Pulmonary Hypertension: Re-imagining Precision Diagnostics and Therapeutics via microRNA Biology

by Erin Schikowski and Stephen Y. Chan

Abstract
Pulmonary hypertension (PH) is a rare and progressive disease whose origins remain elusive, making it difficult to diagnose and treat early. Translational implementation of microRNA (miRNA) biology has the potential to accelerate diagnosis and risk stratification, as circulating levels of specific miRNAs correlate with disease severity and can be easily quantified by PCR. Using miRNA biomarkers may also allow us to subclassify patients into different, as yet undefined, disease subtypes based on precise molecular signatures, anticipated rate of disease progression, and likelihood of response to specific treatment. In addition to diagnostic applications, RNA species have also been considered for therapeutic use (e.g., siRNA, lncRNA). Along these lines, the therapeutic potential of miRNAs has been most often studied for prevention or reversal of PH. Targeted treatment involving inhibition of over‑expressed miRNAs as well as replacing under-expressed miRNAs with molecular mimics have shown promise in early experimental studies. Utilizing a systems or network biology approach could help identify entire miRNA circuits that can be targeted for a more robust therapeutic effect. However, important challenges remain in developing future treatments, including the need for targeted tissue delivery, avoidance of off-target effects, and individualization of treatment to account for inter-individual variations in miRNA expression and response to therapy.

Introduction
Most diseases result from complex pathobiological processes not reducible to a single dysregulated gene or molecular pathway, yet modern advancements in fields like oncology have allowed for the development of targeted therapies based on precise genetic mutations, amplifications, and changes to the tumor microenvironment.¹ The story is different for many other diseases that are still diagnosed using primarily clinical and other less precise laboratory, radiographic, or hemodynamic criteria. Pulmonary hypertension (PH) is one such disease where diagnosis depends heavily on clinical evaluation and hemodynamic measurements, where cutoffs have historically been more arbitrary and remain in flux.² This article seeks to review the current state of diagnostics, risk stratification, and treatment for PH, and to re-imagine precision diagnostics and therapy based on recent discoveries involving microRNAs (miRNAs).

Broadly defined, P

rs to heterogeneous set of vascular conditions characterized by increased pulmonary artery pressure. The World Symposium on Pulmonary Hypertension (WSPH) classifies PH into Groups I-V, based on both clinical assessment and invasive hemodynamic monitoring.2 Group I PH, also called pulmonary arterial hypertension (PAH), is a rare and progressive disease characterized by pulmonary arteriolar remodeling, occlusive vasculopathy, and increased pulmonary vascular resistance ultimately leading to right heart failure and death.³ Patients present with exertional dyspnea, peripheral edema, syncope, and, as a result of increased right ventricular afterload, frequently progress to right heart failure and death. Groups II-V are more prevalent and comprise PH secondary to left heart failure, chronic lung disease causing hypoxemia, thromboembolic disease, and other systemic conditions. Unlike patients with Group I PH, patients with Groups II-V PH have significant heterogeneity in both clinical presentation and response to vasodilator therapy.⁴

Current diagnostic criteria distinguishing Group I from Groups II-V PH rely on nuanced clinical evaluation and invasive hemodynamic evaluation via right heart catheterization (RHC). In practice, diagnosis is often challenging due to overlap among the groups and relies on expert opinion. Group I PH is defined by mean pulmonary artery pressure (mPAP) > 20 mmHg, pulmonary vascular resistance (PVR) > 3 Wood units, and pulmonary capillary wedge pressure (PCWP) < 15 mmHg.⁵ One must also rule out clinically other causes of PH including left heart failure, chronic lung disease resulting in hypoxemia, venous thromboembolic disease, and systemic disorders like sarcoidosis, chronic renal insufficiency, and myeloproliferative diseases. Currently, no molecular profiling or precision tool can be used to differentiate among these; rather, clinicians use a combination of patient history, physical exam, functional exercise testing, routine laboratory testing, imaging, and hemodynamics to make a determination.

