Effect of thymosin ß4 on lipopolysaccharide­stimulated brain microvascular endothelial cell remodeling: A possible role in blood­brain barrier injury.

War veterans, in particular, are more prone to mental illness as they are more likely to have encountered multiple traumatic brain injuries (TBIs) whilst serving on active duty in war zone areas. A TBI is known to cause mortality or serious neurological disabilities among survivors and elicits a number of pathological processes, including neuroinflammation and blood brain barrier (BBB) disruption, leading to secondary brain damage and subsequent impairment of the neurovascular unit. Although several drugs exhibit promising effects for TBI, the repertoire of currently available therapeutic strategies remains limited. Thymosin 4 (Tß4) is a 43-amino acid G-acting sequestering peptide that confers neuroprotective potential in TBI models. However, its role in BBB function remains unclear. Further research into the mechanism of BBB disruption induced by TBI and its specific role in neurovascular pathophysiology is necessary. In the present study, the protective effects of Tß4 in lipopolysaccharide (LPS)-stimulated gene expression of several tight junction proteins, inflammatory genes, apoptotic genes, and adhesion genes in human brain microvascular endothelial cells (hBMVECs), one of the pivotal cell types in the BBB, were reported. The results suggested that pretreatment with Tß4 reversed the LPS-induced damage of BBB components in hBMVECs. Furthermore, these results identified neuregulin 1 as a possible target for Tß4. Therefore, it is proposed that Tß4-mediated cellular signaling in hBMVEC may be vital for understanding the association between the BBB and TBI pathophysiology, which warrants further investigation.

Involvement of Nuclear Factor-κB in Inflammation and Neuronal Plasticity Associated with Post-Traumatic Stress Disorder.

Post-traumatic stress disorder (PTSD) is a debilitating psychiatric condition which develops either due to stress or witnessing a traumatic situation. PTSD is characterized by acute and chronic stress response exhibit anxiety, fear, and an increased inflammatory etiology. Inflammation contributes a critical role in several parts of the brain that control fear and flashback cognatic function. It is known that impairment of the neurological circuit leads to the development of PTSD. Evidence has suggested that dysregulation of the sympathetic nervous system and hypothalamic-pituitary adrenal (HPA) axis and inflammatory responsiveness are pivotal and a greater risk in PTSD. NF-κB, a master regulator for inflammation, has been showed to modulate memory reconsolidation and synaptic plasticity; however, NF-κB's association with PTSD remain elusive. In this review, we provide relevant findings regarding NF-κB activity in various components of brain and describe a potential mechanism linking PTSD using preclinical and clinical models. We envisage NF-κB signaling as a crucial mediator for inflammation, cognitive function, memory restoration and behavioral actions of stress and suggest that it could be used for therapeutic intervention in PTSD.

Inhibition of nuclear factor κB in the lungs protect bleomycin-induced lung fibrosis in mice.

BACKGROUND: Pulmonary fibrosis is a debilitating condition with limited therapeutic avenues. The pathogenicity of pulmonary fibrosis constitutes involvement of cellular proliferation, activation, and transformational changes of fibroblast to myofibroblasts. It is a progressive lung disease and is primarily characterized by aberrant accumulation of extracellular matrix proteins in the lungs with poor prognosis. The inflammatory response in the pathogenesis of lung fibrosis is suggested because of release of several cytokines; however, the underlying mechanism remains undefined. A genetic model is the appropriate way to delineate the underlying mechanism of pulmonary fibrosis. METHODS AND RESULTS: In this report, we have used cc-10 promoter based IκBα mutant mice (IKBM, an inhibitor of NF-κB) which were challenged with bleomycin (BLM). Compared to wild-type (WT) mice, the IKBM mice showed significant reduction in several fibrotic, vascular, and inflammatory genes. Moreover, we have identified a new set of dysregulated microRNAs (miRNAs) by miRNA array analysis in BLM-induced WT mice. Among these miRNAs, let-7a-5p and miR-503-5p were further analyzed. Our data showed that these two miRNAs were upregulated in WT-BLM and were reduced in IKBM-BLM mice. Bioinformatic analyses showed that let-7a-5p and miR-503-5p target for endothelin1 and bone morphogenic receptor 1A (BMPR1A), respectively, and were downregulated in WT-BLM mice indicating a link in pulmonary fibrosis. CONCLUSION: We concluded that inhibition of NF-κB and modulation of let-7a-5p and miR-503-5p contribute a pivotal role in pulmonary fibrosis and may be considered as possible therapeutic target for the clinical management of lung fibrosis.

