Chimpanzee and pig-tailed macaque iPSCs: Improved culture and generation of primate cross-species embryos.

As our closest living relatives, non-human primates uniquely enable explorations of human health, disease, development, and evolution. Considerable effort has thus been devoted to generating induced pluripotent stem cells (iPSCs) from multiple non-human primate species. Here, we establish improved culture methods for chimpanzee (Pan troglodytes) and pig-tailed macaque (Macaca nemestrina) iPSCs. Such iPSCs spontaneously differentiate in conventional culture conditions, but can be readily propagated by inhibiting endogenous WNT signaling. As a unique functional test of these iPSCs, we injected them into the pre-implantation embryos of another non-human species, rhesus macaques (Macaca mulatta). Ectopic expression of gene BCL2 enhances the survival and proliferation of chimpanzee and pig-tailed macaque iPSCs within the pre-implantation embryo, although the identity and long-term contribution of the transplanted cells warrants further investigation. In summary, we disclose transcriptomic and proteomic data, cell lines, and cell culture resources that may be broadly enabling for non-human primate iPSCs research.

Autotaxin loss accelerates intestinal inflammation by suppressing TLR4-mediated immune responses.

Autotaxin (ATX) converts lysophosphatidylcholine and sphingosyl-phosphorylcholine into lysophosphatidic acid and sphingosine 1-phosphate, respectively. Despite the pivotal function of ATX in lipid metabolism, mechanisms by which ATX regulates immune and inflammatory disorders remain elusive. Here, using myeloid cell lineage-restricted Atx knockout mice, we show that Atx deficiency disrupts membrane microdomains and lipid rafts, resulting in the inhibition of Toll-like receptor 4 (TLR4) complex formation and the suppression of adaptor recruitment, thereby inhibiting TLR4-mediated responses in macrophages. Accordingly, TLR4-induced innate immune functions, including phagocytosis and iNOS expression, are attenuated in Atx-deficient macrophages. Consequently, Atx-/- mice exhibit a higher bacterial prevalence in the intestinal mucosa compared to controls. When combined with global Il10-/- mice, which show spontaneous colitis due to the translocation of luminal commensal microbes into the mucosa, myeloid cell lineage-restricted Atx knockout accelerates colitis development compared to control littermates. Collectively, our data reveal that Atx deficiency compromises innate immune responses, thereby promoting microbe-associated gut inflammation.

Alteration in ventricular pressure stimulates cardiac repair and remodeling.

The mammalian heart undergoes complex structural and functional remodeling to compensate for stresses such as pressure overload. While studies suggest that, at best, the adult mammalian heart is capable of very limited regeneration arising from the proliferation of existing cardiomyocytes, how myocardial stress affects endogenous cardiac regeneration or repair is unknown. To define the relationship between left ventricular afterload and cardiac repair, we induced left ventricle pressure overload in adult mice by constriction of the ascending aorta (AAC). One week following AAC, we normalized ventricular afterload in a subset of animals through removal of the aortic constriction (de-AAC). Subsequent monitoring of cardiomyocyte cell cycle activity via thymidine analog labeling revealed that an acute increase in ventricular afterload induced cardiomyocyte proliferation. Intriguingly, a release in ventricular overload (de-AAC) further increases cardiomyocyte proliferation. Following both AAC and de-AAC, thymidine analog-positive cardiomyocytes exhibited characteristics of newly generated cardiomyocytes, including single diploid nuclei and reduced cell size as compared to age-matched, sham-operated adult mouse myocytes. Notably, those smaller cardiomyocytes frequently resided alongside one another, consistent with local stimulation of cellular proliferation. Collectively, our data demonstrate that adult cardiomyocyte proliferation can be locally stimulated by an acute increase or decrease of ventricular pressure, and this mode of stimulation can be harnessed to promote cardiac repair.

Human-Induced Pluripotent Stem Cell Model of Trastuzumab-Induced Cardiac Dysfunction in Patients With Breast Cancer.

BACKGROUND: Molecular targeted chemotherapies have been shown to significantly improve the outcomes of patients who have cancer, but they often cause cardiovascular side effects that limit their use and impair patients' quality of life. Cardiac dysfunction induced by these therapies, especially trastuzumab, shows a distinct cardiotoxic clinical phenotype in comparison to the cardiotoxicity induced by conventional chemotherapies. METHODS: We used the human induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) platform to determine the underlying cellular mechanisms in trastuzumab-induced cardiac dysfunction. We assessed the effects of trastuzumab on structural and functional properties in iPSC-CMs from healthy individuals and performed RNA-sequencing to further examine the effect of trastuzumab on iPSC-CMs. We also generated human induced pluripotent stem cells from patients receiving trastuzumab and examined whether patients' phenotype could be recapitulated in vitro by using patient-specific iPSC-CMs. RESULTS: We found that clinically relevant doses of trastuzumab significantly impaired the contractile and calcium-handling properties of iPSC-CMs without inducing cardiomyocyte death or sarcomeric disorganization. RNA-sequencing and subsequent functional analysis revealed mitochondrial dysfunction and altered the cardiac energy metabolism pathway as primary causes of trastuzumab-induced cardiotoxic phenotype. Human iPSC-CMs generated from patients who received trastuzumab and experienced severe cardiac dysfunction were more vulnerable to trastuzumab treatment than iPSC-CMs generated from patients who did not experience cardiac dysfunction following trastuzumab therapy. It is important to note that metabolic modulation with AMP-activated protein kinase activators could avert the adverse effects induced by trastuzumab. CONCLUSIONS: Our results indicate that alterations in cellular metabolic pathways in cardiomyocytes could be a key mechanism underlying the development of cardiac dysfunction following trastuzumab therapy; therefore, targeting the altered metabolism may be a promising therapeutic approach for trastuzumab-induced cardiac dysfunction.

