Induction of AML cell differentiation using HOXA9/DNA binding inhibitors as a potential therapeutic option for HOXA9-dependent AML.

The mainstay of acute myeloid leukemia (AML) treatment still relies on traditional chemotherapy, with a survival rate of approximately 30% for patients under 65 years of age and as low as 5% for those beyond. This unfavorable prognosis primarily stems from frequent relapses, resistance to chemotherapy, and limited approved targeted therapies for specific AML subtypes. Around 70% of all AML cases show overexpression of the transcription factor HOXA9, which is associated with a poor prognosis, increased chemoresistance, and higher relapse rates. However, direct targeting of HOXA9 in a clinical setting has not been achieved yet. The dysregulation caused by the leukemic HOXA9 transcription factor primarily results from its binding activity to DNA, leading to differentiation blockade. Our previous investigations have identified two HOXA9/DNA binding competitors, namely DB1055 and DB818. We assessed their antileukemic effects in comparison to HOXA9 knockdown or cytarabine treatment. Using human AML cell models, DB1055 and DB818 induced in vitro cell growth reduction, death, differentiation, and common transcriptomic deregulation but did not impact human CD34+ bone marrow cells. Furthermore, DB1055 and DB818 exhibited potent antileukemic activities in a human THP-1 AML in vivo model, leading to the differentiation of monocytes into macrophages. In vitro assays also demonstrated the efficacy of DB1055 and DB818 against AML blasts from patients, with DB1055 successfully reducing leukemia burden in patient-derived xenografts in NSG immunodeficient mice. Our findings indicate that inhibiting HOXA9/DNA interaction using DNA ligands may offer a novel differentiation therapy for the future treatment of AML patients dependent on HOXA9.

The Impact of Maternal Obesity on Adipose Progenitor Cells.

The concept of Developmental Origin of Health and Disease (DOHaD) postulates that adult-onset metabolic disorders may originate from suboptimal conditions during critical embryonic and fetal programming windows. In particular, nutritional disturbance during key developmental stages may program the set point of adiposity and its associated metabolic diseases later in life. Numerous studies in mammals have reported that maternal obesity and the resulting accelerated growth in neonates may affect adipocyte development, resulting in persistent alterations in adipose tissue plasticity (i.e., adipocyte proliferation and storage) and adipocyte function (i.e., insulin resistance, impaired adipokine secretion, reduced thermogenesis, and higher inflammation) in a sex- and depot-specific manner. Over recent years, adipose progenitor cells (APCs) have been shown to play a crucial role in adipose tissue plasticity, essential for its development, maintenance, and expansion. In this review, we aim to provide insights into the developmental timeline of lineage commitment and differentiation of APCs and their role in predisposing individuals to obesity and metabolic diseases. We present data supporting the possible implication of dysregulated APCs and aberrant perinatal adipogenesis through epigenetic mechanisms as a primary mechanism responsible for long-lasting adipose tissue dysfunction in offspring born to obese mothers.

KCNK3 channel is important for the ventilatory response to hypoxia in rats.

To clarify the contribution of KCNK3/TASK-1 channel chemoreflex in response to hypoxia and hypercapnia, we used a unique Kcnk3-deficient rat. We assessed ventilatory variables using plethysmography in Kcnk3-deficient and wild-type rats at rest in response to hypoxia (10% O2) and hypercapnia (4% CO2). Immunostaining for C-Fos, a marker of neuronal activity, was performed to identify the regions of the respiratory neuronal network involved in the observed response.Under basal conditions, we observed increased minute ventilation in Kcnk3-deficient rats, which was associated with increased c-Fos positive cells in the ventrolateral region of the medulla oblongata. Kcnk3-deficient rats show an increase in ventilatory response to hypoxia without changes in response to hypercapnia. In Kcnk3-deficient rats, linked to an increased hypoxia response, we observed a greater increase in c-Fos-positive cells in the first central relay of peripheral chemoreceptors and Raphe Obscurus. This study reports that KCNK3/TASK-1 deficiency in rats induces an inadequate peripheral chemoreflex, alternating respiratory rhythmogenesis, and hypoxic chemoreflex.

Application of Deep Learning Algorithm to Monitor Upper Extremity Task Practice.

