterial hypertension

Background Pulmonary arterial hypertension is most of the time diagnosed late in the course of the disease and necessitates right cardiac catheterization which is an invasive and costly tool. MicroRNAs have a role in the pathogenesis of pulmonary hypertension, systemic sclerosis, and schistosomiasis and their dosages are easy and non-expensive. Therefore, determining their levels in the blood may be helpful in detecting PAH and differentiating its idiopathic form from those caused by systemic sclerosis and schistosomiasis. Purpose of the study To evaluate the role of microRNA (miR) 204 and miR-210 in the diagnosis


Background
Pulmonary hypertension (PH) is a complicated disease.PH is divided into types according to The World Health Organization (WHO) [1].Nowadays, the diagnosis of PH is more easily compared to the past [2,3].
Despite the significant progress in the diagnosis and treatment of PAH, mortality is still high, mainly in cases of terminal stages of the disease (WHO Group IV) [1,5].PAH affects the lung with many types of pulmonary artery cells, such as pulmonary artery endothelial cells (PAECs), pulmonary artery smooth muscle cells (PASMCs), and pulmonary artery adventitious fibroblasts (PAAFs) [6].
Idiopathic PAH (IPAH) is a rare disease with a poor prognosis with a mortality rate of around 50% within 5 years [7].IPAH is characterized by progressively increased mPAP and PVR and there is no specific treatment till now [8].IPAH leads to a decrease in cardiac output due to affection of the pulmonary circulation which leads to constriction of the blood vessels of the lungs.Vasoconstriction, vascular remodeling, and thrombosis lead to an increase in the PVR and this leads to remodeling of the pulmonary vessels and PH.The right ventricular systolic pressure is elevated to overcome the increased PVR in order to maintain the cardiac output [9].
Systemic sclerosis complex (SSc) is a complex disease that is characterized by skin involvement with the involvement of another organ, especially the lungs and the heart [10].
SSc is associated with increased morbidity and mortality.One of the life-threatening complications is PAH.The incidence of PAH in SSc is nearly 5 to 15 cases per million and is more common in women than men [11].PAH's prevalence in SSc is around 7% to 19% and is responsible for about 30% of death [12].In patients with SSc-PAH, the symptoms are usually non-specific (dyspnea, exercise intolerance, and fatigue), and due to that, the diagnosis is unfortunately in the late stages of the disease with poor outcome [13].
Schistosoma-associated pulmonary arterial hypertension (Sch-PAH) is a serious complication of chronic schistosomiasis infection, with a poor prognosis and high mortality [14].
Schistosomiasis affects more than 230 million people in the world, where 10% develop hepatosplenic disease [15].Of these, 5% may have PAH.Current studies assume that Sch-PAH is likely to result from a combination of several factors such as egg embolization into the lung through portosystemic shunts and this leads to the development of PAH.Eggs might result in either direct mechanical obstruction or activation of an immune response with vascular remodeling [16].
Early diagnosis of PH is difficult because signs are nonspecific.Dyspnea on exertion is the commonest symptom but is usually neglected with the appearance of more symptoms such as syncope or chest pain in the late stages of the disease.As a result, there is a delay of more than 2.8 years with worsening symptoms of PH [17].Approximately 75% of patients with PH have advanced symptoms at diagnosis with significant pulmonary vascular remodeling and right ventricular failure.Nowadays, the best tool in the diagnosis of PH is right heart catheterization but because it is an invasive tool, there is a need to develop new non-invasive diagnostic tools, such as biomarkers to avoid the hazards and costs of RHC [18].
Several factors are reported to be related to the pathogenesis of PH such as miRNAs which were considered to have an important role in the pathogenesis of PH. miR-NAs are small endogenous non-coding RNA molecules of ~ 21 to 25 nt [19,20].A single miRNA can regulate hundreds of proteins or genes, and multiple miRNAs can regulate one protein [20].
Nowadays it has been assumed that many miRNAs have a vital role in PAH.In addition, miRNAs could be detected in blood samples and analyzed by polymerase chain reaction test (PCR) [21].This provides an easy and inexpensive method for the diagnosis of PAH [22].So, miRNAs may be used as a good biomarker for the diagnosis of PAH [23].
MiRNAs have a role in several biological processes like cell survival, proliferation, and differentiation [24].The miRNAs are transcribed and processed into various primary miRNAs (pri-miRNAs), which are then converted into miRNAs before leaving the nucleus and processed into mature miRNAs.It is then incorporated into the RNA-induced silencing complex (RISC) and binds to the 3′ untranslated region of the mRNA, resulting in degradation of the mRNA or inhibition of its translation [25], causing a decrease in the expression of the target gene.Nearly 1000 miRNAs can regulate more than 30% of human genes [26].Circulating miRNAs can be used as biomarkers for the diagnosis of many cardiovascular diseases.Identifying the molecular pathways provides a molecular basis for the imbalance between a hyperproliferative and apoptosis-resistant phenotype, mainly in the three major pulmonary artery cells involved, including PAEC, PASMC, and PAAF [27].Left side assessment included the following:

