Tm4sf1-marked Endothelial Subpopulation Is Dysregulated in Pulmonary Arterial Hypertension.

The identification and role of endothelial progenitor cells in pulmonary arterial hypertension (PAH) remain controversial. Single-cell omics analysis can shed light on endothelial progenitor cells and their potential contribution to PAH pathobiology. We aim to identify endothelial cells that may have stem/progenitor potential in rat lungs and assess their relevance to PAH. Differential expression, gene set enrichment, cell-cell communication, and trajectory reconstruction analyses were performed on lung endothelial cells from single-cell RNA sequencing of Sugen-hypoxia, monocrotaline, and control rats. Relevance to human PAH was assessed in multiple independent blood and lung transcriptomic data sets. Rat lung endothelial cells were visualized by immunofluorescence in situ, analyzed by flow cytometry, and assessed for tubulogenesis in vitro. A subpopulation of endothelial cells (endothelial arterial type 2 [EA2]) marked by Tm4sf1 (transmembrane 4 L six family member 1), a gene strongly implicated in cancer, harbored a distinct transcriptomic signature enriched for angiogenesis and CXCL12 signaling. Trajectory analysis predicted that EA2 has a less differentiated state compared with other endothelial subpopulations. Analysis of independent data sets revealed that TM4SF1 is downregulated in lungs and endothelial cells from patients and PAH models, is a marker for hematopoietic stem cells, and is upregulated in PAH circulation. TM4SF1+CD31+ rat lung endothelial cells were visualized in distal pulmonary arteries, expressed hematopoietic marker CD45, and formed tubules in coculture with lung fibroblasts. Our study uncovered a novel Tm4sf1-marked subpopulation of rat lung endothelial cells that may have stem/progenitor potential and demonstrated its relevance to PAH. Future studies are warranted to further elucidate the role of EA2 and Tm4sf1 in PAH.

HNRNPA2B1: RNA-Binding Protein That Orchestrates Smooth Muscle Cell Phenotype in Pulmonary Arterial Hypertension.

BACKGROUND: RNA-binding proteins are master orchestrators of gene expression regulation. They regulate hundreds of transcripts at once by recognizing specific motifs. Thus, characterizing RNA-binding proteins targets is critical to harvest their full therapeutic potential. However, such investigation has often been restricted to a few RNA-binding protein targets, limiting our understanding of their function. In cancer, the RNA-binding protein HNRNPA2B1 (heterogeneous nuclear ribonucleoprotein A2B1; A2B1) promotes the pro-proliferative/anti-apoptotic phenotype. The same phenotype in pulmonary arterial smooth muscle cells (PASMCs) is responsible for the development of pulmonary arterial hypertension (PAH). However, A2B1 function has never been investigated in PAH. METHOD: Through the integration of computational and experimental biology, the authors investigated the role of A2B1 in human PAH-PASMC. Bioinformatics and RNA sequencing allowed them to investigate the transcriptome-wide function of A2B1, and RNA immunoprecipitation and A2B1 silencing experiments allowed them to decipher the intricate molecular mechanism at play. In addition, they performed a preclinical trial in the monocrotaline-induced pulmonary hypertension rat model to investigate the relevance of A2B1 inhibition in mitigating pulmonary hypertension severity. RESULTS: They found that A2B1 expression and its nuclear localization are increased in human PAH-PASMC. Using bioinformatics, they identified 3 known motifs of A2B1 and all mRNAs carrying them. In PAH-PASMC, they demonstrated the complementary nonredundant function of A2B1 motifs because all motifs are implicated in different aspects of the cell cycle. In addition, they showed that in PAH-PASMC, A2B1 promotes the expression of its targets. A2B1 silencing in PAH-PASMC led to a decrease of all tested mRNAs carrying an A2B1 motif and a concomitant decrease in proliferation and resistance to apoptosis. Last, in vivo A2B1 inhibition in the lungs rescued pulmonary hypertension in rats. CONCLUSIONS: Through the integration of computational and experimental biology, the study revealed the role of A2B1 as a master orchestrator of the PAH-PASMC phenotype and its relevance as a therapeutic target in PAH.

Y-Chromosome Gene, Uty, Protects Against Pulmonary Hypertension by Reducing Proinflammatory Chemokines.