Diverse etiologies have been implicated for PAH, ranging from heritable mutations in bone morphogenetic protein receptor 2 (BMPR2) to drugs and toxins and infections, including schistosomiasis and HIV.^6-8 However, the molecular origins of disease remain largely undefined, as patients present with advanced pathology not easily traceable to a single, precise mechanism. Nonetheless, patients behave similarly in terms of clinical presentation and response to pulmonary vasodilator therapy.

The prevailing understanding of disease pathogenesis involves pulmonary arteriolar remodeling driven by endothelial and smooth muscle cell proliferation resulting in medial hypertrophy, intimal proliferative and fibrotic changes, perivascular inflammation, and thrombotic lesions.⁴ In advanced disease, the pathophysiology is multifaceted and complex involving the nitric oxide, prostacyclin‑thromboxane, and endothelin-1 pathways. These end-stage pathways are well understood and have led to effective therapies, but they fail to target the molecular origins of disease and, thus, fail to reverse or cure it.^9,10 To better identify the central hubs of activity that drive PH pathogenesis, molecular classifiers are needed that will diagnose disease earlier and subclassify patients based on the molecular pathophysiology, natural history of disease, and response to therapy. Ultimately, the goal would be developing more effective targeted diagnostics and therapies with the hope of classifying condition by specific classification group and then regressing or curing early disease before it becomes advanced.

One possible molecular classifier that could be used in this regard is non-coding RNA, specifically microRNA (miRNA). MiRNAs are short non-coding RNAs that inhibit gene expression by binding to messenger RNAs (mRNAs) causing degradation, destabilization, and translation repression (Figure 1).^11,12 MiRNAs are thought to regulate 30% of all protein coding genes in mammals and are crucial to many aspects of normal physiology ranging from cell survival, differentiation, and proliferation to metabolism and cell-cell communication.^13-15 Because of the pleiotropy mediated by miRNAs, it has been difficult to harness these molecules for precision diagnostics and therapeutics. Nonetheless, studies have shown that circulating miRNAs can be used as diagnostic biomarkers for other cardiac diseases such as acute myocardial infarction.^16,17 More progress is being made in the field of PAH, as we now know there are numerous dysregulated miRNAs involved in pulmonary artery smooth muscle cell differentiation, proliferation, and apoptosis among other processes.¹² Some studies have even shown that modulation of specific miRNAs (including miRNA-21, miRNA-130/301, miRNA-145, miRNA-17, and miRNA-20a, among others) can prevent or reverse existing disease (Table 1).^18-23

Diagnosis & Classification
There is a critical need for early detection as patients diagnosed earlier with better functional capacity (e.g., New York Heart Association [NYHA] or World Health Organization [WHO] functional class I/II) have improved survival.^24-26 However, this is challenging because many patients are asymptomatic or have only mild, non-specific symptoms early in the disease course. Even for symptomatic patients, time from symptom onset to diagnosis commonly takes over two years, a number not significantly changed from the 1980s.^27,28 Currently recommended screening modalities include echocardiography, but only for patients who are already symptomatic or with specific high-risk disease such as systemic sclerosis. Unfortunately, this method of screening is operator dependent and imprecise.^24,29 Yet, we know that there are changes in metabolism, redox status, cellular damage, and activation of inflammatory pathways that precede the clinical manifestation of PAH and that miRNAs regulate many of these phenomena.^10,30-33