MicroRNAs as biomarker and novel therapeutic target for posttraumatic stress disorder in Veterans.

Posttraumatic stress disorder (PTSD) is a common psychiatric disorder for military Veterans, characterized by hyperarousal, intrusive thoughts, flashbacks, hypervigilance, and distress after experiencing traumatic events. Some of the known physiological effects of PTSD include hypothalamic-pituitary-adrenal (HPA)-axis imbalance, a cortical function resulting in neuronal deficit and changes in behavior. Moreover, excessive discharge of inflammatory molecules and a dysregulated immune system are implicated in the pathophysiology of PTSD. Due to complex nature of this disorder, the biological underpinnings of PTSD remain inexplicable. Investigating novel biomarkers to understanding the pathogenesis of PTSD may reflect the underlying molecular network for therapeutic use and treatment. Circulatory microRNAs (miRNAs) and exosomes are evolving biomarkers that have shown a key role in psychiatric and neurological disorders including PTSD. Given the unique nature of combat trauma, as well as evidence that a large portion of Veterans do not benefit from frontline treatments, focus on veterans specifically is warranted. In the present review, we delineate the identification and role of several miRNAs in PTSD among veterans. An association of miRNA with HPA-axis regulation through FKBP5, a key modulator in PTSD is discussed as an emerging molecule in psychiatric diseases. We conclude that miRNAs may be used as circulatory biomarker detection in Veterans with PTSD.

Retraction Note: Role of the NF-κB signaling cascade and NF-κB-targeted genes in failing human hearts.

The Editor-in-Chief has retracted this article [1] because a number of lanes in Figs. 3, 4 and 6 of this article are duplicated.

Deficiency of MicroRNA miR-1954 Promotes Cardiac Remodeling and Fibrosis.

Background Cardiac fibrosis occurs because of disruption of the extracellular matrix network leading to myocardial dysfunction. Angiotensin II (AngII) has been implicated in the development of cardiac fibrosis. Recently, microRNAs have been identified as an attractive target for therapeutic intervention in cardiac pathologies; however, the underlying mechanism of microRNAs in cardiac fibrosis remains unclear. Next-generation sequencing analysis identified a novel characterized microRNA, miR-1954, that was significantly reduced in AngII-infused mice. The finding led us to hypothesize that deficiency of miR-1954 triggers cardiac fibrosis. Methods and Results A transgenic mouse was created using α-MHC (α-myosin heavy chain) promoter and was challenged with AngII infusion. AngII induced cardiac hypertrophy and remodeling. The in vivo overexpression of miR-1954 showed significant reduction in cardiac mass and blood pressure in AngII-infused mice. Further analysis showed significant reduction in cardiac fibrotic genes, hypertrophy marker genes, and an inflammatory gene and restoration of a calcium-regulated gene (Atp2a2 [ATPase sarcoplasmic/endoplasmic reticulum Ca2+ transporting 2]; also known as SERCA2), but no changes were observed in apoptotic genes. THBS1 (thrombospondin 1) is indicated as a target gene for miR-1954. Conclusions Our findings provide evidence, for the first time, that miR-1954 plays a critical role in cardiac fibrosis by targeting THBS1. We conclude that promoting the level of miR-1954 would be a promising strategy for the treatment of cardiac fibrosis.

The role of Thymosin ß4 in angiotensin II-induced cardiomyocytes growth.

INTRODUCTION: Thymosin beta-4 (Tß4) is an actin sequestering protein and is furthermore involved in diverse biological processes including cell proliferation, differentiation, wound healing, stem- or progenitor cell differentiation, and modulates inflammatory mediators. Tß4 also attenuates fibrosis. However, the role of Tß4 in cardiomyocytes hypertrophy is unknown. AREAS COVERED: In this review, we will discuss the role of Tß4 in cardiac remodeling that specifically includes cardiac hypertrophy and fibrosis only. Our review will further cover a new signaling pathway, the wingless and integrated-1 (Wnt) pathway in cardiac remodeling. In rat neonatal and adult cardiomyocytes stimulated with angiotensin II (Ang II), we showed that Tß4 has the ability to reduce cell sizes, attenuate hypertrophy marker genes expression, along with a panel of WNT-associated gene expressions induced by Ang II. Selected target gene WNT1-inducible-signaling pathway protein 1 (WISP-1) was identified by Tß4. Data further confirmed that WISP-1 overexpression promoted cardiomyocytes growth and was reversed by Tß4 pretreatment. EXPERT OPINION: Our data suggested that Tß4 protects cardiomyocytes from hypertrophic response by targeting WISP-1. The new role of Tß4 in cardiac hypertrophy advances our understanding, and the mechanism of action of Tß4 may provide a solid foundation for the treatment of cardiac disease.