Rationale: Idiopathic pulmonary arterial hypertension (PAH) is a terminal pulmonary vascular disease characterized by increased pressure, right ventricular failure, and death. PAH exhibits a striking sex bias and is up to four times more prevalent in females. Understanding the molecular basis behind sex differences could help uncover novel therapies. Objectives: We previously discovered that the Y chromosome is protective against hypoxia-induced experimental pulmonary hypertension (PH), which may contribute to sex differences in PAH. Here, we identify the gene responsible for Y-chromosome protection, investigate key downstream autosomal genes, and demonstrate a novel preclinical therapy. Methods: To test the effect of Y-chromosome genes on PH development, we knocked down each Y-chromosome gene expressed in the lung by means of intratracheal instillation of siRNA in gonadectomized male mice exposed to hypoxia and monitored changes in right ventricular and pulmonary artery hemodynamics. We compared the lung transcriptome of Uty knockdown mouse lungs to those of male and female PAH patient lungs to identify common downstream pathogenic chemokines and tested the effects of these chemokines on human pulmonary artery endothelial cells. We further inhibited the activity of these chemokines in two preclinical pulmonary hypertension models to test the therapeutic efficacy. Measurements and Main Results: Knockdown of the Y-chromosome gene Uty resulted in more severe PH measured by increased right ventricular pressure and decreased pulmonary artery acceleration time. RNA sequencing revealed an increase in proinflammatory chemokines Cxcl9 and Cxcl10 as a result of Uty knockdown. We found CXCL9 and CXCL10 significantly upregulated in human PAH lungs, with more robust upregulation in females with PAH. Treatment of human pulmonary artery endothelial cells with CXCL9 and CXCL10 triggered apoptosis. Inhibition of Cxcl9 and Cxcl10 expression in male Uty knockout mice and CXCL9 and CXCL10 activity in female rats significantly reduced PH severity. Conclusions:Uty is protective against PH. Reduction of Uty expression results in increased expression of proinflammatory chemokines Cxcl9 and Cxcl10, which trigger endothelial cell death and PH. Inhibition of CLXC9 and CXLC10 rescues PH development in multiple experimental models.

Single-Cell Study of Two Rat Models of Pulmonary Arterial Hypertension Reveals Connections to Human Pathobiology and Drug Repositioning.

Rationale: The cellular and molecular landscape and translational value of commonly used models of pulmonary arterial hypertension (PAH) are poorly understood. Single-cell transcriptomics can enhance molecular understanding of preclinical models and facilitate their rational use and interpretation.Objectives: To determine and prioritize dysregulated genes, pathways, and cell types in lungs of PAH rat models to assess relevance to human PAH and identify drug repositioning candidates.Methods: Single-cell RNA sequencing was performed on the lungs of monocrotaline (MCT), Sugen-hypoxia (SuHx), and control rats to identify altered genes and cell types, followed by validation using flow-sorted cells, RNA in situ hybridization, and immunofluorescence. Relevance to human PAH was assessed by histology of lungs from patients and via integration with human PAH genetic loci and known disease genes. Candidate drugs were predicted using Connectivity Map.Measurements and Main Results: Distinct changes in genes and pathways in numerous cell types were identified in SuHx and MCT lungs. Widespread upregulation of NF-κB signaling and downregulation of IFN signaling was observed across cell types. SuHx nonclassical monocytes and MCT conventional dendritic cells showed particularly strong NF-κB pathway activation. Genes altered in SuHx nonclassical monocytes were significantly enriched for PAH-associated genes and genetic variants, and candidate drugs predicted to reverse the changes were identified. An open-access online platform was developed to share single-cell data and drug candidates (http://mergeomics.research.idre.ucla.edu/PVDSingleCell/).Conclusions: Our study revealed the distinct and shared dysregulation of genes and pathways in two commonly used PAH models for the first time at single-cell resolution and demonstrated their relevance to human PAH and utility for drug repositioning.

Role of miRNA-1 and miRNA-21 in Acute Myocardial Ischemia-Reperfusion Injury and Their Potential as Therapeutic Strategy.

