BMPR2 Loss Activates AKT by Disrupting DLL4/NOTCH1 and PPARγ Signaling in Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a progressive cardiopulmonary disease characterized by pathologic vascular remodeling of small pulmonary arteries. Endothelial dysfunction in advanced PAH is associated with proliferation, apoptosis resistance, and endothelial to mesenchymal transition (EndoMT) due to aberrant signaling. DLL4, a cell membrane associated NOTCH ligand, plays a pivotal role maintaining vascular integrity. Inhibition of DLL4 has been associated with the development of pulmonary hypertension, but the mechanism is incompletely understood. Here we report that BMPR2 silencing in pulmonary artery endothelial cells (PAECs) activated AKT and suppressed the expression of DLL4. Consistent with these in vitro findings, increased AKT activation and reduced DLL4 expression was found in the small pulmonary arteries of patients with PAH. Increased NOTCH1 activation through exogenous DLL4 blocked AKT activation, decreased proliferation and reversed EndoMT. Exogenous and overexpression of DLL4 induced BMPR2 and PPRE promoter activity, and BMPR2 and PPARG mRNA in idiopathic PAH (IPAH) ECs. PPARγ, a nuclear receptor associated with EC homeostasis, suppressed by BMPR2 loss was induced and activated by DLL4/NOTCH1 signaling in both BMPR2-silenced and IPAH ECs, reversing aberrant phenotypic changes, in part through AKT inhibition. Directly blocking AKT or restoring DLL4/NOTCH1/PPARγ signaling may be beneficial in preventing or reversing the pathologic vascular remodeling of PAH.

Identification of Prognostic Genes in Acute Myeloid Leukemia Microenvironment: A Bioinformatic and Experimental Analysis.

Acute myeloid leukemia (AML) is a lethal hematologic malignancy with a variable prognosis that is highly dependent on the bone marrow microenvironment. Consequently, a better understanding of the AML microenvironment is crucial for early diagnosis, risk stratification, and personalized therapy. In recent years, the role of bioinformatics as a powerful tool in clarifying the complexities of cancer has become more prominent. Gene expression profile and clinical data of 173 AML patients were downloaded from the TCGA database, and the xCell algorithm was applied to calculate the microenvironment score (MS). Then, the correlation of MS with FAB classification, and CALGB cytogenetic risk category was investigated. Differentially expressed genes (DEGs) were identified, and the correlation analysis of DEGs with patient survival was done using univariate cox. The prognostic value of candidate prognostic DEGs was confirmed based on the GEO database. In the last step, real-time PCR was used to compare the expression of the top three prognostic genes between patients and the control group. During TCGA data analysis, 716 DEGs were identified, and survival analysis results showed that 152 DEGs had survival-related changes. In addition, the prognostic value of 31 candidate prognostic genes was confirmed by GEO data analysis. Finally, the expression analysis of FLVCR2, SMO, and CREB5 genes, the most related genes to patients' survival, was significantly different between patients and control groups. In summary, we identified key microenvironment-related genes that influence the survival of AML patients and may serve as prognostic and therapeutic targets.

Combination MEG3 lncRNA and Ciprofloxacin dramatically decreases cell migration and viability as well as induces apoptosis in GC cells in vitro.

Gastric cancer (GC) is a prominent cause of cancer-related mortality worldwide. Long noncoding RNA (lncRNA) maternal expression gene3 (MEG3) participates in numerous signaling pathways by targeting the miRNA-mRNA axis. Studies on human tumors have demonstrated that the antibiotic Ciprofloxacin induces cell cycle changes, programmed cell death, and growth suppression. In this study, we transfected MEG3 lncRNA and Ciprofloxacin into the MKN-45 GC cell line. qRT-PCR was employed to evaluate the effects on the specific microRNA and mRNA. The wound healing test, MTT assay, and flow cytometry were used to assess the impact of their administration on cell migration, viability, and apoptosis, respectively. Research showed that miR-147 expression fell even more after MEG3 lncRNA transfection, leading to an increase in B-cell lymphoma 2 (BCL-2) levels. Ciprofloxacin transfection did not significantly affect the axis, except for MEG3, which led to its slight upregulation. MEG3 lncRNA inhibited the migration of MKN-45 cells compared to the control group. When MEG3 lncRNA was coupled with Ciprofloxacin, there was a significant reduction in cell migration compared to untreated groups and controls. MTT assay and flow cytometry demonstrated that MEG3 lncRNA decreased cell viability and triggered apoptosis. Simultaneous administration of MEG3 lncRNA and Ciprofloxacin revealed a significant reduction in cell viability caused by increased apoptosis obtained from MTT or flow cytometry assays. Modulating the miR-147-BCL-2 axis decreases cell migration and survival while promoting cell death. In conclusion, combining MEG3 lncRNA with Ciprofloxacin may be an effective therapeutic approach for GC treatment by influencing the miR-14-BCl-2 axis, resulting in reduced cell viability, migration, and increased apoptosis.