Circulating miRNAs make for promising non‑invasive screening tests because samples can be obtained and quantified by PCR from peripheral blood and other body fluids.12 One study of pediatric patients with PAH ranging from WHO functional class I-III showed that the expression of 19 circulating miRNAs significantly correlated with indexed pulmonary vascular resistance, and that miRNA-623 was a good classifier for vascular responsiveness to acute oxygen (O2) and inhaled nitric oxide (iNO).³⁴ A different study used PCR to quantify serum miRNA-509-3p in patients with both congenital heart disease and known PAH; these researchers found that the level of circulating miRNA-509-3p was significantly lower in the PAH group. Based on the expression of serum miRNA-509-3p, the area under the curve (AUC) for single-factor diagnosis of PAH was 0.694, comparable to that of echocardiography at 0.81. Combining the two modalities resulted in an AUC of 0.844, thereby improving confidence in the results.³⁵ Future studies looking at a more expansive panel of miRNA may allow us to identify other miRNA with even better diagnostic utility. Ultimately, a systems or network biology approach using higher levels of computational analysis will be needed to identify a signature of miRNAs that are functionally related and can be used together as a screening test, rather than relying on a single miRNA.36 Overlaying genomic data may also lead to improvement in their diagnostic and prognostic performance.³⁷

Using new diagnostic markers could enable us to subclassify patients into different (even as yet undefined) subtypes of PAH. At present, PAH subtypes are determined according to the revised WSPH Classification, which relies on presumed etiology: idiopathic (IPAH), familial (FPAH), associated (APAH), pulmonary veno‑occlusive disease (PVOD), pulmonary capillary hemangiomatosis (PCH), and persistent pulmonary hypertension of the newborn.² Associated PAH, in particular, is a poorly defined category including connective tissue disorders, congenital systemic‑topulmonary shunts, portal hypertension, infection with HIV, drugs and toxins, thyroid disorders, and myeloproliferative disorders. Instead, one can imagine future classifications based on precise molecular abnormalities in conjunction with standard clinical markers, anticipated rate of disease progression, and likelihood of response to specific therapies. For example, one study showed that in patients with Group 1 PH only, the concentration of miRNA‑204 increased sequentially along the pulmonary vasculature suggesting differences in muscle‑specific pathobiology between Group 1 and Group 2 PH.³⁸

Nonetheless, there are several challenges to using miRNAs for screening and diagnostic purposes. First, more work is needed to determine whether circulating levels of miRNAs reflect dynamic intracellular changes. Proving a correlation between intracellular and extracellular concentrations, or using extracellular, microvesicle‑encapsulated miRNAs, would make them more amenable for use as “liquid biopsies” similar to what is being explored in oncology.³⁹ Second, miRNAs as the diagnostic alone are typically not cell‑ or tissue‑specific, making it difficult to diagnose localized disease based on serum levels of miRNAs. Third, miRNA levels are dynamically altered in normal physiologic activities and have high inter‑individual variability.⁴⁰ Rather than having a normal cutoff, it may be necessary to determine individual baselines and monitor these levels over time (e.g., tracking patients in at‑risk categories). Finally, the full basic science of miRNA expression, degradation, secretion, and excretion remains incomplete. Having a better understanding of these processes may allow us to track extracellular miRNA for PH not only in the serum, but also in the urine and stool, which could be sampled for diagnostic purposes.⁴¹

Risk Stratification & Prognosis
Current risk stratification models include demographics like age and sex, functional class as defined by NYHA and WHO, exercise capacity measured by the 6‑minute walk test (6MWT), plasma brain natriuretic peptide (BNP), echocardiogram measurements such as peak tricuspid regurgitant jet velocity (TRV), and hemodynamic parameters obtained during right heart catheterization (RHC).^42,43 A more formal risk assessment calculator is available via the Registry to Evaluate Early and Long‑term Disease Management (REVEAL Registry), which stratifies patients as low (predicted survival ≥ 95%), average (90% to < 95%), moderate (85% to < 90%), high (70% to < 85%), and very high (< 70%) risk with corresponding 1‑year survival estimates.⁴⁴ None of these methods of risk stratification incorporate biomarkers specific to PAH.