Coronary artery disease remains the leading cause of death. Acute myocardial infarction (MI) is characterized by decreased blood flow to the coronary arteries, resulting in cardiomyocytes death. The most effective strategy for treating an MI is early and rapid myocardial reperfusion, but restoring blood flow to the ischemic myocardium can induce further damage, known as ischemia-reperfusion (IR) injury. Novel therapeutic strategies are critical to limit myocardial IR injury and improve patient outcomes following reperfusion intervention. miRNAs are small non-coding RNA molecules that have been implicated in attenuating IR injury pathology in pre-clinical rodent models. In this review, we discuss the role of miR-1 and miR-21 in regulating myocardial apoptosis in ischemia-reperfusion injury in the whole heart as well as in different cardiac cell types with special emphasis on cardiomyocytes, fibroblasts, and immune cells. We also examine therapeutic potential of miR-1 and miR-21 in preclinical studies. More research is necessary to understand the cell-specific molecular principles of miRNAs in cardioprotection and application to acute myocardial IR injury.

Sex differences underlying preexisting cardiovascular disease and cardiovascular injury in COVID-19.

The novel 2019 coronavirus disease (COVID-19), resulting from severe acute respiratory syndrome coronarvirus-2 (SARS-CoV-2) infection, typically leads to respiratory failure in severe cases; however, cardiovascular injury is reported to contribute to a substantial proportion of COVID-19 deaths. Preexisting cardiovascular disease (CVD) is among the most common risk factors for hospitalization and death in COVID-19 patients, and the pathogenic mechanisms of COVID-19 disease progression itself may promote the development of cardiovascular injury, increasing risk of in-hospital death. Sex differences in COVID-19 are becoming more apparent as mounting data indicate that males seem to be disproportionately at risk of severe COVID-19 outcome due to preexisting CVD and COVID-19-related cardiovascular injury. In this review, we will provide a basic science perspective on current clinical observations in this rapidly evolving field and discuss the interplay sex differences, preexisting CVD and COVID-19-related cardiac injury.

Pulmonary hypertension secondary to pulmonary fibrosis: clinical data, histopathology and molecular insights.

Pulmonary hypertension (PH) developing secondarily in pulmonary fibrosis (PF) patients (PF-PH) is a frequent co-morbidity. The high prevalence of PH in PF patients is very concerning since the presence of PH is a strong predictor of mortality in PF patients. Until recently, PH was thought to arise solely from fibrotic destruction of the lung parenchyma, leading to hypoxic vasoconstriction and loss of vascular bed density. Thus, potential cellular and molecular dysregulation of vascular remodeling as a driver of PF-PH has been under-investigated. The recent demonstrations that there is no correlation between the severity of the fibrosis and development of PH, along with the finding that significant vascular histological and molecular differences exist between patients with and without PH have shifted the etiological paradigm of PF-PH. This review aims to provide a comprehensive translational overview of PH in PF patients from clinical diagnosis and outcome to the latest understanding of the histology and molecular pathophysiology of PF-PH.

The Role of Estrogen Receptors in Cardiovascular Disease.

Cardiovascular Diseases (CVDs) are the leading cause of death globally. More than 17 million people die worldwide from CVD per year. There is considerable evidence suggesting that estrogen modulates cardiovascular physiology and function in both health and disease, and that it could potentially serve as a cardioprotective agent. The effects of estrogen on cardiovascular function are mediated by nuclear and membrane estrogen receptors (ERs), including estrogen receptor alpha (ERα), estrogen receptor beta (ERß), and G-protein-coupled ER (GPR30 or GPER). Receptor binding in turn confers pleiotropic effects through both genomic and non-genomic signaling to maintain cardiovascular homeostasis. Each ER has been implicated in multiple pre-clinical cardiovascular disease models. This review will discuss current reports on the underlying molecular mechanisms of the ERs in regulating vascular pathology, with a special emphasis on hypertension, pulmonary hypertension, and atherosclerosis, as well as in regulating cardiac pathology, with a particular emphasis on ischemia/reperfusion injury, heart failure with reduced ejection fraction, and heart failure with preserved ejection fraction.

Intralipid protects the heart in late pregnancy against ischemia/reperfusion injury via Caveolin2/STAT3/GSK-3ß pathway.