Expression Changes of SIRT1 and FOXO3a Significantly Correlate with Oxidative Stress Resistance Genes in AML Patients.

The increased metabolism in acute myeloid leukemia (AML) malignant cells resulted in the production of high levels of free radicals, called oxidative stress conditions. To avoid this situation, malignant cells produce a considerable amount of antioxidant agents, which will lead to the release of a continuous low level of reactive oxygen species (ROS), causing genomic damage and subsequent clonal evolution. SIRT1 has a key role in driving the adaptation to this condition, mainly through the deacetylation of FOXO3a that affects the expression of oxidative stress resistance target genes such as Catalase and Manganese superoxide dismutase (MnSOD). The aim of this study is to simultaneously investigate the expression of SIRT1, FOXO3a, and free radical-neutralizing enzymes such as Catalase and MnSOD in AML patients and measure their simultaneous change in relation to each other. The gene expression was analyzed using Real Time-PCR in 65 AML patients and 10 healthy controls. Our finding revealed that expression of SIRT1, FOXO3a, MnSOD and Catalase was significantly higher in AML patients in comparison to healthy controls. Also, there was a significant correlation between the expression of SIRT1 and FOXO3a, as well as among the expression of FOXO3a, MnSOD and Catalase genes in patients. According to the results, the expression of genes involved in oxidative stress resistance was higher in AML patients, which possibly contributed to the development of malignant clones. Also, the correlation between the expression of SIRT1 and FOXO3a gene reflects the importance of these two genes in increased oxidative stress resistance of cancer cells.

Inflammation and DKK1-induced AKT activation contribute to endothelial dysfunction following NR2F2 loss.

NR2F2 is expressed in endothelial cells (ECs) and Nr2f2 knockout produces lethal cardiovascular defects. In humans, reduced NR2F2 expression is associated with cardiovascular diseases including congenital heart disease and atherosclerosis. Here, NR2F2 silencing in human primary ECs led to inflammation, endothelial-to-mesenchymal transition (EndMT), proliferation, hypermigration, apoptosis-resistance, and increased production of reactive oxygen species. These changes were associated with STAT and AKT activation along with increased production of DKK1. Co-silencing DKK1 and NR2F2 prevented NR2F2-loss-induced STAT and AKT activation and reversed EndMT. Serum DKK1 concentrations were elevated in patients with pulmonary arterial hypertension (PAH) and DKK1 was secreted by ECs in response to in vitro loss of either BMPR2 or CAV1, which are genetic defects associated with the development of PAH. In human primary ECs, NR2F2 suppressed DKK1, whereas its loss conversely induced DKK1 and disrupted endothelial homeostasis, promoting phenotypic abnormalities associated with pathologic vascular remodeling. Activating NR2F2 or blocking DKK1 may be useful therapeutic targets for treating chronic vascular diseases associated with EC dysfunction.NEW & NOTEWORTHY NR2F2 loss in the endothelial lining of blood vessels is associated with cardiovascular disease. Here, NR2F2-silenced human endothelial cells were inflammatory, proliferative, hypermigratory, and apoptosis-resistant with increased oxidant stress and endothelial-to-mesenchymal transition. DKK1 was induced in NR2F2-silenced endothelial cells, while co-silencing NR2F2 and DKK1 prevented NR2F2-loss-associated abnormalities in endothelial signaling and phenotype. Activating NR2F2 or blocking DKK1 may be useful therapeutic targets for treating vascular diseases associated with endothelial dysfunction.

Effect of Acute Myeloid Leukemia-derived Extracellular Vesicles on Bone Marrow Mesenchymal Stromal Cells: Expression of Poor Prognosis Genes.