Upper extremity hemiplegia is a serious problem affecting the lives of many people post-stroke. Motor recovery requires high repetitions and quality of task-specific practice. Sufficient practice cannot be completed during therapy sessions, requiring patients to perform additional task practices at home on their own. Adherence to and quality of these home task practices are often limited, which is likely a factor reducing rehabilitation effectiveness post-stroke. However, home adherence is typically measured by self-reports that are known to be inconsistent with objective measurement. The objective of this study was to develop algorithms to enable the objective identification of task type and quality. Twenty neurotypical participants wore an IMU sensor on the wrist and performed four representative tasks in prescribed fashions that mimicked correct, compensatory, and incomplete movement qualities typically seen in stroke survivors. LSTM classifiers were trained to identify the task being performed and its movement quality. Our models achieved an accuracy of 90.8% for task identification and 84.9%, 81.1%, 58.4%, and 73.2% for movement quality classification for the four tasks for unseen participants. The results warrant further investigation to determine the classification performance for stroke survivors and if quantity and quality feedback from objective monitoring facilitates effective task practice at home, thereby improving motor recovery.

Angiopoietin-Like Proteins: Cardiovascular Biology and Therapeutic Targeting for the Prevention of Cardiovascular Diseases.

Despite the best pharmacologic tools available, cardiovascular diseases (CVDs) remain a major cause of morbidity and mortality in developed countries. After 2 decades of research, new therapeutic targets, such as angiopoietin-like proteins (ANGPTLs), are emerging. ANGPTLs belong to a family of 8 members, from ANGPTL1 to ANGPTL8; they have structural homology with angiopoietins and are secreted in the circulation. ANGPTLs display a multitude of physiological and pathologic functions; they contribute to inflammation, angiogenesis, cell death, senescence, hematopoiesis, and play a role in repair, maintenance, and tissue homeostasis. ANGPTLs-particularly the triad ANGPTL3, 4, and 8-have an established role in lipid metabolism through the regulation of triacylglycerol trafficking according to the nutritional status. Some ANGPTLs also contribute to glucose metabolism. Therefore, dysregulation in ANGPTL expression associated with abnormal circulating levels are linked to a plethora of CVD and metabolic disorders including atherosclerosis, heart diseases, diabetes, but also obesity and cancers. Because ANGPTLs bind to different receptors according to the cell type, antagonists are therapeutically inadequate. Recently, direct inhibitors of ANGPTLs, mainly ANGPTL3, have been developed, and specific monoclonal antibodies and antisense oligonucleotides are currently being tested in clinical trials. The aim of the current review is to provide an up-to-date preclinical and clinical overview on the function of the 8 members of the ANGPTL family in the cardiovascular system, their contribution to CVD, and the therapeutic potential of manipulating some of them.

Importance of the Microenvironment and Mechanosensing in Adipose Tissue Biology.

The expansion of adipose tissue is an adaptive mechanism that increases nutrient buffering capacity in response to an overall positive energy balance. Over the course of expansion, the adipose microenvironment undergoes continual remodeling to maintain its structural and functional integrity. However, in the long run, adipose tissue remodeling, typically characterized by adipocyte hypertrophy, immune cells infiltration, fibrosis and changes in vascular architecture, generates mechanical stress on adipose cells. This mechanical stimulus is then transduced into a biochemical signal that alters adipose function through mechanotransduction. In this review, we describe the physical changes occurring during adipose tissue remodeling, and how they regulate adipose cell physiology and promote obesity-associated dysfunction in adipose tissue.

SUR1 As a New Therapeutic Target for Pulmonary Arterial Hypertension.