MiRNAs may be useful for risk stratification as these specific effectors have been shown to indicate severity of disease. In one study of patients with groups I-IV PH, researchers studied serum samples obtained from the distal pulmonary artery during RHC to identify novel downregulated miRNAs (miRNA-451, miRNA-1246) and upregulated miRNAs (miRNA‑23b, miRNA-130a, and miRNA-191). Patients were stratified as having mild, moderate, or severe PH on the basis of mPAP (mild > 25 mmHg, moderate > 35 mmHg, and severe >40 mmHg). Levels of miRNA-1 were lower in patients with severe PH, and levels of miRNA-130a, miRNA-191, miRNA-204, and miRNA-208b were higher in patients with severe PH.45 Moreover, many of these miRNAs are known to target genes involved in profibrotic processes. For example, the miRNA-29 family acts on over a dozen extracellular matrix genes with targets including collagen, fibrillins, and elastin.⁴⁶ Additionally, miRNA-1 has target genes for Notch3, which is upregulated in PH.⁴⁵ Further work is needed to determine whether these miRNAs could be used to characterize treatment response after exposure to vasodilator therapy, which could alter how we define responders vs non-responders.

At present, response to vasodilator therapy is defined by decrease in mPAP by > 10 mmHg to an mPAP < 40 mmHg with preserved cardiac output after exposure to inhaled NO.^47,48 Kyehfets et al recently showed that the level of miRNA-623 could be used to predict with 88% probability whether a patient would respond to pulmonary vasodilator challenge as measured by improvement in indexed PVR. In the same study, downregulation of miRNA-627 was also shown to explain at least some of the variability in the magnitude of improvement in indexed PVR.³⁴ In the future, additional studies could be done evaluating whether specific miRNAs can predict a patient’s response to other vasodilator therapies beyond NO.

Novel Therapeutics
Enthusiasm regarding the development of new targeted therapies has been increasing, as we learn more about the myriad roles of miRNAs in PAH. As noted above, specific miRNAs can be either over- or under-expressed in PAH. Multiple technologies have been successfully developed to inhibit overexpressed miRNA including antisense oligonucleotides or nucleic acid molecules with masking, sponging, eraser, and decoy functionality.^49-52 For example, synthetic circular RNA has been shown to function as a miRNA-21 sponge that suppresses gastric carcinoma cell proliferation.⁵³ For miRNAs that are under-expressed, molecular mimics can be used to replace or substitute the lost miRNAs (Figure 2). In prostate cancer cells, for example, miRNA mimics for tumor suppressor miRNA-15a were used to arrest growth and apoptosis, and also resulted in volume regression of the tumors.^54,55

Several therapies involving miRNAs have the potential to prevent or reverse PAH. In a study published earlier this year, researchers used miRNA-150-5p complexed with a lipid carrier to deliver miRNA-150 directly to vascular endothelium and found that this reduced vascular remodeling and improved pulmonary vascular hemodynamics in a mouse model of PAH.[32] A different study, also published this year, showed that inhaled delivery of miRNA-483 using a lentiviral vector suppressed PAH-related gene expression, resulted in lower mPAP, and improved right ventricular hypertrophy in a rat model of PAH.⁵⁶ Finally, work produced by our group showed that oropharyngeal administration of antisense oligonucleotides targeting miRNA-130/301 led to coordinated and systems-wide alterations across multiple pathogenic pathways and cell types to ameliorate PH.^23,30 Delivering entire miRNA circuits, rather than a single miRNA, would likely produce a more robust effect, and further work is needed in this regard.

As our knowledge advances, other treatment strategies are likely to emerge that leverage the emerging notions of long-range endocrine delivery of endogenous miRNAs. For example, miRNA-210 has been implicated as a causative factor in the development of Groups 1 and III PH via repression of iron-sulfur cluster assembly proteins in pulmonary arterial endothelial cells (PAECs). Furthermore, we know extracellular miRNA-210 can be taken up by cultured PAECs.⁵⁷ Recent work by this group demonstrated that long-range extracellular transport of miRNA-210, with uptake into PAECs in rodent models of disease, promotes PH.⁵⁷ As blood levels of miRNAs are delivered to tissues for activity, then depleting or increasing miRNAs in the blood may serve as an indirect but effective method to alter tissue-specific miRNA expression. This provides the groundwork for translational studies seeking to develop novel bloodbased therapies for diseases like PH, where traditional limitations have existed for direct administration of therapies to more cloistered tissue compartments.