Purpose of the study
• LV, LA dimensions, and functions • Regional wall motions.
The right-sided assessment included the following: • RV, RA dimensions and functions.

Chest X-ray
Plain chest X-ray was done as a work-up to rule out group 3 PH.

Statistical analysis
SPSS version 18.0 for Windows from SPSS, Inc. IL, USA, was used for data entry and analysis.Qualitative data presented by numbers and percentages was assessed by using chi-square.Continuous data were expressed as mean and standard deviation.We compared between two means by using t-test and more than two means were assessed by using ANOVA.Mann-Whitney test was used when data are not normally distributed and used for comparing two or more independent samples.Receiver-operating characteristic (ROC) analysis was used to obtain the area under the curve (AUC) and the corresponding 95% CI.The maximum cut-off point was measured which corresponds to the highest Youden index for each biomarker.The AUC for each significant biomarker was assessed by using the ROC curve.The sensitivity, specificity, positive predictive values (PPV), and negative predictive values (NPV) were calculated for the identified cut-off points for each biomarker.P value < 0.05 is considered significant and < 0.01 is considered highly significant.

Results
Sixty patients with PAH and 30 apparently healthy controls were included in the study.
-In comparison between the PAH group and controls as regards age, sex, and body mass index (BMI), there was no significant difference (Tables 1 and 2).
• Plasma expression of miR-204 is downregulated in patients with PAH with a highly significant difference between PAH and control (P = 0.003) while plasma   expression of miR-210 was upregulated in patients with PAH with a highly significant difference between PAH and control (P < 0.001) (Table 3, Figs. 1 and 2).• ROC curve was done in order to evaluate the predictive value of miR-204 and miR-210 in the diagnosis of PAH.The cut-off predictive value of miR-204 was ≤ 0.15 µM with 70% sensitivity, and 85% specificity with AUC (0.749).The cut-off predictive value of miR-210 was ≥ 1.16 µM with 93.33% sensitivity and 85% specificity with AUC (0.917).These values showed that the miR-210 has excellent performance for prediction of PAH (Table 4, Fig. 3).
• In the comparison between IPAH, SSc-PAH, and Sch-PAH regarding RHC, echocardiographic, and clinical parameters, we found that there was no significant difference between the 3 diseases (Table 5).• In the comparison between IPAH, SSc-PAH, and Sch-PAH regarding miR-204, we found that miR-204 could not differentiate between the 3 diseases (p = 0.263) while regarding miR-210 there was a statistically significant difference between SSc-PAH and IPAH (P = 0.012) and between SSc-PAH and Sch-PAH (p = 0.035) (Table 6, Fig. 4).