OBJECTIVE: Acute myeloid leukemia (AML) is a heterogeneous clonal disorder resulting from a complex interplay between leukemic cells and supporting factors from their microenvironment. In this context, extracellular vesicles (EVs) have been shown to play an essential role in forming a tumor-protective microenvironment. In this study, we examined the influence of AML-derived EVs on cellular and molecular characterization of bone marrow mesenchymal stromal cells (BM-MSCs), particularly alteration in the expression of genes (IL-6, Gas-6, and Galectin-3) relating to relapse and chemoresistance. METHODS: MSCs were co-cultured with different concentrations of AML-EVs. Our data has been achieved by MTT assay, ROS assay, proliferation assay and apoptosis assay. RT-qPCR was also performed for gene expression analysis. RESULTS: Our results demonstrated that AML-EVs impact the MSCs characterization in a concentration-dependent manner. We revealed higher viability, increased Ki-67 and BCL-2, and lower ROS levels in MSCs treated with a 40 µg/mL dose of EVs. On the other hand, the rate of MSCs apoptosis and BAX expression exposed to a 60 µg/mL dose of EVs were increased compared with the control group. In addition, RT-qPCR results showed that the expression of IL-6, Gas-6, and Galectin-3 was significantly up-regulated in treated MSCs with a 40 µg/mL dose of EVs. CONCLUSION: Because the overexpression of IL-6, Gas-6, and Galectin-3 has contributed to chemoresistance and relapse, our findings suggest that AML-EVs propel MSCs to express these genes, which in turn could guard leukemic cells from chemotherapy-inflicted damages and eventually lead to relapse.

Recent findings on chimeric antigen receptor (CAR)-engineered immune cell therapy in solid tumors and hematological malignancies.

Advancements in adoptive cell therapy over the last four decades have revealed various new therapeutic strategies, such as chimeric antigen receptors (CARs), which are dedicated immune cells that are engineered and administered to eliminate cancer cells. In this context, CAR T-cells have shown significant promise in the treatment of hematological malignancies. However, many obstacles limit the efficacy of CAR T-cell therapy in both solid tumors and hematological malignancies. Consequently, CAR-NK and CAR-M cell therapies have recently emerged as novel therapeutic options for addressing the challenges associated with CAR T-cell therapies. Currently, many CAR immune cell trials are underway in various human malignancies around the world to improve antitumor activity and reduce the toxicity of CAR immune cell therapy. This review will describe the comprehensive literature of recent findings on CAR immune cell therapy in a wide range of human malignancies, as well as the challenges that have emerged in recent years.

Biological causes of immunogenic cancer cell death (ICD) and anti-tumor therapy; Combination of Oncolytic virus-based immunotherapy and CAR T-cell therapy for ICD induction.

Chimeric antigen receptor (CAR) T-cell therapy is a promising and rapidly expanding therapeutic option for a wide range of human malignancies. Despite the ongoing progress of CAR T-cell therapy in hematologic malignancies, the application of this therapeutic strategy in solid tumors has encountered several challenges due to antigen heterogeneity, suboptimal CAR T-cell trafficking, and the immunosuppressive features of the tumor microenvironment (TME). Oncolytic virotherapy is a novel cancer therapy that employs competent or genetically modified oncolytic viruses (OVs) to preferentially proliferate in tumor cells. OVs in combination with CAR T-cells are promising candidates for overcoming the current drawbacks of CAR T-cell application in tumors through triggering immunogenic cell death (ICD) in cancer cells. ICD is a type of cellular death in which danger-associated molecular patterns (DAMPs) and tumor-specific antigens are released, leading to the stimulation of potent anti-cancer immunity. In the present review, we discuss the biological causes of ICD, different types of ICD, and the synergistic combination of OVs and CAR T-cells to reach potent tumor-specific immunity.

Design, synthesis and biological evaluations of a long-acting, hypoxia-activated prodrug of fasudil, a ROCK inhibitor, to reduce its systemic side-effects.