MicroRNA-130a, a Potential Antifibrotic Target in Cardiac Fibrosis.

BACKGROUND: Cardiac fibrosis occurs because of disruption of the extracellular matrix network leading to myocardial dysfunction. Angiotensin II has been implicated in the development of cardiac fibrosis. Recently, microRNAs have been identified as an attractive target for therapeutic intervention in cardiac pathologies; however, the underlying mechanism of microRNAs in cardiac fibrosis remains unclear. MicroRNA-130a (miR-130a) has been shown to participate in angiogenesis and cardiac arrhythmia; however, its role in cardiac fibrosis is unknown. METHODS AND RESULTS: In this study, we found that miR-130a was significantly upregulated in angiotensin II-infused mice. The in vivo inhibition of miR-130a by locked nucleic acid- based anti-miR-130a in mice significantly reduced angiotensin II-induced cardiac fibrosis. Upregulation of miR-130a was confirmed in failing human hearts. Overexpressing miR-130a in cardiac fibroblasts promoted profibrotic gene expression and myofibroblasts differentiation, and the inhibition of miR-130a reversed the processes. Using the constitutive and dominant negative constructs of peroxisome proliferator-activated receptor γ 3-'untranslated region (UTR), data revealed that the protective mechanism was associated with restoration of peroxisome proliferator-activated receptor γ level leading to the inhibition of angiotensin II-induced cardiac fibrosis. CONCLUSIONS: Our findings provide evidence that miR-130a plays a critical role in cardiac fibrosis by directly targeting peroxisome proliferator-activated receptor γ. We conclude that inhibition of miR-130a would be a promising strategy for the treatment of cardiac fibrosis.

MicroRNAs: New Players in the Pathobiology of Preeclampsia.

Our understanding of how microRNAs (miRNAs) regulate gene networks and affect different molecular pathways leading to various human pathologies has significantly improved over the years. In contrary, the role of miRNAs in pregnancy-related hypertensive disorders such as preeclampsia (PE) is only beginning to emerge. Recent papers highlight that adverse pregnancy outcomes are associated with aberrant expression of several miRNAs. Presently, efforts are underway to determine the biologic function of these placental miRNAs which can shed light on their contribution to these pregnancy-related disease conditions. The discovery that miRNAs are stable in circulation coupled with the fact that the placenta is capable of releasing them to the circulation in exosomes generates a lot of enthusiasm to use them as biomarkers. In this review, we will summarize the recent findings of our understanding of miRNA regulation in relation to PE, a hypertensive disorder of pregnancy. Particular emphasis will be given to the role of key miRNA molecules such as miR-210 and miR-155 that are known to be consistently dysregulated in women with PE.

NF-κB mediated miR-130a modulation in lung microvascular cell remodeling: Implication in pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is a progressive pulmonary vascular disease which is associated with pulmonary arterial endothelial cells (PAEC) dysfunction and pulmonary arterial smooth muscle cells proliferation. Moreover, inflammation is contributing a critical role in EC dysfunction and remains elusive. Nuclear factor-kappa B (NF-κB) is a master transcriptional regulator in various cardiovascular pathologies; but, NF-κB's role in EC dysfunction in unknown. Our previous study using cardiac and lung specific IκBα mutant mice (3M and IKBM) showed that PAH induced right ventricular hypertrophy (RVH) was prevented in monocrotaline (MCT) treated 3M and IKBM mice, compared to the wild-type mice. Recently, microRNAs (miRNAs) have emerged as a new class of post-transcriptional regulators in vascular remodeling; but, NF-κB regulated miRNA modulation in EC dysfunction is unknown. Using miRNA array analysis, we identified miR-130a which is upregulated in MCT-induced PAH mouse model, as a possible candidate to study. We showed that overexpressing miR-130a in lung microvascular endothelial cells (MVEC) promoted activation of α-smooth muscle actin, a critical component in endothelial-to-mesenchymal transition in EC remodeling. In this study, we demonstrated that bone morphogenetic protein receptor 2 (BMPR2) was a target for miR-130a and miR-130a was regulated by NF-κB which controlled apoptosis and vascular genes in MVEC. The findings reveal that NF-κB-mediated miR-130a modulation is critical in lung vascular remodeling.