A Premature Termination Codon Mutation in MYBPC3 Causes Hypertrophic Cardiomyopathy via Chronic Activation of Nonsense-Mediated Decay.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is frequently caused by mutations in myosin-binding protein C3 ( MYBPC3) resulting in a premature termination codon (PTC). The underlying mechanisms of how PTC mutations in MYBPC3 lead to the onset and progression of HCM are poorly understood. This study's aim was to investigate the molecular mechanisms underlying the pathogenesis of HCM associated with MYBPC3 PTC mutations by utilizing human isogenic induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). METHODS: Isogenic iPSC lines were generated from HCM patients harboring MYBPC3 PTC mutations (p.R943x; p.R1073P_Fsx4) using genome editing. Comprehensive phenotypic and transcriptome analyses were performed in the iPSC-CMs. RESULTS: We observed aberrant calcium handling properties with prolonged decay kinetics and elevated diastolic calcium levels in the absence of structural abnormalities or contracile dysfunction in HCM iPSC-CMs as compared to isogenic controls. The mRNA expression levels of MYBPC3 were significantly reduced in mutant iPSC-CMs, but the protein levels were comparable among isogenic iPSC-CMs, suggesting that haploinsufficiency of MYBPC3 does not contribute to the pathogenesis of HCM in vitro. Furthermore, truncated MYBPC3 peptides were not detected. At the molecular level, the nonsense-mediated decay pathway was activated, and a set of genes involved in major cardiac signaling pathways was dysregulated in HCM iPSC-CMs, indicating an HCM gene signature in vitro. Specific inhibition of the nonsense-mediated decay pathway in mutant iPSC-CMs resulted in reversal of the molecular phenotype and normalization of calcium-handling abnormalities. CONCLUSIONS: iPSC-CMs carrying MYBPC3 PTC mutations displayed aberrant calcium signaling and molecular dysregulations in the absence of significant haploinsufficiency of MYBPC3 protein. Here we provided the first evidence of the direct connection between the chronically activated nonsense-mediated decay pathway and HCM disease development.

Genome Editing of Induced Pluripotent Stem Cells to Decipher Cardiac Channelopathy Variant.

BACKGROUND: The long QT syndrome (LQTS) is an arrhythmogenic disorder of QT interval prolongation that predisposes patients to life-threatening ventricular arrhythmias such as Torsades de pointes and sudden cardiac death. Clinical genetic testing has emerged as the standard of care to identify genetic variants in patients suspected of having LQTS. However, these results are often confounded by the discovery of variants of uncertain significance (VUS), for which there is insufficient evidence of pathogenicity. OBJECTIVES: The purpose of this study was to demonstrate that genome editing of patient-specific induced pluripotent stem cells (iPSCs) can be a valuable approach to delineate the pathogenicity of VUS in cardiac channelopathy. METHODS: Peripheral blood mononuclear cells were isolated from a carrier with a novel missense variant (T983I) in the KCNH2 (LQT2) gene and an unrelated healthy control subject. iPSCs were generated using an integration-free Sendai virus and differentiated to iPSC-derived cardiomyocytes (CMs). RESULTS: Whole-cell patch clamp recordings revealed significant prolongation of the action potential duration (APD) and reduced rapidly activating delayed rectifier K+ current (IKr) density in VUS iPSC-CMs compared with healthy control iPSC-CMs. ICA-105574, a potent IKr activator, enhanced IKr magnitude and restored normal action potential duration in VUS iPSC-CMs. Notably, VUS iPSC-CMs exhibited greater propensity to proarrhythmia than healthy control cells in response to high-risk torsadogenic drugs (dofetilide, ibutilide, and azimilide), suggesting a compromised repolarization reserve. Finally, the selective correction of the causal variant in iPSC-CMs using CRISPR/Cas9 gene editing (isogenic control) normalized the aberrant cellular phenotype, whereas the introduction of the homozygous variant in healthy control cells recapitulated hallmark features of the LQTS disorder. CONCLUSIONS: The results suggest that the KCNH2T983I VUS may be classified as potentially pathogenic.

Pluripotent Stem Cell-Derived Cardiomyocytes as a Platform for Cell Therapy Applications: Progress and Hurdles for Clinical Translation.