ROCK, one of the downstream regulators of Rho, controls actomyosin cytoskeleton organization, stress fiber formation, smooth muscle contraction, and cell migration. ROCK plays an important role in the pathologies of cerebral and coronary vasospasm, hypertension, cancer, and arteriosclerosis. Pharmacological-induced systemic inhibition of ROCK affects both the pathological and physiological functions of Rho-kinase, resulting in hypotension, increased heart rate, decreased lymphocyte count, and eventually cardiovascular collapse. To overcome the adverse effects of systemic ROCK inhibition, we developed a bioreductive prodrug of a ROCK inhibitor, fasudil, that functions selectively under hypoxic conditions. By masking fasudil's active site with a bioreductive 4-nitrobenzyl group, we synthesized a prodrug of fasudil that is inactive in normoxia. Reduction of the protecting group initiated by hypoxia reveals an electron-donating substituent that leads to fragmentation of the parent molecule. Under normoxia the fasudil prodrug displayed significantly reduced activity against ROCK compared to its parent compound, but under severe hypoxia the prodrug was highly effective in suppressing ROCK activity. Under hypoxia the prodrug elicited an antiproliferative effect on disease-afflicted pulmonary arterial smooth muscle cells and pulmonary arterial endothelial cells. The prodrug displayed a long plasma half-life, remained inactive in the blood, and produced no drop in systemic blood pressure when compared with fasudil-treated controls. Due to its selective nature, our hypoxia-activated fasudil prodrug could be used to treat diseases where tissue-hypoxia or hypoxic cells are the pathological basis of the disease.

Toll-like receptors (TLRs) in cancer; with an extensive focus on TLR agonists and antagonists.

At the forefront of the battle against pathogens or any endogenously released molecules, toll-like receptors (TLRs) play an important role as the most noble pattern recognition receptors. The ability of these receptors in distinguishing "self" and "non-self" antigens is a cornerstone in the innate immunity system; however, misregulation links inflammatory responses to the development of human cancers. It has been known for some time that aberrant expression and regulation of TLRs not only endows cancer cells an opportunity to escape from the immune system but also supports them through enhancing proliferation and angiogenesis. Over the past decades, cancer research studies have witnessed a number of preclinical and clinical breakthroughs in the field of TLR modulators and some of the agents have exceptionally performed well in advanced clinical trials. In the present review, we have provided a comprehensive review of different TLR agonists and antagonists and discuss their limitations, toxicities, and challenges to outline their future incorporation in cancer treatment strategies.

The effects of green cardamom supplementation on blood pressure and endothelium function in type 2 diabetic patients: A study protocol for a randomized controlled clinical trial.

INTRODUCTION: Cardamom possesses antioxidant, anti-inflammation, and blood pressure lowering properties, which might improve endothelial function in type 2 diabetic patients. However, no study has examined the effect of cardamom on diabetic patients. The present study aimed to examine the effects of 10-week green cardamom intake on blood pressure, concentrations of inflammatory and endothelial function biomarkers in type 2 diabetes mellitus patients, and its potential mechanisms. METHODS AND ANALYSIS DESIGN: Eighty overweight or obese patients with type 2 diabetes mellitus (aged 30-60 years) will be recruited into the trial and will assign to receive either cardamom (3 g/day, 6 capsules) or placebo (rusk powder, 6 capsules) for a period of 10 weeks. Systolic blood pressure and diastolic blood pressure, asymmetric dimethylarginine, and nitric oxide will be measured. Serum inflammatory markers namely interleukin 6, tumor necrosis factor-α, high-sensitivity C-reactive protein, and factors related to endothelial function including intercellular adhesion molecule-1, vascular cell adhesion molecule 1, CD62 antigen-like family member E, and cluster of differentiation 163 will be measured at baseline and at the end of the trial. Sociodemographic, International Physical Activity Questionnaire, and three 24-hour dietary recall questionnaires will be collected for each participant. ETHICS AND DISSEMINATION: The study has been approved by The Ethics Committee of Tehran University of Medical Sciences (IR.TUMS.REC.1395.2700). Each participant will sign a written informed consent at the beginning of the study. At the end of the study, results will be published timely manner. TRIAL REGISTRATION NUMBER: (http://www.irct.ir, identifier: IRCT-2016042717254N5) Date of registration: 2016-11-23.

Fasudil and DETA NONOate, Loaded in a Peptide-Modified Liposomal Carrier, Slow PAH Progression upon Pulmonary Delivery.