Discussion
PAH is a rare but progressive cardiopulmonary disease characterized by elevated PVR due to pulmonary vasoconstriction and pulmonary vascular remodeling which finally leads to right heart failure and death.To date, the RHC remains the best diagnostic method for the diagnosis of PH.However, it is an invasive and expensive procedure for patients with PH.Therefore, the development of a non-invasive and less expensive method may be important for the diagnosis of PH [30].
Nowadays some markers are used to diagnose PH like brain natriuretic peptide (BNP) and N-terminal pro-Btype natriuretic peptide (NT-proBNP) but they could not be of significant value in the early stages of the disease.MiRNAs are non-coding, small RNA molecules circulating in blood, tissues, and plasma.They are dysregulated in PAH and play an important role in the disease progress [31].It has been assumed that many miRNAs play a vital role in PAH.In addition, miRNAs could be detected in blood samples and analyzed by PCR [21].This provides an easy and inexpensive diagnostic method [22].So, miR-NAs may be used as a good biomarker for the diagnosis of PAH [23].
In our study, we studied two miRNAs (204 and 210) that might be potential biomarkers in the diagnosis of patients with PAH.
We found that miR-204 was downregulated in patients with PAH with a highly significant difference between PAH and control (P = 0.003) so it might be a useful tool to diagnose patients with PAH.
Also, we found that miR-204 with a cut-off value ≤ 0.15 µM might help in early diagnosis patients with PAH with 70% sensitivity, 85% specificity, and AUC (0.749).
Several studies were performed and showed that miR-204 was downregulated in patients with PH where platelet-derived growth factor (PDGF), endothelin-1, and angiotensin II can reduce the expression of miR-204, accelerate proliferation, and lead to resistance to apoptosis through the activation of the signal transducer and activator of transcription 3-T cell nuclear factor (STAT-NFAT) pathway via upregulation of tyrosine-protein phosphatase 2 (SHP2) protein, hypoxia-inducible factor 1-alpha (HIF-1α) and Runt-related transcription factor 2 gene (RUNX2) in PASMCs which leads finally to PASMC proliferation and higher resistance to apoptosis [32][33][34].
Liu et al. found that decreased expression of miR-204 by hypoxia in a rat model of chronic hypoxia and human PAECs played a dichotomous role in promoting autophagy and attenuating endothelial-mesenchymal transition, which finally improved hypoxia-induced PH [35].
Sarrion et al. reported that blood levels of miR-204 decreased around 5 times in patients with IPAH as compared to the control [36].Also, Courboulin et al. found that downregulation of miR-204 is associated with PAH in 13 PAH patients and found that activation of STAT3 inhibited miR-204 expression and that miR-204 directly targets SHP2 expression, thus by upregulating SHP2 and downregulating miR-204-activation of Src kinase and NFAT leading to cell proliferation and ultimately PH [37].
In our study miR-204 failed to differentiate between idiopathic PAH, SSc-PAH, and Sch-PAH.
Regarding miR-210, we found that it was upregulated in patients with PAH with a highly significant difference between it and the control (< 0.001) so it might be a useful tool in diagnosis of PAH.Also, we found that miR-210 with a cut-off value ≥ 1.16 µM has 93.33% sensitivity, 85.0% specificity, and AUC (0.917) which means that it might be an excellent tool for early diagnosis of PAH.
Scientists investigated potential interactions between miR-210 and mitogen-activated protein phosphatase kinase 1 (MKP-1) and its effect on cell proliferation in hypoxic human PASMCs [38].
Gou D et al. found that miR-210 is induced in human PASMCs and mouse lungs via the HIF-1 pathway and hypoxia and upregulation of miR-210 leads to suppression of E2F transcription factor 3 (E2F3) and inhibit apoptosis, resulting in hyperplasia of PASMCs [39].Also, Jingsi Zhao et al. also found that the transport of miR-210 into pulmonary vascular endothelial cells leads to PH [40].
In humans, hypoxia potentiates miR-210, which suppresses the expression of the iron-sulfur cluster-assembly protein ISCU1/2 and controls mitochondrial metabolic functions [41].Iron homeostasis and mitochondrial dysfunction are major factors in the development of PH [42].
Also in our study, we found that regarding miR-210 there is a significant difference between SSc-PAH and IPAH (P = 0.012) and between SSc-PAH and Sch-PAH (P = 0.035) but it failed to differentiate between IPAH and Sch-PAH so miR-210 might be a useful tool to diagnose SSc-PAH and differentiate it from IPAH and Sch-PAH.
The mortality and therapeutic response in SSc-PAH are worse than idiopathic pulmonary arterial hypertension (IPAH) and might partially be due to its multifaceted underlying mechanisms and the multisystem nature [43].
TGF-ß signaling plays an important role in the pathogenesis of SSc-PAH.TGF-β signal activation leads to the development of PAH, and TGF-ß signaling regulates several processes like cellular angiogenesis and proliferation [44].Gilbane AJ et al. found reduced BMPR2 protein in patients with SSc-PAH, and raised proteasomal degradation of BMPR2 in a relevant mouse model and that that TGF-β might affect signaling of BMP by degradation of its receptor and promote the PAH susceptibility in SSc, which might provide a unifying mechanism from other types of PAH [45].
In recent studies, miRNAs were assumed as novel biomarkers in SSc fibrosis with the ability to modulate several fibrotic-related genes [46].The expression of profibrotic and anti-fibrotic miRNAs in SSc may play an important role in the disease [47].
Several studies have found a close interaction between miRNAs and the progress of SSc-PAH.Many studies found that microRNA-21 (miR-21) might play an important role in PH [48], where in terms of TGF-β signaling, miR-suppress the anti-fibrotic signaling molecule SMAD7 expression and promotes pro-fibrotic activity in spinal fibroblasts [49].It also provides a pro-fibrotic effect in SSc dermal fibroblasts via Smad7 [50,51].Kawashita et al. found that miR-29a has decreased expression in patients with SSc, which may have a role in the early diagnosis of SSc-PAH [52].MiR-29a levels have been associated with increased right ventricular systolic pressure in SSc patients, which may play a role in the pathogenesis of PH.
Moreover, a reduced level of miR-29a-3p was found in pulmonary adventitial fibroblasts with activation of hypoxia or cultured pulmonary adventitial fibroblasts with upregulation of HIF-1 which leads to PASMC proliferation and PH [53].
Christmann RB found that miR-155 may play a role in the development of pulmonary fibrosis in SSc.Upregulation of miR-155 in patients with SSc-associated interstitial lung disease is associated with impaired respiratory function and increased pulmonary fibrosis [54].