Mutations in ABCC8 have been identified in pulmonary arterial hypertension (PAH). ABCC8 encodes SUR1, a regulatory subunit of the ATP-sensitive potassium channel Kir6.2. However, the pathophysiological role of the SUR1/Kir6.2 channel in PAH is unknown. We hypothesized that activation of SUR1 could be a novel potential target for PAH. We analyzed the expression of SUR1/Kir6.2 in the lungs and pulmonary artery (PA) in human PAH or experimental pulmonary hypertension (PH). The contribution of SUR1 in human or rat PA tone was evaluated, and we measured the consequences of in vivo activation of SUR1 in control and PH rats. SUR1 and Kir6.2 protein expression was not reduced in the lungs or human pulmonary arterial endothelial cells and smooth muscle cells from PAH or experimentally induced PH. We showed that pharmacological activation of SUR1 by three different SUR1 activators (diazoxide, VU0071063, and NN414) leads to PA relaxation. Conversely, the inhibition of SUR1/Kir6.2 channels causes PA constriction. In vivo, long- and short-term activation of SUR1 with diazoxide reversed monocrotaline-induced PH in rats. In addition, in vivo diazoxide application (short protocol) reduced the severity of PH in chronic-hypoxia rats. Moreover, 3 weeks of diazoxide exposure in control rats had no cardiovascular effects. Finally, in vivo, activation of SUR1 with NN414 reduced monocrotaline-induced PH in rats. In PAH and experimental PH, the expression of SUR1/Kir6.2 was still present. In vivo pharmacological SUR1 activation by two different molecules alleviated experimental PH, providing proof of concept that SUR1 activation should be considered for PAH and evaluated more thoroughly.

A novel CD34-specific T-cell engager efficiently depletes acute myeloid leukemia and leukemic stem cells in vitro and in vivo.

Less than a third of patients with acute myeloid leukemia (AML) are cured by chemotherapy and/or hematopoietic stem cell transplantation, highlighting the need to develop more efficient drugs. The low efficacy of standard treatments is associated with inadequate depletion of CD34+ blasts and leukemic stem cells, the latter a drug-resistant subpopulation of leukemia cells characterized by the CD34+CD38- phenotype. To target these drug-resistant primitive leukemic cells better, we have designed a CD34/CD3 bi-specific T-cell engager (BTE) and characterized its anti-leukemia potential in vitro, ex vivo and in vivo. Our results show that this CD34-specific BTE induces CD34-dependent T-cell activation and subsequent leukemia cell killing in a dose-dependent manner, further corroborated by enhanced T-cell-mediated killing at the singlecell level. Additionally, the BTE triggered efficient T-cell-mediated depletion of CD34+ hematopoietic stem cells from peripheral blood stem cell grafts and CD34+ blasts from AML patients. Using a humanized AML xenograft model, we confirmed that the CD34-specific BTE had in vivo efficacy by depleting CD34+ blasts and leukemic stem cells without side effects. Taken together, these data demonstrate that the CD34-specific BTE has robust antitumor effects, supporting development of a novel treatment modality with the aim of improving outcomes of patients with AML and myelodysplastic syndromes.

Angptl2 is a Marker of Cellular Senescence: The Physiological and Pathophysiological Impact of Angptl2-Related Senescence.

Cellular senescence is a cell fate primarily induced by DNA damage, characterized by irreversible growth arrest in an attempt to stop the damage. Senescence is a cellular response to a stressor and is observed with aging, but also during wound healing and in embryogenic developmental processes. Senescent cells are metabolically active and secrete a multitude of molecules gathered in the senescence-associated secretory phenotype (SASP). The SASP includes inflammatory cytokines, chemokines, growth factors and metalloproteinases, with autocrine and paracrine activities. Among hundreds of molecules, angiopoietin-like 2 (angptl2) is an interesting, although understudied, SASP member identified in various types of senescent cells. Angptl2 is a circulatory protein, and plasma angptl2 levels increase with age and with various chronic inflammatory diseases such as cancer, atherosclerosis, diabetes, heart failure and a multitude of age-related diseases. In this review, we will examine in which context angptl2 was identified as a SASP factor, describe the experimental evidence showing that angptl2 is a marker of senescence in vitro and in vivo, and discuss the impact of angptl2-related senescence in both physiological and pathological conditions. Future work is needed to demonstrate whether the senescence marker angptl2 is a potential clinical biomarker of age-related diseases.

Right Ventricle Remodeling Metabolic Signature in Experimental Pulmonary Hypertension Models of Chronic Hypoxia and Monocrotaline Exposure.