S100A8/MYD88/NF-қB: a novel pathway involved in cardiomyocyte hypertrophy driven by thyroid hormone.

Recent studies have evidenced the involvement of inflammation-related pathways to the development of cardiac hypertrophy and other consequences on the cardiovascular system, including the calcium-binding protein S100A8. However, this has never been investigated in the thyroid hormone (TH)-prompted cardiac hypertrophy. Thus, we aimed to test whether S100A8 and related signaling molecules, myeloid differentiation factor-88 (MyD88) and nuclear factor kappa B (NF-қB), could be associated with the cardiomyocyte hypertrophy induced by TH. Our results demonstrate that the S100A8/MyD88/NF-қB signaling pathway is activated in cardiomyocytes following TH stimulation. The knockdown of S100A8 and MyD88 indicates the contribution of those molecules to cardiomyocyte hypertrophy in response to TH, as evaluated by cell surface area, leucine incorporation assay, and gene expression. Furthermore, S100A8 and MyD88 are crucial mediators of NF-қB activation, which is also involved in the hypertrophic growth of TH-treated cardiomyocytes. Supporting the in vitro data, the contribution of NF-қB for TH-induced cardiac hypertrophy is confirmed in vivo, by using transgenic mice with cardiomyocyte-specific suppression of NF-қB. These data identify a novel pathway regulated by TH that mediates cardiomyocyte hypertrophy. However, the potential role of this new pathway in short and long-term cardiac effects of TH remains to be further investigated. KEY MESSAGES: Inflammation-related signaling is activated by T3 in cardiomyocytes. S100A8 and MyD88 have a crucial role in cardiomyocyte hypertrophy by T3. S100A8 and MyD88 mediate NF-қB activation by T3. NF-қB contributes to T3-induced cardiac hypertrophy in vitro and in vivo.

Thymosin ß4 Prevents Angiotensin II-Induced Cardiomyocyte Growth by Regulating Wnt/WISP Signaling.

Thymosin beta-4 (Tß4) is a ubiquitous protein with many properties relating to cell proliferation and differentiation that promotes wound healing and modulates inflammatory mediators. However, the role of Tß4 in cardiomyocyte hypertrophy is currently unknown. The purpose of this study was to determine the cardio-protective effect of Tß4 in angiotensin II (Ang II)-induced cardiomyocyte growth. Neonatal rat ventricular cardiomyocytes (NRVM) were pretreated with Tß4 followed by Ang II stimulation. Cell size, hypertrophy marker gene expression and Wnt signaling components, ß-catenin, and Wnt-induced secreted protein-1 (WISP-1) were evaluated by quantitative real-time PCR, Western blotting and fluorescent microscopy. Pre-treatment of Tß4 resulted in reduction of cell size, hypertrophy marker genes and Wnt-associated gene expression, and protein levels; induced by Ang II in cardiomyocyte. WISP-1 was overexpressed in NRVM and, the effect of Tß4 in Ang II-induced cardiomyocyte growth was evaluated. WISP-1 overexpression promoted cardiomyocytes growth and was reversed by pretreatment with Tß4. This is the first report which demonstrates that Tß4 targets Wnt/WISP-1 to protect Ang II-induced cardiomyocyte growth. J. Cell. Physiol. 231: 1737-1744, 2016. © 2015 Wiley Periodicals, Inc.

Modulation of miRNAs in Pulmonary Hypertension.