BACKGROUND: We recently demonstrated that the heart of late pregnant (LP) rodents is more prone to ischemia/reperfusion (I/R) injury compared to non-pregnant rodents. Lipids, particularly polyunsaturated fatty acids, have received special attention in the field of cardiovascular research. Here, we explored whether Intralipid (ITLD) protects the heart against I/R injury in LP rodents and investigated the mechanisms underlying this protection. METHODS AND RESULTS: In-vivo female LP rat hearts or ex-vivo isolated Langendorff-perfused LP mouse hearts were subjected to ischemia followed by reperfusion with PBS or ITLD (one bolus of 5mg/kg of 20% in in-vivo and 1% in ex-vivo). Myocardial infarct size, mitochondrial calcium retention capacity, genome-wide expression profiling, pharmacological inhibition and co-immunoprecipitation were performed. One bolus of ITLD at reperfusion significantly reduced the in-vivo myocardial infarct size in LP rats (23.3±2% vs. 55.5±3.4% in CTRL, p<0.01). Postischemic administration of ITLD also protected the LP hearts against I/R injury ex-vivo. ITLD significantly increased the threshold for the opening of the mitochondrial permeability transition pore in response to calcium overload (nmol-calcium/mg-mitochondrial protein: 290±17 vs. 167±10 in CTRL, p<0.01) and significantly increased phosphorylation of STAT3 (1.8±0.08 vs. 1±0.16 in CTRL, p<0.05) and GSK-3ß (2.63±0.55 vs. 1±0.0.34 in CTRL, p<0.05). The ITLD-induced cardioprotection was fully abolished by Stattic, a specific inhibitor of STAT3. Transcriptome analysis revealed caveolin 2 (Cav2) was significantly upregulated by ITLD in hearts of LP rats under I/R injury. Co-immunoprecipitation experiments showed that Cav2 interacts with STAT3. CONCLUSIONS: ITLD protects the heart in late pregnancy against I/R injury by inhibiting the mPTP opening through Cav2/STAT3/GSK-3ß pathway.

Potassium Channel Subfamily K Member 3 (KCNK3) Contributes to the Development of Pulmonary Arterial Hypertension.

BACKGROUND: Mutations in the KCNK3 gene have been identified in some patients suffering from heritable pulmonary arterial hypertension (PAH). KCNK3 encodes an outward rectifier K(+) channel, and each identified mutation leads to a loss of function. However, the pathophysiological role of potassium channel subfamily K member 3 (KCNK3) in PAH is unclear. We hypothesized that loss of function of KCNK3 is a hallmark of idiopathic and heritable PAH and contributes to dysfunction of pulmonary artery smooth muscle cells and pulmonary artery endothelial cells, leading to pulmonary artery remodeling: consequently, restoring KCNK3 function could alleviate experimental pulmonary hypertension (PH). METHODS AND RESULTS: We demonstrated that KCNK3 expression and function were reduced in human PAH and in monocrotaline-induced PH in rats. Using a patch-clamp technique in freshly isolated (not cultured) pulmonary artery smooth muscle cells and pulmonary artery endothelial cells, we found that KCNK3 current decreased progressively during the development of monocrotaline-induced PH and correlated with plasma-membrane depolarization. We demonstrated that KCNK3 modulated pulmonary arterial tone. Long-term inhibition of KCNK3 in rats induced distal neomuscularization and early hemodynamic signs of PH, which were related to exaggerated proliferation of pulmonary artery endothelial cells, pulmonary artery smooth muscle cell, adventitial fibroblasts, and pulmonary and systemic inflammation. Lastly, in vivo pharmacological activation of KCNK3 significantly reversed monocrotaline-induced PH in rats. CONCLUSIONS: In PAH and experimental PH, KCNK3 expression and activity are strongly reduced in pulmonary artery smooth muscle cells and endothelial cells. KCNK3 inhibition promoted increased proliferation, vasoconstriction, and inflammation. In vivo pharmacological activation of KCNK3 alleviated monocrotaline-induced PH, thus demonstrating that loss of KCNK3 is a key event in PAH pathogenesis and thus could be therapeutically targeted.

Autonomic nervous system involvement in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a chronic pulmonary vascular disease characterized by increased pulmonary vascular resistance (PVR) leading to right ventricular (RV) failure. Autonomic nervous system involvement in the pathogenesis of PAH has been demonstrated several years ago, however the extent of this involvement is not fully understood. PAH is associated with increased sympathetic nervous system (SNS) activation, decreased heart rate variability, and presence of cardiac arrhythmias. There is also evidence for increased renin-angiotensin-aldosterone system (RAAS) activation in PAH patients associated with clinical worsening. Reduction of neurohormonal activation could be an effective therapeutic strategy for PAH. Although therapies targeting adrenergic receptors or RAAS signaling pathways have been shown to reverse cardiac remodeling and improve outcomes in experimental pulmonary hypertension (PH)-models, the effectiveness and safety of such treatments in clinical settings have been uncertain. Recently, novel direct methods such as cervical ganglion block, pulmonary artery denervation (PADN), and renal denervation have been employed to attenuate SNS activation in PAH. In this review, we intend to summarize the multiple aspects of autonomic nervous system involvement in PAH and overview the different pharmacological and invasive strategies used to target autonomic nervous system for the treatment of PAH.