Fig. 1 Fig. 2
Fig. 1 Comparison between the PAH group and controls regarding miR-204

Fig. 3
Fig. 3 ROC of Predictive value of miR-204 and miR-210 in PAH

Fig. 4
Fig. 4 Comparison between IPAH, SSc-PAH, and Sch-PAH regarding miR-210 All the studied groups were subjected to the following: Wood units in the absence of other causes of precapillary PH like lung diseases, CTEPH.4.Transthoracic echocardiographyTransthoracic echocardiography by using the Acuson Siemens machine was performed in the left lateral decubitus position in all patients.
age range from 22 to 60 years, 16 males, 14 females All patients were documented to have PAH by RHC.We excluded patients with uncertain diagnoses, patients with end-stage PH, and patients with malignancy or any other systemic diseases.tors with patients in the supine position after proper catheter calibration.Pressure waveforms from the RA, PA, and pulmonary capillary wedge pressure (PCWP) positions were measured at end-expiration.Patients considered to present PAH had a mPAP > 20 mmHg, PCWP ≤ 15 mmHg, and a PVR > 2

Table 1
Comparison between the PAH group and controls regarding age, sex, and BMI

Table 2
Descriptive statistics of clinical, echocardiographic, and RHC findings in PAH patients

Table 3
Comparison between the PAH group and controls regarding miR-204 and miR 210 P < 0.01 highly significant (HS)

Table 4
Predictive values of miR-204 and miR-210 in PAH

Table 5
Comparison between IPAH, SSc-PAH, and Sch-PAH regarding RHC, echocardiographic and clinical parameters