MicroRNAs (miRNAs) have emerged as a new class of posttranscriptional regulators of many cardiac and vascular diseases. They are a class of small, noncoding RNAs that contributes crucial roles typically through binding of the 3'-untranslated region of mRNA. A single miRNA may influence several signaling pathways associated with cardiac remodeling by targeting multiple genes. Pulmonary hypertension (PH) is a rare disorder characterized by progressive obliteration of pulmonary (micro) vasculature that results in elevated vascular resistance, leading to right ventricular hypertrophy (RVH) and RV failure. The pathology of PH involves vascular cell remodeling including pulmonary arterial endothelial cell (PAEC) dysfunction and pulmonary arterial smooth muscle cell (PASMC) proliferation. There is no cure for this disease. Thus, novel intervention pathways that govern PH induced RVH may result in new treatment modalities. Current therapies are limited to reverse the vascular remodeling. Recent studies have demonstrated the roles of various miRNAs in the pathogenesis of PH and pulmonary disorders. This review provides an overview of recent discoveries on the role of miRNAs in the pathogenesis of PH and discusses the potential for miRNAs as therapeutic targets and biomarkers of PH at clinical setting.

Thymosin Beta 4 protects mice from monocrotaline-induced pulmonary hypertension and right ventricular hypertrophy.

Pulmonary hypertension (PH) is a progressive vascular disease of pulmonary arteries that impedes ejection of blood by the right ventricle. As a result there is an increase in pulmonary vascular resistance and pulmonary arterial pressure causing right ventricular hypertrophy (RVH) and RV failure. The pathology of PAH involves vascular cell remodeling including pulmonary arterial endothelial cell (PAEC) dysfunction and pulmonary arterial smooth muscle cell (PASMC) proliferation. Current therapies are limited to reverse the vascular remodeling. Investigating a key molecule is required for development of new therapeutic intervention. Thymosin beta-4 (Tß4) is a ubiquitous G-actin sequestering protein with diverse biological function and promotes wound healing and modulates inflammatory responses. However, it remains unknown whether Tß4 has any protective role in PH. The purpose of this study is to evaluate the whether Tß4 can be used as a vascular-protective agent. In monocrotaline (MCT)-induced PH mouse model, we showed that mice treated with Tß4 significantly attenuated the systolic pressure and RVH, compared to the MCT treated mice. Our data revealed for the first time that Tß4 selectively targets Notch3-Col 3A-CTGF gene axis in preventing MCT-induced PH and RVH. Our study may provide pre-clinical evidence for Tß4 and may consider as vasculo-protective agent for the treatment of PH induced RVH.

Cardiac-specific suppression of NF-κB signaling prevents diabetic cardiomyopathy via inhibition of the renin-angiotensin system.

Activation of NF-κB signaling in the heart may be protective or deleterious depending on the pathological context. In diabetes, the role of NF-κB in cardiac dysfunction has been investigated using pharmacological approaches that have a limitation of being nonspecific. Furthermore, the specific cellular pathways by which NF-κB modulates heart function in diabetes have not been identified. To address these questions, we used a transgenic mouse line expressing mutated IκB-α in the heart (3M mice), which prevented activation of canonical NF-κB signaling. Diabetes was developed by streptozotocin injections in wild-type (WT) and 3M mice. Diabetic WT mice developed systolic and diastolic cardiac dysfunction by the 12th week, as measured by echocardiography. In contrast, cardiac function was preserved in 3M mice up to 24 wk of diabetes. Diabetes induced an elevation in cardiac oxidative stress in diabetic WT mice but not 3M mice compared with nondiabetic control mice. In diabetic WT mice, an increase in the phospholamban/sarco(endo)plasmic reticulum Ca(2+)-ATPase 2 ratio and decrease in ryanodine receptor expression were observed, whereas diabetic 3M mice showed an opposite effect on these parameters of Ca(2+) handling. Significantly, renin-angiotensin system activity was suppressed in diabetic 3M mice compared with an increase in WT animals. In conclusion, these results demonstrate that inhibition of NF-κB signaling in the heart prevents diabetes-induced cardiac dysfunction through preserved Ca(2+) handling and inhibition of the cardiac renin-angiotensin system.

Can microRNAs emerge as biomarkers in distinguishing HFpEF versus HFrEF?