INTRODUCTION: Over time and despite optimal medical management of patients with pulmonary hypertension (PH), the right ventricle (RV) function deteriorates from an adaptive to maladaptive phenotype, leading to RV failure (RVF). Although RV function is well recognized as a prognostic factor of PH, no predictive factor of RVF episodes has been elucidated so far. We hypothesized that determining RV metabolic alterations could help to understand the mechanism link to the deterioration of RV function as well as help to identify new biomarkers of RV failure. METHODS: In the current study, we aimed to characterize the metabolic reprogramming associated with the RV remodeling phenotype during experimental PH induced by chronic-hypoxia-(CH) exposure or monocrotaline-(MCT) exposure in rats. Three weeks after PH initiation, we hemodynamically characterized PH (echocardiography and RV catheterization), and then we used an untargeted metabolomics approach based on liquid chromatography coupled to high-resolution mass spectrometry to analyze RV and LV tissues in addition to plasma samples from MCT-PH and CH-PH rat models. RESULTS: CH exposure induced adaptive RV phenotype as opposed to MCT exposure which induced maladaptive RV phenotype. We found that predominant alterations of arginine, pyrimidine, purine, and tryptophan metabolic pathways were detected on the heart (LV+RV) and plasma samples regardless of the PH model. Acetylspermidine, putrescine, guanidinoacetate RV biopsy levels, and cytosine, deoxycytidine, deoxyuridine, and plasmatic thymidine levels were correlated to RV function in the CH-PH model. It was less likely correlated in the MCT model. These pathways are well described to regulate cell proliferation, cell hypertrophy, and cardioprotection. These findings open novel research perspectives to find biomarkers for early detection of RV failure in PH.

Optimisation of the Synthesis and Cell Labelling Conditions for [89Zr]Zr-oxine and [89Zr]Zr-DFO-NCS: a Direct In Vitro Comparison in Cell Types with Distinct Therapeutic Applications.

BACKGROUND: There is a need to better characterise cell-based therapies in preclinical models to help facilitate their translation to humans. Long-term high-resolution tracking of the cells in vivo is often impossible due to unreliable methods. Radiolabelling of cells has the advantage of being able to reveal cellular kinetics in vivo over time. This study aimed to optimise the synthesis of the radiotracers [89Zr]Zr-oxine (8-hydroxyquinoline) and [89Zr]Zr-DFO-NCS (p-SCN-Bn-Deferoxamine) and to perform a direct comparison of the cell labelling efficiency using these radiotracers. PROCEDURES: Several parameters, such as buffers, pH, labelling time and temperature, were investigated to optimise the synthesis of [89Zr]Zr-oxine and [89Zr]Zr-DFO-NCS in order to reach a radiochemical conversion (RCC) of >95 % without purification. Radio-instant thin-layer chromatography (iTLC) and radio high-performance liquid chromatography (radio-HPLC) were used to determine the RCC. Cells were labelled with [89Zr]Zr-oxine or [89Zr]Zr-DFO-NCS. The cellular retention of 89Zr and the labelling impact was determined by analysing the cellular functions, such as viability, proliferation, phagocytotic ability and phenotypic immunostaining. RESULTS: The optimised synthesis of [89Zr]Zr-oxine and [89Zr]Zr-DFO-NCS resulted in straightforward protocols not requiring additional purification. [89Zr]Zr-oxine and [89Zr]Zr-DFO-NCS were synthesised with an average RCC of 98.4 % (n = 16) and 98.0 % (n = 13), respectively. Cell labelling efficiencies were 63.9 % (n = 35) and 70.2 % (n = 30), respectively. 89Zr labelling neither significantly affected the cell viability (cell viability loss was in the range of 1-8 % compared to its corresponding non-labelled cells, P value > 0.05) nor the cells' proliferation rate. The phenotype of human decidual stromal cells (hDSC) and phagocytic function of rat bone-marrow-derived macrophages (rMac) was somewhat affected by radiolabelling. CONCLUSIONS: Our study demonstrates that [89Zr]Zr-oxine and [89Zr]Zr-DFO-NCS are equally effective in cell labelling. However, [89Zr]Zr-oxine was superior to [89Zr]Zr-DFO-NCS with regard to long-term stability, cellular retention, minimal variation between cell types and cell labelling efficiency.

Involvement of CFTR in the pathogenesis of pulmonary arterial hypertension.

