ADAR1-Mediated RNA Editing Regulates Innate Immunity in Pulmonary Hypertension.

pulmonary hypertension (PH), diagnosed when mean pulmonary arterial pressure exceeds the upper limits of normal (i.e., >25 mmHg) at rest (2), occurs in a variety of clinical situations and is associated with a broad spectrum of histological patterns and abnormalities. PH is currently classified into five distinct World Health Organization (WHO) groups, based on common clinical parameters, potential etiological mechanisms, and responses to treatment (22). Although any form of PH can contribute to increased patient morbidity and mortality, pulmonary arterial hypertension (PAH) (WHO group 1) is a particularly severe and progressive form associated with right heart failure and premature death (1). At present, therapeutic approaches to stabilize or reverse this debilitating condition involve treatment with one or a combination of up to three specific classes of agents, including prostacyclin analogs, endothelin-1 receptor antagonists, and/or phosphodiesterase-5 inhibitors. Retrospective (meta)analyses of these therapeutic strategies have demonstrated a reduction in mortality with their use (7, 12); however, many experts believe that current PAH treatment is inadequate given the persistently high mortality rate and functional hemodynamic impairment in many patients. These observations have led to continued intensive investigation into pathogenetic mechanisms and many proposals for additional alternative new therapies (20, 24). Among the potential new therapies, increasing interest in the role of endothelial progenitor cells (EPCs) as a cell-based therapy has emerged. However, issues remain regarding what group of PH patients are most likely to benefit from treatment, at what point in the disease is treatment most likely to be successful, and what types of cells should be utilized for therapy.

Pulmonary hypertension (PH) is a debilitating progressive disease characterized by increased pulmonary arterial pressures, leading to right ventricular (RV) failure, heart failure and, eventually, death. Based on the underlying conditions, PH patients can be subdivided into the following five groups: (1) pulmonary arterial hypertension (PAH), (2) PH due to left heart disease, (3) PH due to lung disease, (4) chronic thromboembolic PH (CTEPH), and (5) PH with unclear and/or multifactorial mechanisms. Currently, even with PAH-specific drug treatment, prognosis for PAH and CTEPH patients remains poor, with mean five-year survival rates of 57%–59% and 53%–69% for PAH and inoperable CTEPH, respectively. Therefore, more insight into the pathogenesis of PAH and CTEPH is highly needed, so that new therapeutic strategies can be developed. Recent studies have shown increased presence and activation of innate and adaptive immune cells in both PAH and CTEPH patients. Moreover, extensive biomarker research revealed that many inflammatory and immune markers correlate with the hemodynamics and/or prognosis of PAH and CTEPH patients. Increased evidence of the pathological role of immune cells in innate and adaptive immunity has led to many promising pre-clinical interventional studies which, in turn, are leading to innovative clinical trials which are currently being performed. A combination of immunomodulatory therapies might be required besides current treatment based on vasodilatation alone, to establish an effective treatment and prevention of progression for this disease. In this review, we describe the recent progress on our understanding of the involvement of the individual cell types of the immune system in PH. We summarize the accumulating body of evidence for inflammation and immunity in the pathogenesis of PH, as well as the use of inflammatory biomarkers and immunomodulatory therapy in PAH and CTEPH.

Chapter 5 - Molecular and immunological basis of pulmonary arterial hypertension and pulmonary veno-occlusive disease