MicroRNAs (miRNAs) are short strands of approximately 21-25 nucleotides. MiRNAs are emerging as important biomarker candidates for various cardiovascular diseases. These small molecules are being currently investigated for diagnosis, prognosis and more importantly as therapeutic targets. This review tries to explore the possibility of identifying miRNAs that are specific to Heart Failure with reduced Ejection Fraction (HFrEF) and Heart Failure with preserved Ejection Fraction (HFpEF) as both conditions carry equal morbidity and mortality risks, but drastically differ in their underlying pathophysiology. The concept of circulating miRNAs as biomarkers needs further investigation because the mechanism of their release into circulation still remains elusive; and, the biological correlation between circulatory miRNA and the relevant organ/tissue expression has not been established. A growing body of evidence indicates that miRNA may "shuttle" in between intracellular compartments for paracrine activities. Generating different panels of miRNAs may be useful in distinguishing HFrEF vs HFpEF. The use of antisense oligonucleotides to silence miRNAs would be another avenue towards establishing target-driven therapeutics in the context of personalized medicine.

NF-κB-mediated integrin-linked kinase regulation in angiotensin II-induced pro-fibrotic process in cardiac fibroblasts.

AIMS: Cardiac fibrosis is a final outcome of many clinical conditions that lead to cardiac failure and is characterized by a progressive substitution of cellular elements by extracellular-matrix proteins, such as collagen type I, collagen type II, connective tissue growth factor (CTGF), etc. The aim of this study was to identify the mechanisms responsible for angiotensin II (Ang II)-stimulated cardiac fibrosis using rat neonatal cardiac fibroblasts. MAIN METHODS: Neonatal fibroblasts were transfected with IκBα mutant, constitutively active (ca) integrin-linked kinase (ILK), dominant negative of ILK and small interfering RNA (siRNA) of ILK in the presence and absence of Ang-II stimulation. The pro-fibrotic gene expression and protein levels were determined by quantitative real time PCR and western blotting using their specific probes and antibodies. NF-κB translocation was determined by immunocytochemistry and confocal microscopy images were analyzed. KEY FINDINGS: Our results indicate that overexpression of ILK promotes a pro-fibrotic process by upregulating collagen type I and CTGF genes via activation of nuclear factor-κB (NF-κB) in cardiac fibroblasts. Inactivation of either NF-κB by the super-repressor IκBα or ILK by siRNA significantly attenuates the pro-fibrotic process. Moreover, ILK overexpression triggers NF-κB-p65 translocation to the nucleus, and ILK inhibition prevents the translocation in cardiac fibroblasts stimulated with Ang II. SIGNIFICANCE: Our data suggest that the Ang II-stimulated pro-fibrotic process is regulated by a complex mechanism involving crosstalk between ILK and NF-κB activation. This dual mechanism may play a critical role in the progression of cardiac fibrosis.

Inhibition of nuclear factor-κB in the lungs prevents monocrotaline-induced pulmonary hypertension in mice.

Pulmonary arterial hypertension (PAH) is a devastating cardiopulmonary disorder with significant morbidity and mortality in patients with various lung and heart diseases. PAH is characterized by vascular obstruction which leads to a sustained increased pulmonary vascular resistance, vascular remodeling, and right ventricular hypertrophy and failure. Limited PAH therapies indicate that novel approaches are urgently needed for the treatment of PAH. Nuclear factor-κB (NF-κB) has been shown to play an important role in different cardiac pathologies; however, the role of NF-κB remains limited in the setting of PAH. Here, we investigated whether NF-κB inhibition in the lungs using Club (Clara) cell-10 promoter driving IκBα mutant had any effect in monocrotaline (MCT)-induced PAH mouse model. Our data revealed that MCT-induced PAH and right ventricular hypertrophy were associated with NF-κB activation, inflammatory response, and altered expression of bone morphogenetic protein receptor 2, inhibitor of differentiation, and Notch-3 signaling molecules in wild-type mice; and all these alterations were prevented in IκBα mutant mice treated with MCT. Moreover, endothelial cell apoptosis and endothelial-to-mesenchymal transition occurred in the lungs of MCT-treated wild-type mice and were restored in IκBα mutant+MCT mice, indicating an association with NF-κB signaling. In lung microvascular endothelial cells, IκBα (AA) mutant plasmid restored the decreased bone morphogenetic protein receptor 2 protein level and reversed the endothelial-to-mesenchymal transition process induced by transforming growth factor-ß1. We conclude that NF-κB regulates bone morphogenetic protein receptor 2-inhibitor of differentiation-Notch-3 axis genes and the subsequent endothelial cell apoptosis and endothelial-to-mesenchymal transition events in the lungs, providing new mechanistic information about MCT-induced PAH and right ventricular hypertrophy.