Despite its many promises, there are challenges to precision therapy design and application using miRNA. Clinical trials have demonstrated several barriers including dose‑limiting and immune-mediated toxicities, failure to produce sufficient therapeutic efficacy, metabolic instability, and off-target effects.⁵⁸ Because a single miRNA can regulate hundreds of genes, off-target effects are perhaps the biggest challenge, limiting therapeutic specificity.⁵⁹ Successful therapy often relies on tissue-dependent gene expression, and when target genes are affected in nontarget tissue this may result in unwanted side effects or toxicity. Moreover, the lipid and polymer nanoparticles used to promote cellular uptake and endosomal escape may accumulate in the liver, kidney, and spleen.⁶⁰ Finally, given interindividual variations in miRNA expression, it is possible that each person may respond differently to specific miRNAs, and an individualized approach may be needed. How we determine successful individual response to therapy remains undefined in many cases. Nonetheless, successes for RNA therapies in other fields have been attained, leading to approval of drugs like patisiran for transthyretinmediated amyloidosis.⁶¹ Thus, we are hopeful that new developments in our understanding of miRNAs can be used to improve molecular profiling, diagnostic precision, and targeted therapy in PH as well as other diseases in need of similar diagnostic and therapeutic breakthroughs for unmet medical needs.

Conclusions
Pulmonary hypertension is a progressive and heterogeneous set of diseases where molecular origins remain elusive, making it difficult to diagnose and treat early. MicroRNA biology has the potential to help subclassify the disease into more pathobiologically relevant categories and thereby advance our approach to developing precision diagnoses, risk stratification strategies, and therapeutic treatments. Treatments targeting single miRNAs have proven successful in early experimental studies; utilizing a systems or network biology approach could help identify entire miRNA circuits that can be targeted for even more robust therapeutic effects.

Stephen Chan, MD, PhD, is a Professor of Medicine (Cardiology) at the University of Pittsburgh School of Medicine and serves as the Director for the Vascular Medicine Institute and the Director for the Center of Pulmonary Vascular Biology and Medicine. Dr. Chan devotes a majority of his time to leading a basic science and translational research laboratory studying the molecular mechanisms of pulmonary vascular disease and pulmonary hypertension (PH) – a disease where reductionistic studies have primarily focused on only end-stage molecular effectors. To capitalize on the emerging discipline of “network medicine,” the Chan laboratory utilizes a combination of network-based bioinformatics with unique experimental reagents derived from genetically altered rodent and human subjects to accelerate systems-wide discovery in PH. In doing so, Dr. Chan’s published work was the first to identify the systems-level importance of microRNAs as a root cause for pulmonary hypertension, controlling metabolism, inflammation, and vascular stiffness. Dr. Chan’s recent work also delves into the computational biology of -omics datasets in order to predict unique pathogenic pathways important in PH. Dr. Chan serves as Chair of the NIH Respiratory Integrative Biology and Translational Research (RIBT) study section, holds multiple grants from the NIH, is an elected member of the American Society for Clinical Investigation, and holds an Established Investigator Award from the American Heart Association.

Erin Schikowski, MD is a resident physician at University of Pittsburgh Medical Center in Pittsburgh, PA. Her research interests include socioeconomic determinants of health, as well as sex- and race-based differences in hemodynamic risk factors for pulmonary arterial hypertension. Erin received her medical degree from the University of Rochester Medical Center.

Corresponding Author: Stephen Y. Chan, MD, PhD Center for Pulmonary Vascular Biology and Medicine Pittsburgh Heart, Lung, and Blood Vascular Medicine Institute Division of Cardiology, Department of Medicine University of Pittsburgh Medical Center 200 Lothrop Street BST1704.2 Pittsburgh, PA USA 15261 Tel: 412-383-6990 Fax: 412-624-9160 Email: chansy@pitt.edu

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