Introduction: Pulmonary arterial hypertension (PAH) is a devastating disease characterized by obliterative pulmonary vascular remodeling and progressive elevation of pulmonary vascular resistance that leads to right heart failure and premature death. RNA modifications such as N6-methyladenosine (m 6 A) has have recently been discovered as essential regulators of gene expression but its function in regulating the development of PAH remain unclear. Hypothesis: Fat mass and obesity-associated protein (FTO), as a major m 6 A eraser, acts as a key regulator of endothelial cell (EC) dysfunction and pulmonary vascular remodeling and, thus, is a novel therapeutic target for PAH. Methods: To determine the FTO expression on the idiopathic PAH (IPAH) patients, we performed Western blotting and immunofluorescence on lung tissue from IPAH patients. A novel mouse model with Tie2-Cre-mediated deletion of Fto in endothelial cells was generated and treated with hypoxia for 3 weeks to induce experimental PAH. Measurement of right ventricular systolic pressure (RVSP) and weight ratio of right ventricular versus left ventricular plus septum (RV/LV+S), an indicator of RV hypertrophy were determined. To evaluate the translational potential of targeting FTO in PAH, rats were exposed to monocrotaline to induce pulmonary hypertension and then were treated with FTO inhibitor FB23-2. RVSP, RV/(LV+S) ratio and histology assessment were measured subsequently. Results: FTO expression level is markedly elevated in the endothelial cells of IPAH patients. The Tie2-Cre-Fto (CKO) mice exhibited inhibited PAH under hypoxia treatment as evident by reduced RVSP and lower RV/(LV+S) ratio. Histological and immunofluorescent staining showed that media layer of pulmonary arteries had less cell proliferation and reduced pulmonary vascular remodeling. Moreover, preclinical MCT-rat PAH model showed attenuated PAH phenotype as well as pulmonary vascular remodeling with pharmacological inhibition of FTO. Conclusions: These studies demonstrate that FTO expression markedly elevates in the EC of IPAH patients. Tie2-Cre mediated loss of FTO protects mice from hypoxia-induced PAH. Thus, targeting FTO is a promising therapeutic strategy for PAH.

Pulmonary hypertension (PH) represents a grave condition associated with high morbidity and mortality, emphasizing a desperate need for innovative and targeted therapeutic strategies. Cumulative evidence suggests that inflammation and dysregulated immunity interdependently affect maladaptive organ perfusion and congestion as hemodynamic hallmarks of the pathophysiology of PH. The role of altered cellular and humoral immunity in PH gains increasing attention, especially in pulmonary arterial hypertension (PAH), revealing novel mechanistic insights into the underlying immunopathology. Whether these immunophysiological aspects display a universal character and also hold true for other types of PH (e.g., PH associated with left heart disease, PH-LHD), or whether there are unique immunological signatures depending on the underlying cause of disease are points of consideration and discussion. Inflammatory mediators and cellular immune circuits connect the local inflammatory landscape in the lung and heart through inter-organ communication, involving, e.g., the complement system, sphingosine-1-phosphate (S1P), cytokines and subsets of, e.g., monocytes, macrophages, natural killer (NK) cells, dendritic cells (DCs), and T- and B-lymphocytes with distinct and organ-specific pro- and anti-inflammatory functions in homeostasis and disease. Perivascular macrophage expansion and monocyte recruitment have been proposed as key pathogenic drivers of vascular remodeling, the principal pathological mechanism in PAH, pinpointing toward future directions of anti-inflammatory therapeutic strategies. Moreover, different B- and T-effector cells as well as DCs may play an important role in the pathophysiology of PH as an imbalance of T-helper-17-cells (TH17) activated by monocyte-derived DCs, a potentially protective role of regulatory T-cells (Treg) and autoantibody-producing plasma cells occur in diverse PH animal models and human PH. This article highlights novel aspects of the innate and adaptive immunity and their interaction as disease mediators of PH and its specific subtypes, noticeable inflammatory mediators and summarizes therapeutic targets and strategies arising thereby.