We investigated the feasibility of a combination therapy comprising fasudil, a Rho-kinase inhibitor, and DETA NONOate (diethylenetriamine NONOate, DN), a long-acting nitric oxide donor, both loaded in liposomes modified with a homing peptide, CAR (CARSKNKDC), in the treatment of pulmonary arterial hypertension (PAH). We first prepared and characterized unmodified and CAR-modified liposomes of fasudil and DN. Using individual drugs alone or a mixture of fasudil and DN as controls, we studied the efficacy of the two liposomal preparations in reducing mean pulmonary arterial pressure (mPAP) in monocrotaline (MCT) and SUGEN-hypoxia-induced PAH rats. We also conducted morphometric studies (degree of muscularization, arterial medial wall thickness, and collagen deposition) after treating the PAH rats with test and control formulations. When the rats were treated acutely and chronically, the reduction in mPAP was more pronounced in the liposomal formulation-treated rats than in plain drug-treated rats. CAR-modified liposomes were more selective in reducing mPAP than unmodified liposomes of the drugs. Both drugs, formulated in CAR-modified liposomes, reduced the degree of muscularization, medial arterial wall thickness, and collagen deposition more than the combination of plain drugs did. As seen with the in vivo data, CAR-modified liposomes of fasudil or DN increased the levels of the vasodilatory signaling molecule, cGMP, in the smooth muscle cells of PAH-afflicted human pulmonary arteries. Overall, fasudil and DN, formulated in liposomes, could be used as a combination therapy for a better management of PAH.

The effects of secondhand smoke exposure on infant growth: a prospective cohort study.

Mother's and infant exposure to cigarette smoke is one of the most important public health problems. There is no study in Iran evaluating the impact of cigarette smoke on infant growth and development. The purpose of this study was to determine the effects of cigarette. This prospective cohort study was conducted on 51 cigarette smoke-exposed infants (exposed group) and 51 non-exposed infants (non-exposed group). They were evaluated for weight, height and head circumference three times; five to seven days, two months and four months after birth. Urine samples were also collected in each turn. Exposure to secondhand smoke was assessed through questionnaires and urinary cotinine levels. The analysis was performed using an independent t-test, Mann-Whitney U test, chi-square and Fisher's exact and Kappa tests. Mean urinary cotinine level in the exposed group was 38.57±2.85 ng/mg creatinine at baseline, 86.95±1.16 at two months and 63.32±2.08 at four months of age. These indicated a gradual reduction of exposure from two to four months. The weight and height of the exposed group were significantly lower than the non-exposed group (P< 0.001) at two and four months after birth. The results of the present study showed that the exposure to secondhand smoke during infancy may lead to weight and height growth reduction in the first four months of life.

The Prevalence of Occult Celiac Disease among Patients with Functional Dyspepsia: A Study from the Western Region of Iran.

Objective. The prevalence of Celiac Disease (CD) is high in Iran, and evaluation of CD is not part of the routine screening procedure for dyspeptic patients; therefore, cases of occult CD may be missed. This study aimed to investigate the prevalence of occult CD among dyspeptic patients who presented at a gastroenterology clinic in the Western region of Iran. Methods. In this descriptive, cross-sectional prospective study, patients who had a history of at least 12 weeks of upper abdominal discomfort were eligible to participate in the study during a 14-month recruitment period. Patients with a clinical or paraclinical data in favor of organic causes were excluded from the study. Enrolled patients were screened for IgA antiendomysium antibody (EMA) and IgA antitissue transglutaminase antibody (tTG). Those who screened positive for EMA/tTG received a confirmatory diagnostic biopsy for Marsh classification of CD. Results. From 225 potential participants with dyspepsia, 55 patients were excluded due to having explainable organic causes. The study sample included 170 patients with "functional dyspepsia." Mean age of participants was 31 years and 55.8% were female. Twelve patients (7%) had positive tests (EMA/tTG), of which 10 were female (83.4%). According to Rome II criteria, all twelve patients with positive tests had "dysmotility type dyspepsia." Based on Marsh classification, six patients were consistent with "Marsh I," four with "Marsh II," and two with the "Marsh III" classification. Conclusions. In this study, the prevalence of CD in dyspeptic patients was high. As a result, this study suggests that screening by serology tests (EMA/tTG) is justifiable for the detection of CD among functional dyspeptic patients in the tertiary centers in our country.