INTRODUCTION: A reduction in pulmonary artery relaxation is a key event in the pathogenesis of pulmonary arterial hypertension (PAH). Cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction in airway epithelial cells plays a central role in cystic fibrosis; CFTR is also expressed in pulmonary arteries and has been shown to control endothelium-independent relaxation. AIM AND OBJECTIVES: We aimed to delineate the role of CFTR in PAH pathogenesis through observational and interventional experiments in human tissues and animal models. METHODS AND RESULTS: Reverse-transcriptase quantitative PCR, confocal imaging and electron microscopy showed that CFTR expression was reduced in pulmonary arteries from patients with idiopathic PAH (iPAH) and in rats with monocrotaline-induced pulmonary hypertension (PH). Moreover, using myography on human, pig and rat pulmonary arteries, we demonstrated that CFTR activation induces pulmonary artery relaxation. CFTR-mediated pulmonary artery relaxation was reduced in pulmonary arteries from iPAH patients and rats with monocrotaline- or chronic hypoxia-induced PH. Long-term in vivo CFTR inhibition in rats significantly increased right ventricular systolic pressure, which was related to exaggerated pulmonary vascular cell proliferation in situ and vessel neomuscularisation. Pathologic assessment of lungs from patients with severe cystic fibrosis (F508del-CFTR) revealed severe pulmonary artery remodelling with intimal fibrosis and medial hypertrophy. Lungs from homozygous F508delCftr rats exhibited pulmonary vessel neomuscularisation. The elevations in right ventricular systolic pressure and end diastolic pressure in monocrotaline-exposed rats with chronic CFTR inhibition were more prominent than those in vehicle-exposed rats. CONCLUSIONS: CFTR expression is strongly decreased in pulmonary artery smooth muscle and endothelial cells in human and animal models of PH. CFTR inhibition increases vascular cell proliferation and strongly reduces pulmonary artery relaxation.

Kcnk3 dysfunction exaggerates the development of pulmonary hypertension induced by left ventricular pressure overload.

AIMS: Pulmonary hypertension (PH) is a common complication of left heart disease (LHD, Group 2 PH) leading to right ventricular (RV) failure and death. Several loss-of-function (LOF) mutations in KCNK3 were identified in pulmonary arterial hypertension (PAH, Group 1 PH). Additionally, we found that KCNK3 dysfunction is a hallmark of PAH at pulmonary vascular and RV levels. However, the role of KCNK3 in the pathobiology of PH due to LHD is unknown. METHODS AND RESULTS: We evaluated the role of KCNK3 on PH induced by ascending aortic constriction (AAC), in WT and Kcnk3-LOF-mutated rats, by echocardiography, RV catheterization, histology analyses, and molecular biology experiments. We found that Kcnk3-LOF-mutation had no consequence on the development of left ventricular (LV) compensated concentric hypertrophy in AAC, while left atrial emptying fraction was impaired in AAC-Kcnk3-mutated rats. AAC-animals (WT and Kcnk3-mutated rats) developed PH secondary to AAC and Kcnk3-mutated rats developed more severe PH than WT. AAC-Kcnk3-mutated rats developed RV and LV fibrosis in association with an increase of Col1a1 mRNA in right ventricle and left ventricle. AAC-Kcnk3-mutated rats developed severe pulmonary vascular (pulmonary artery as well as pulmonary veins) remodelling with intense peri-vascular and peri-bronchial inflammation, perivascular oedema, alveolar wall thickening, and exaggerated lung vascular cell proliferation compared to AAC-WT-rats. Finally, in lung, right ventricle, left ventricle, and left atrium of AAC-Kcnk3-mutated rats, we found a strong increased expression of Il-6 and periostin expression and a reduction of lung Ctnnd1 mRNA (coding for p120 catenin), contributing to the exaggerated pulmonary and heart remodelling and pulmonary vascular oedema in AAC-Kcnk3-mutated rats. CONCLUSIONS: Our results indicate that Kcnk3-LOF is a key event in the pathobiology of PH due to AAC, suggesting that Kcnk3 channel dysfunction could play a potential key role in the development of PH due to LHD.

Targeted proteomics of right heart adaptation to pulmonary arterial hypertension.