Early apoptosis of pulmonary artery endothelial cells (PAECs) is a driver of vascular remodeling and pulmonary hypertension (PH), but its regulation is poorly defined. Adenosine deaminase acting on RNA 1 (ADAR1, gene name ADAR) is an RNA editing enzyme that converts adenosine to inosine (A-to-I) in RNA transcripts and participates in RNA metabolism. While deficiency in ADAR1-mediated RNA editing stimulates cellular innate immunity signaling and can promote apoptosis, the exact ADAR1 RNA editing targets and downstream mechanisms regulating PAEC survival are unknown. We sought to define the functions and targets of ADAR1-dependent RNA editing that control pulmonary endothelial pathophenotypes in PH. ADAR1 or Nocturnin (NOCT) expression and A-to-I RNA editing levels were evaluated in human PAH lungs by immunofluorescent staining and single cell RNA sequencing, respectively. Mice carrying a human missense ADAR mutation and genetic deletion of Noct with interleukin-6 (il6) transgene were studied in chronic hypoxia-induced PH in vivo models. ADAR1 expression was downregulated in the pulmonary vascular endothelium and in lung tissue of human and mouse PH. Global A-to-I RNA editing was decreased in lungs from PAH patients and hypoxic PH mice. In vitro, hypoxia, a PH trigger, downregulated ADAR1 in PAECs. Circadian gene NOCT was identified as a direct ADAR1 target which carries two active A-to-I RNA editing sites in the 3'UTR. In human PAH lungs, NOCT editing levels were reduced, while NOCT protein level increased. Correspondingly, in vitro, ADAR silencing increased NOCT mRNA levels, thus inducing dsRNA-MDA5 sensing interferon signaling and PAEC apoptosis. Importantly, silencing of NOCT reversed these changes. Forced NOCT expression phenocopied the effect of ADAR1 knockdown, upregulating interferon signaling molecules and increasing apoptosis. Chronically hypoxic PH mice carrying human ADAR mutation displayed worsened PH. Forced adeno-associated virus (AAV) expression of Adar improved monocrotaline-induced PH in rats. Genetic deletion of Noct mitigated PH in hypoxic il6-expressing transgenic PH mice, emphasizing the crucial role of NOCT in PH pathogenesis. Hypoxia-induced ADAR1 deficiency upregulates NOCT expression to induce PAEC interferon signaling activation, PAEC apoptosis, and PH. This study provides impetus to target the ADAR1-NOCT axis for more effective diagnostics and therapeutics for PH.

Recently, a paper by Kim et al. [1] in Nature Medicine magazine in January, 2013 showed that apelin (also known as APLN) inhibits fibroblast growth factor 2 (FGF2) and FGF receptor 1 (FGFR1) expression to ameliorate pulmonary hypertension by regulating the expression of miR-424 and miR-503. This study revealed the molecular mechanism of apelin in inhibiting the process of pulmonary arterial hypertension (PAH) and discovered the role of apelin in regulating miRNA generation for the first time. miRNA functions in the transcriptional regulation of gene expression to control cellular processes. miRNA is a key regulatory factor of protein expression, but the generation and regulation mechanism of miRNA is still unclear. These novel findings bring us inspiration for further research, especially on the mechanism of miRNA generation. Experiments on revealing endogenous active substance, which regulates the generation of miRNAs or revealing miRNAs that regulate the expression of apelin, may bring more breakthroughs in the future. PAH is characterized by vascular remodeling associated with obliteration of pulmonary arterioles and formation of plexiform lesions composed of hyperproliferative endothelial and vascular smooth muscle cells. Recent studies have suggested that apelin is a novel PAH endothelial function homeostasis-related factor. Alastalo et al. [2] found that apelin expression is decreased in endothelial cells of the pulmonary hypertension. However, the exact mechanism remains poorly understood. FGF2 is highly expressed in PAH and plays an important role in the progress of PAH by promoting proliferation [3] and inhibiting apoptosis [4] in endothelial cells and smooth muscle cells. miRNA is a fundamental factor of numerous cellular events by regulating RNA modification, transcription, and translation. Current studies of miRNA showed its critical function in the development of PAH. Morphological changes of plexiform vasculopathy in the end-stage PAH lung are reflected by alterations at the miRNA level [5]. Kim et al. [1] integrated these isolated observations into a mechanism and identified the miRNA-FGF signaling axis that is apelin-dependent in the maintenance of pulmonary vascular homeostasis. Previous studies found that hypoxia induces endothelial function injury. Accumulating evidence revealed that hypoxia also induces the expression of apelin [6,7],which is reduced in PAH. Actually, apelin expression and secretion, which are strongly induced under hypoxic conditions, are the early response [7]. Mechanisms that maintain sustained expression of apelin may contribute to preventing injuries caused by hypoxia and restoring the function of endothelial cells in PAH. These findings supported the development of novel therapeutic strategies to augment apelin, as well as to inhibit FGF2 signaling. In portal vein hypertension, another vascular disease, apelin/APJ presents a novel therapeutic target. APJ antagonist F13A effectively decreased the formation of portosystemic collateral vessels [8]. In atherosclerosis (AS), increasing evidence tends to prove that apelin is a novel therapeutic target for AS [9]. Although no effective treatment for PAH is available at present, certain medicines are available to mitigate disease progression. Considering its protective effect on vasodilatory and endothelial cells, apelin may be a more efficacious target for PAH therapy. In past decades, apelin was suggested to involve in numerous physiological processes, including vasodilation, systole, salt and water balance, as well as pathophysiological processes such as high blood pressure, cancer, and so on. There must be many other undiscovered functions of apelin. The findings of the intimate relationship between apelin and miRNA provide much fresh thinking for the study of apelin. The study on miR-424 and miR-503 will help to discover additional features of apelin. Park et al. [10] found that the high expression of miR-424 and miR-503 is significantly implicated in chemoresistance and tumor progression in ovarian cancer, which probably regulates cancer stem cell processes. These results indicated that apelin probably plays a vital role in epithelial mesenchymal transition in cancers. A recent study revealed that miR-503 makes women predispose to lupus [11]. The relationship between apelin and lupus has not been explored. More evidence is required to confirm whether apelin controls the effects of miR-424 and miR-503 in the above process. There are some controversial reports of apelin/APJ effects on AS. Chun et al. [12] found that apelin decreases AS formation by blocking AngII actions in ApoE-KO mice. But Hashimoto et al. [13] Acta Biochim Biophys Sin 2013, 45: 896–898 |a The Author 2013. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. DOI: 10.1093/abbs/gmt090. Advance Access Publication 28 August 2013