Bromodomain-Containing Protein 4: The Epigenetic Origin of Pulmonary Arterial Hypertension.

RATIONALE: Pulmonary arterial hypertension (PAH) is a vasculopathy characterized by enhanced pulmonary artery (PA) smooth muscle cell (PASMC) proliferation and suppressed apoptosis. Decreased expression of microRNA-204 has been associated to this phenotype. By a still elusive mechanism, microRNA-204 downregulation promotes the expression of oncogenes, including nuclear factor of activated T cells, B-cell lymphoma 2, and Survivin. In cancer, increased expression of the epigenetic reader bromodomain-containing protein 4 (BRD4) sustains cell survival and proliferation. Interestingly, BRD4 is a predicted target of microRNA-204 and has binding sites on the nuclear factor of activated T cells promoter region. OBJECTIVE: To investigate the role of BRD4 in PAH pathogenesis. METHODS AND RESULTS: BRD4 is upregulated in lungs, distal PAs, and PASMCs of patients with PAH compared with controls. With mechanistic in vitro experiments, we demonstrated that BRD4 expression in PAH is microRNA-204 dependent. We further studied the molecular downstream targets of BRD4 by inhibiting its activity in PAH-PASMCs using a clinically available inhibitor JQ1. JQ1 treatment in PAH-PASMCs increased p21 expression, thus triggering cell cycle arrest. Furthermore, BRD4 inhibition, by JQ1 or siBRD4, decreased the expression of 3 major oncogenes, which are overexpressed in PAH: nuclear factor of activated T cells, B-cell lymphoma 2, and Survivin. Blocking this oncogenic signature led to decreased PAH-PASMC proliferation and increased apoptosis in a BRD4-dependent manner. Indeed, pharmacological JQ1 or molecular (siRNA) inhibition of BRD4 reversed this pathological phenotype in addition to restoring mitochondrial membrane potential and to increasing cells spare respiratory capacity. Moreover, BRD4 inhibition in vivo reversed established PAH in the Sugen/hypoxia rat model. CONCLUSIONS: BRD4 plays a key role in the pathological phenotype in PAH, which could offer new therapeutic perspectives for patients with PAH.

Role for Runt-related Transcription Factor 2 in Proliferative and Calcified Vascular Lesions in Pulmonary Arterial Hypertension.

RATIONALE: Pulmonary arterial hypertension (PAH) is characterized by excessive proliferation of pulmonary artery smooth muscle cells (PASMCs). This is sustained in time by the down-regulation of microRNA (miR)-204. In systemic vascular diseases, reduced miR-204 expression promotes vascular biomineralization by augmenting the expression of the transcription factor Runt-related transcription factor 2 (RUNX2). Implication of RUNX2 in PAH-related vascular remodeling and presence of calcified lesions in PAH remain unexplored. OBJECTIVES: We hypothesized that RUNX2 is up-regulated in lungs of patients with PAH, contributing to vascular remodeling and calcium-related biomineralization. METHODS: We harvested human lung tissues in which we assessed calcification lesions and RUNX2 expression. We also isolated PASMCs from these tissues for in vitro analyses. Using a bidirectional approach, we investigated the role for RUNX2 in cell proliferation, apoptosis, and calcification capacity. Ectopic delivery of small interfering RNA against RUNX2 was used in an animal model of PAH to evaluate the therapeutic potential of RUNX2 inhibition in this disease. MEASUREMENTS AND MAIN RESULTS: Patients with PAH display features of calcified lesions within the distal pulmonary arteries (PAs). We show that RUNX2 is up-regulated in lungs, distal PAs, and primary cultured human PASMCs isolated from PAH and compared with patients without PAH. RUNX2 expression histologically correlates with vascular remodeling and calcification. Using in vitro gain- and loss-of-function approaches, we mechanistically demonstrate that miR-204 diminution promotes RUNX2 up-regulation and that sustained RUNX2 expression activates hypoxia-inducible factor-1α, leading to aberrant proliferation, resistance to apoptosis, and subsequent transdifferentiation of PAH-PASMCs into osteoblast-like cells. In the PAH Sugen/hypoxia rat model, molecular RUNX2 inhibition reduces PA remodeling and prevents calcification, thus improving pulmonary hemodynamic parameters and right ventricular function. CONCLUSIONS: RUNX2 plays a pivotal role in the pathogenesis of PAH, contributing to the development of proliferative and calcified PA lesions. Inhibition of RUNX2 may therefore represent an attractive therapeutic strategy for PAH.