No prior proteomic screening study has centred on the right ventricle (RV) in pulmonary arterial hypertension (PAH). This study investigates the circulating proteomic profile associated with right heart maladaptive phenotype (RHMP) in PAH.Plasma proteomic profiling was performed using multiplex immunoassay in 121 (discovery cohort) and 76 (validation cohort) PAH patients. The association between proteomic markers and RHMP, defined by the Mayo right heart score (combining RV strain, New York Heart Association (NYHA) class and N-terminal pro-brain natriuretic peptide (NT-proBNP)) and Stanford score (RV end-systolic remodelling index, NYHA class and NT-proBNP), was assessed by partial least squares regression. Biomarker expression was measured in RV samples from PAH patients and controls, and pulmonary artery banding (PAB) mice.High levels of hepatocyte growth factor (HGF), stem cell growth factor-ß, nerve growth factor and stromal derived factor-1 were associated with worse Mayo and Stanford scores independently from pulmonary resistance or pressure in both cohorts (the validation cohort had more severe disease features: lower cardiac index and higher NT-proBNP). In both cohorts, HGF added value to the REVEAL score in the prediction of death, transplant or hospitalisation at 3 years. RV expression levels of HGF and its receptor c-Met were higher in end-stage PAH patients than controls, and in PAB mice than shams.High plasma HGF levels are associated with RHMP and predictive of 3-year clinical worsening. Both HGF and c-Met RV expression levels are increased in PAH. Assessing plasma HGF levels might identify patients at risk of heart failure who warrant closer follow-up and intensified therapy.

Proteomic Analysis of KCNK3 Loss of Expression Identified Dysregulated Pathways in Pulmonary Vascular Cells.

The physiopathology of pulmonary arterial hypertension (PAH) is characterized by pulmonary artery smooth muscle cell (PASMC) and endothelial cell (PAEC) dysfunction, contributing to pulmonary arterial obstruction and PAH progression. KCNK3 loss of function mutations are responsible for the first channelopathy identified in PAH. Loss of KCNK3 function/expression is a hallmark of PAH. However, the molecular mechanisms involved in KCNK3 dysfunction are mostly unknown. To identify the pathological molecular mechanisms downstream of KCNK3 in human PASMCs (hPASMCs) and human PAECs (hPAECs), we used a Liquid Chromatography-Tandem Mass Spectrometry-based proteomic approach to identify the molecular pathways regulated by KCNK3. KCNK3 loss of expression was induced in control hPASMCs or hPAECs by specific siRNA targeting KCNK3. We found that the loss of KCNK3 expression in hPAECs and hPASMCs leads to 326 and 222 proteins differentially expressed, respectively. Among them, 53 proteins were common to hPAECs and hPASMCs. The specific proteome remodeling in hPAECs in absence of KCNK3 was mostly related to the activation of glycolysis, the superpathway of methionine degradation, and the mTOR signaling pathways, and to a reduction in EIF2 signaling pathways. In hPASMCs, we found an activation of the PI3K/AKT signaling pathways and a reduction in EIF2 signaling and the Purine Nucleotides De Novo Biosynthesis II and IL-8 signaling pathways. Common to hPAECs and hPASMCs, we found that the loss of KCNK3 expression leads to the activation of the NRF2-mediated oxidative stress response and a reduction in the interferon pathway. In the hPAECs and hPASMCs, we found an increased expression of HO-1 (heme oxygenase-1) and a decreased IFIT3 (interferon-induced proteins with tetratricopeptide repeats 3) (confirmed by Western blotting), allowing us to identify these axes to understand the consequences of KCNK3 dysfunction. Our experiments, based on the loss of KCNK3 expression by a specific siRNA strategy in control hPAECs and hPASMCs, allow us to identify differences in the activation of several signaling pathways, indicating the key role played by KCNK3 dysfunction in the development of PAH. Altogether, these results allow us to better understand the consequences of KCNK3 dysfunction and suggest that KCNK3 loss of expression acts in favor of the proliferation and migration of hPASMCs and promotes the metabolic shift and apoptosis resistance of hPAECs.

In vivo miR-138-5p inhibition alleviates monocrotaline-induced pulmonary hypertension and normalizes pulmonary KCNK3 and SLC45A3 expression.