Immune mechanisms of pulmonary intravascular coagulopathy in COVID-19 pneumonia.

Hereditary hemorrhagic telangiectasia (HHT) or Rendu-Osler-Weber disease is a genetic disorder with autosomal dominance and variable penetrance, characterized by epistaxis, telangiectasia and visceral manifestations of the disease. The estimated minimal prevalence is 1/10,000 inhabitants. The diagnosis is established on clinical criteria, and may be further confirmed by the identification of causative mutations in either the ENG or the ACVRL1 gene coding for endoglin and ALK1, respectively. Pulmonary vascular manifestations of HHT include pulmonary arteriovenous malforma- tions (PAVMs; especially in patients with ENG mutations) and less frequently pulmonary hypertension (especially in patients with ACVRL1 mutations). In 15–33% of patients with HHT, PAVMs consist of abnormal communications between pulmonary arteries and pulmonary veins, causing right-to-left shunting, and thus, frequently hypoxemia and dyspnea on exertion, although PAVMs may remain asymptomatic and frequently undiagnosed unless complications occur. PAVMs result in severe and frequent complications often at a young age, which may reveal the diagnosis, e.g. transient ischemic attack and cerebral stroke (10–19% of patients), systemic severe infections and abscesses (including cerebral abscess in 5–19% of patients), and rarely massive hemoptysis or hemothorax. Infections in HHT are related to the right-to-left shunting that bypasses the pulmonary capillaries and facilitates the passage of septic or aseptic emboli into the systemic and especially cerebral circulation, and potentially to minor defects in innate immunity. Treatment of PAVMs based on transcatheter coil vaso-occlusion of the feeding artery significantly decreases right-to-left shunting, hypoxemia and dyspnea on exertion, and reduces the risk of systemic complications. Long-term follow-up is warranted after transcatheter vaso-occlusion of PAVMs due to frequent recanalization of treated PAVMs and development or growth of untreated PAVMs. Patients with HHT should be informed of the risk of PAVM and potentially severe complications occurring in heretofore asymptomatic subjects. All adult patients with HHT should be proposed systematic screening for PAVM, by contrast echocardiography (preceded by anteroposterior chest radiograph) or computed tomography of the chest. Pulmonary hypertension is rare in HHT, and may be due either to systemic arteriovenous shunting in the liver increasing cardiac output or be clinically and histologically indistinguishable from idiopathic pulmonary arterial hypertension. Pulmonary hypertension is detected by systematic examination of right cardiac cavities and tricuspid regurgitation flow at echocardiography, and the diagnosis is established by right heart catheterization.