Downregulation of MicroRNA-126 Contributes to the Failing Right Ventricle in Pulmonary Arterial Hypertension.

BACKGROUND: Right ventricular (RV) failure is the most important factor of both morbidity and mortality in pulmonary arterial hypertension (PAH). However, the underlying mechanisms resulting in the failed RV in PAH remain unknown. There is growing evidence that angiogenesis and microRNAs are involved in PAH-associated RV failure. We hypothesized that microRNA-126 (miR-126) downregulation decreases microvessel density and promotes the transition from a compensated to a decompensated RV in PAH. METHODS AND RESULTS: We studied RV free wall tissues from humans with normal RV (n=17), those with compensated RV hypertrophy (n=8), and patients with PAH with decompensated RV failure (n=14). Compared with RV tissues from patients with compensated RV hypertrophy, patients with decompensated RV failure had decreased miR-126 expression (quantitative reverse transcription-polymerase chain reaction; P<0.01) and capillary density (CD31(+) immunofluorescence; P<0.001), whereas left ventricular tissues were not affected. miR-126 downregulation was associated with increased Sprouty-related EVH1 domain-containing protein 1 (SPRED-1), leading to decreased activation of RAF (phosphorylated RAF/RAF) and mitogen-activated protein kinase (MAPK); (phosphorylated MAPK/MAPK), thus inhibiting the vascular endothelial growth factor pathway. In vitro, Matrigel assay showed that miR-126 upregulation increased angiogenesis of primary cultured endothelial cells from patients with decompensated RV failure. Furthermore, in vivo miR-126 upregulation (mimic intravenous injection) improved cardiac vascular density and function of monocrotaline-induced PAH animals. CONCLUSIONS: RV failure in PAH is associated with a specific molecular signature within the RV, contributing to a decrease in RV vascular density and promoting the progression to RV failure. More importantly, miR-126 upregulation in the RV improves microvessel density and RV function in experimental PAH.

Novel biomarkers for pulmonary arterial hypertension.

Pulmonary arterial hypertension is a deadly disease characterized by elevated pulmonary arterial pressures leading to right ventricular hypertrophy and failure. The confirmatory gold standard test is the invasive right heart catheterization. The disease course is monitored by pulmonary artery systolic pressure measurement via transthoracic echocardiography. A simple non-invasive test to frequently monitor the patients is much needed. Search for a novel biomarker that can be detected by a simple test is ongoing and many different options are being studied. Here we review some of the new and unique pre-clinical options for potential pulmonary hypertension biomarkers. These biomarkers can be broadly categorized based on their association with endothelial cell dysfunction, inflammation, epigenetics, cardiac function, oxidative stress, metabolism,extracellular matrix, and volatile compounds in exhaled breath condensate. A biomarker that can be detected in blood, urine or breath condensate and correlates with disease severity, progression and response to therapy may result in significant cost reduction and improved patient outcomes.

Functions of RNA-Binding Proteins in Cardiovascular Disease.

Gene expression is under tight regulation from the chromatin structure that regulates gene accessibility by the transcription machinery to protein degradation. At the transcript level, this regulation falls on RNA-binding proteins (RBPs). RBPs are a large and diverse class of proteins involved in all aspects of a transcript's lifecycle: splicing and maturation, localization, stability, and translation. In the past few years, our understanding of the role of RBPs in cardiovascular diseases has expanded. Here, we discuss the general structure and function of RBPs and the latest discoveries of their role in pulmonary and systemic cardiovascular diseases.

Myocardial fibrosis and calcification are attenuated by microRNA-129-5p targeting Asporin and Sox9 in cardiac fibroblasts.