BACKGROUND: The pathogenesis of pulmonary arterial hypertension (PAH) involves many signalling pathways. MicroRNAs are potential candidates involved in simultaneously coordinating multiple genes under such multifactorial conditions. METHODS AND RESULTS: MiR-138-5p is overexpressed in pulmonary arterial smooth muscle cells (PASMCs) from PAH patients and in lungs from rats with monocrotaline-induced pulmonary hypertension (MCT-PH). MiR-138-5p is predicted to regulate the expression of the potassium channel KCNK3, whose loss is associated with the development and progression of PAH. We hypothesized that, in vivo, miR-138-5p inhibition would restore KCNK3 lung expression and subsequently alleviate PAH. Nebulization-based delivery of anti-miR-138-5p to rats with established MCT-PH significantly reduced the right ventricular systolic pressure and significantly improved the pulmonary arterial acceleration time (PAAT). These haemodynamic improvements were related to decrease pulmonary vascular remodelling, lung inflammation and pulmonary vascular cell proliferation in situ. In vivo inhibition of miR-138-5p restored KCNK3 mRNA expression and SLC45A3 protein expression in the lungs. CONCLUSIONS: We confirmed that in vivo inhibition of miR-138-5p reduces the development of PH in experimental MCT-PH. The possible curative mechanisms involve at least the normalization of lung KCNK3 as well as SLC45A3 expression.

Comparison of Human and Experimental Pulmonary Veno-Occlusive Disease.

Pulmonary veno-occlusive disease (PVOD) occurs in humans either as a heritable form (hPVOD) due to biallelic inactivating mutations of EIF2AK4 (encoding GCN2) or as a sporadic form in older age (sPVOD). The chemotherapeutic agent mitomycin C (MMC) is a potent inducer of PVOD in humans and in rats (MMC-PVOD). Here, we compared human hPVOD and sPVOD, and MMC-PVOD pathophysiology at the histological, cellular, and molecular levels to unravel common altered pathomechanisms. MMC exposure in rats was associated primarily with arterial and microvessel remodeling, and secondarily by venous remodeling, when PVOD became symptomatic. In all forms of PVOD tested, there was convergent GCN2-dependent but eIF2α-independent pulmonary protein overexpression of HO-1 (heme oxygenase 1) and CHOP (CCAAT-enhancer-binding protein [C/EBP] homologous protein), two downstream effectors of GCN2 signaling and endoplasmic reticulum stress. In human PVOD samples, CHOP immunohistochemical staining mainly labeled endothelial cells in remodeled veins and arteries. Strong HO-1 staining was observed only within capillary hemangiomatosis foci, where intense microvascular proliferation occurs. HO-1 and CHOP stainings were not observed in control and pulmonary arterial hypertension lung tissues, supporting the specificity for CHOP and HO-1 involvement in PVOD pathobiology. In vivo loss of GCN2 (EIF2AK4 mutations carriers and Eif2ak4-/- rats) or in vitro GCN2 inhibition in cultured pulmonary artery endothelial cells using pharmacological and siRNA approaches demonstrated that GCN2 loss of function negatively regulates BMP (bone morphogenetic protein)-dependent SMAD1/5/9 signaling. Exogenous BMP9 was still able to reverse GCN2 inhibition-induced proliferation of pulmonary artery endothelial cells. In conclusion, we identified CHOP and HO-1 inhibition, and BMP9, as potential therapeutic options for PVOD.

CRISPR/Cas9-Based Gene Engineering of Human Natural Killer Cells: Protocols for Knockout and Readouts to Evaluate Their Efficacy.

Natural killer (NK) cells are cytotoxic lymphocytes of our immune system with the ability to identify and kill certain virally infected and tumor-transformed cells. During the past 15 years, it has become increasingly clear that NK cells are involved in tumor immune surveillance and that they can be utilized to treat cancer patients. However, their ability to induce durable responses in settings of adoptive cell therapy needs to be further improved. One possible approach is to genetically engineer NK cells to augment their cytotoxicity per se, but also their ability to persist in vivo and home to the tumor-bearing tissue. In recent years, investigators have explored the potential of viral transduction and mRNA electroporation to modify NK cells. Although these methods have generated promising data, they are associated with certain limitations. With the increasing advances in the CRISPR/Cas9 technology, investigators have now turned their attention toward using this technology with NK cells as an alternative method. In this book chapter, we introduce NK cells and provide an historical overview of techniques to genetically engineer lymphocytes. Further, we elucidate protocols for inducing double-strand breaks in NK cells via CRISPR/Cas9 together with readouts to address its efficacy and functional outcome. We also discuss the pros and cons of the described readouts. The overall aim of this book chapter is to help introduce the CRISPR/Cas9 technology to the broader audience of NK cell researchers.