AimsThe ability of the right ventricle (RV) to adapt to increased afterload is the major determinant of survival in patients with pulmonary hypertension (PH). In this study, we explored the effect of genetic background on RV adaptation and survival in a rat model of severe pulmonary arterial hypertension (PAH).Methods and resultsPH was induced by a single injection of SU5416 (SU) in age-matched Sprague Dawley (SD) or Fischer rats, followed by a 3-week exposure to chronic hypoxia (SUHx). SD and Fischer rats exhibited similar elevations in RV systolic pressure, number of occlusive pulmonary vascular lesions, and RV hypertrophy (RV/LV+S) in response to SUHx. However, no Fischer rats survived beyond 7 weeks compared with complete survival for SD rats. This high early mortality of Fischer rats was associated with significantly greater RV dilatation and reduced ejection fraction, cardiac output, and exercise capacity at 4 weeks post-SU. Moreover, microarray analysis revealed that over 300 genes were uniquely regulated in the RV in the severe PAH model in the Fischer compared with SD rats, mainly related to angiogenesis and vascular homoeostasis, fatty acid metabolism, and innate immunity. A focused polymerase chain reaction array confirmed down-regulation of angiogenic genes in the Fischer compared with SD RV. Furthermore, Fischer rats demonstrated significantly lower RV capillary density compared with SD rats in response to SUHx.ConclusionFischer rats are prone to develop RV failure in response to increased afterload. Moreover, the high mortality in the SUHx model of severe PAH was caused by a failure of RV adaptation associated with lack of adequate microvascular angiogenesis, together with metabolic and immunological responses in the hypertrophied RV.

Essential role of neutrophils in the monocrotaline-induced pulmonary arterial hypertension in rats.

Challenges and opportunities in targeting epigenetic mechanisms for pulmonary arterial hypertension treatment.

BackgroundEvidence suggests a role of both innate and adaptive immunity in the development of pulmonary arterial hypertension. The complement system is a key sentry of the innate immune system and bridges innate and adaptive immunity. To date there are no studies addressing a role for the complement system in pulmonary arterial hypertension.Methodology/Principal FindingsImmunofluorescent staining revealed significant C3d deposition in lung sections from IPAH patients and C57Bl6/J wild-type mice exposed to three weeks of chronic hypoxia to induce pulmonary hypertension. Right ventricular systolic pressure and right ventricular hypertrophy were increased in hypoxic vs. normoxic wild-type mice, which were attenuated in C3−/− hypoxic mice. Likewise, pulmonary vascular remodeling was attenuated in the C3−/− mice compared to wild-type mice as determined by the number of muscularized peripheral arterioles and morphometric analysis of vessel wall thickness. The loss of C3 attenuated the increase in interleukin-6 and intracellular adhesion molecule-1 expression in response to chronic hypoxia, but not endothelin-1 levels. In wild-type mice, but not C3−/− mice, chronic hypoxia led to platelet activation as assessed by bleeding time, and flow cytometry of platelets to determine cell surface P-selectin expression. In addition, tissue factor expression and fibrin deposition were increased in the lungs of WT mice in response to chronic hypoxia. These pro-thrombotic effects of hypoxia were abrogated in C3−/− mice.ConclusionsHerein, we provide compelling genetic evidence that the complement system plays a pathophysiologic role in the development of PAH in mice, promoting pulmonary vascular remodeling and a pro-thrombotic phenotype. In addition we demonstrate C3d deposition in IPAH patients suggesting that complement activation plays a role in the development of PAH in humans.

Circadian molecular clock disruption in chronic pulmonary diseases.

Involvement of interleukin-1 receptor (IL1R1) and myeloid differentiation primary response gene 88 (MyD88) signaling in pulmonary hypertension (PH)