Myocardial fibrosis and calcification associate with adverse outcomes in nonischemic heart failure. Cardiac fibroblasts (CF) transition into myofibroblasts (MF) and osteogenic fibroblasts (OF) to promote myocardial fibrosis and calcification. However, common upstream mechanisms regulating both CF-to-MF transition and CF-to-OF transition remain unknown. microRNAs are promising targets to modulate CF plasticity. Our bioinformatics revealed downregulation of miR-129-5p and upregulation of its targets small leucine-rich proteoglycan Asporin (ASPN) and transcription factor SOX9 as common in mouse and human heart failure (HF). We experimentally confirmed decreased miR-129-5p and enhanced SOX9 and ASPN expression in CF in human hearts with myocardial fibrosis and calcification. miR-129-5p repressed both CF-to-MF and CF-to-OF transition in primary CF, as did knockdown of SOX9 and ASPN. Sox9 and Aspn are direct targets of miR-129-5p that inhibit downstream ß-catenin expression. Chronic Angiotensin II infusion downregulated miR-129-5p in CF in WT and TCF21-lineage CF reporter mice, and it was restored by miR-129-5p mimic. Importantly, miR-129-5p mimic not only attenuated progression of myocardial fibrosis, calcification marker expression, and SOX9 and ASPN expression in CF but also restored diastolic and systolic function. Together, we demonstrate miR-129-5p/ASPN and miR-129-5p/SOX9 as potentially novel dysregulated axes in CF-to-MF and CF-to-OF transition in myocardial fibrosis and calcification and the therapeutic relevance of miR-129-5p.

Involvement of Low-Density Lipoprotein Receptor in the Pathogenesis of Pulmonary Hypertension.

Background Recently, we and others have reported a causal role for oxidized lipids in the pathogenesis of pulmonary hypertension (PH). However, the role of low-density lipoprotein receptor (LDL-R) in PH is not known. Methods and Results We examined the role of LDL-R in the development of PH and determined the efficacy of high-density lipoprotein mimetic peptide 4F in mitigating PH. Explanted human lungs and plasma from patients with PH and control subjects were analyzed for gene expression, histological characteristics, and lipoprotein oxidation. Male LDL-R null (LDL-R knockout) mice (12-15 months old) were fed chow, Western diet (WD), WD with 4F, and WD with scramble peptide for 12 weeks. Serial echocardiography, cardiac catheterization, oxidized LDL assay, real-time quantitative reverse transcription-polymerase chain reaction, and histological analysis were performed. The effect of LDL-R knockdown and oxidized LDL on human pulmonary artery smooth muscle cell proliferation was assessed in vitro. LDL-R and CD36 expression levels were significantly downregulated in the lungs of patients with PH. Patients with PH also had increased lung lipid deposits, oxidized LDL, E06 immunoreactivity, and plasma oxidized LDL/LDL ratio. LDL-R knockout mice on WD developed PH, right ventricular hypertrophy, right ventricular dysfunction, pulmonary vascular remodeling, fibrosis, and lipid deposition in lungs, aortic atherosclerosis, and left ventricular dysfunction, which were prevented by 4F. Interestingly, PH in WD group preceded left ventricular dysfunction. Oxidized LDL or LDL-R knockdown significantly increased proliferation of human pulmonary artery smooth muscle cells in vitro. Conclusions Human PH is associated with decreased LDL-R in lungs and increased oxidized LDL in lungs and plasma. WD-fed LDL-R knockout mice develop PH and right ventricular dysfunction, implicating a role for LDL-R and oxidized lipids in PH.

Radiation mitigation of the intestinal acute radiation injury in mice by 1-[(4-nitrophenyl)sulfonyl]-4-phenylpiperazine.

The objective of the study was to identify the mechanism of action for a radiation mitigator of the gastrointestinal (GI) acute radiation syndrome (ARS), identified in an unbiased high-throughput screen. We used mice irradiated with a lethal dose of radiation and treated with daily injections of the radiation mitigator 1-[(4-nitrophenyl)sulfonyl]-4-phenylpiperazine to study its effects on key pathways involved in intestinal stem cell (ISC) maintenance. RNASeq, quantitative reverse transcriptase-polymerase chain reaction, and immunohistochemistry were performed to identify pathways engaged after drug treatment. Target validation was performed with competition assays, reporter cells, and in silico docking. 1-[(4-Nitrophenyl)sulfonyl]-4-phenylpiperazine activates Hedgehog signaling by binding to the transmembrane domain of Smoothened, thereby expanding the ISC pool, increasing the number of regenerating crypts and preventing the GI-ARS. We conclude that Smoothened is a target for radiation mitigation in the small intestine that could be explored for use in radiation accidents as well as to mitigate normal tissue toxicity during and after radiotherapy of the abdomen.