Cardiovascular diseases are the leading cause of morbidity and mortality worldwide. Regenerative therapy has been applied to restore lost cardiac muscle and cardiac performance. Induced pluripotent stem cells (iPSCs) can provide an unlimited source of cardiomyocytes and therefore play a key role in cardiac regeneration. Despite initial encouraging results from pre-clinical studies, progress toward clinical applications has been hampered by issues such as tumorigenesis, arrhythmogenesis, immune rejection, scalability, low graft-cell survival, and poor engraftment. Here, we review recent developments in iPSC research on regenerating injured heart tissue, including novel advances in cell therapy and potential strategies to overcome current obstacles in the field.

Modeling human diseases with induced pluripotent stem cells: from 2D to 3D and beyond.

The advent of human induced pluripotent stem cells (iPSCs) presents unprecedented opportunities to model human diseases. Differentiated cells derived from iPSCs in two-dimensional (2D) monolayers have proven to be a relatively simple tool for exploring disease pathogenesis and underlying mechanisms. In this Spotlight article, we discuss the progress and limitations of the current 2D iPSC disease-modeling platform, as well as recent advancements in the development of human iPSC models that mimic in vivo tissues and organs at the three-dimensional (3D) level. Recent bioengineering approaches have begun to combine different 3D organoid types into a single '4D multi-organ system'. We summarize the advantages of this approach and speculate on the future role of 4D multi-organ systems in human disease modeling.

The Xenobiotic Transporter Mdr1 Enforces T Cell Homeostasis in the Presence of Intestinal Bile Acids.

CD4+ T cells are tightly regulated by microbiota in the intestine, but whether intestinal T cells interface with host-derived metabolites is less clear. Here, we show that CD4+ T effector (Teff) cells upregulated the xenobiotic transporter, Mdr1, in the ileum to maintain homeostasis in the presence of bile acids. Whereas wild-type Teff cells upregulated Mdr1 in the ileum, those lacking Mdr1 displayed mucosal dysfunction and induced Crohn's disease-like ileitis following transfer into Rag1-/- hosts. Mdr1 mitigated oxidative stress and enforced homeostasis in Teff cells exposed to conjugated bile acids (CBAs), a class of liver-derived emulsifying agents that actively circulate through the ileal mucosa. Blocking ileal CBA reabsorption in transferred Rag1-/- mice restored Mdr1-deficient Teff cell homeostasis and attenuated ileitis. Further, a subset of ileal Crohn's disease patients displayed MDR1 loss of function. Together, these results suggest that coordinated interaction between mucosal Teff cells and CBAs in the ileum regulate intestinal immune homeostasis.

Circulating cathelicidin levels correlate with mucosal disease activity in ulcerative colitis, risk of intestinal stricture in Crohn's disease, and clinical prognosis in inflammatory bowel disease.

BACKGROUND: Cathelicidin (LL-37) is an antimicrobial peptide known to be associated with various autoimmune diseases. We attempt to determine if cathelicidin can accurately reflect IBD disease activity. We hypothesize that serum cathelicidin correlates with mucosal disease activity, stricture, and clinical prognosis of IBD patients. METHODS: Serum samples were collected from two separate cohorts of patients at the University of California, Los Angeles. Cohort 1 consisted of 50 control, 23 UC, and 28 CD patients. Cohort 2 consisted of 20 control, 57 UC, and 67 CD patients. LL-37 levels were determined by ELISA. Data from both cohorts were combined for calculation of accuracies in indicating mucosal disease activity, relative risks of stricture, and odds ratios of predicting disease development. RESULTS: Serum cathelicidin levels were inversely correlated with Partial Mayo Scores of UC patients and Harvey-Bradshaw Indices of CD patients. Among IBD patients with moderate or severe initial disease activity, the patients with high initial LL-37 levels had significantly better recovery than the patients with low initial LL-37 levels after 6-18 months, suggesting that high LL-37 levels correlate with good prognosis. Co-evaluation of LL-37 and CRP levels was more accurate than CRP alone or LL-37 alone in the correlation with Mayo Endoscopic Score of UC patients. Low LL-37 levels indicated a significantly elevated risk of intestinal stricture in CD patients. CONCLUSION: Co-evaluation of LL-37 and CRP can indicate mucosal disease activity in UC patients. LL-37 can predict future clinical activity in IBD patients and indicate risk of intestinal stricture in CD patients.

Informatics-Based Discovery of Disease-Associated Immune Profiles.

Advances in flow and mass cytometry are enabling ultra-high resolution immune profiling in mice and humans on an unprecedented scale. However, the resulting high-content datasets challenge traditional views of cytometry data, which are both limited in scope and biased by pre-existing hypotheses. Computational solutions are now emerging (e.g., Citrus, AutoGate, SPADE) that automate cell gating or enable visualization of relative subset abundance within healthy versus diseased mice or humans. Yet these tools require significant computational fluency and fail to show quantitative relationships between discrete immune phenotypes and continuous disease variables. Here we describe a simple informatics platform that uses hierarchical clustering and nearest neighbor algorithms to associate manually gated immune phenotypes with clinical or pre-clinical disease endpoints of interest in a rapid and unbiased manner. Using this approach, we identify discrete immune profiles that correspond with either weight loss or histologic colitis in a T cell transfer model of inflammatory bowel disease (IBD), and show distinct nodes of immune dysregulation in the IBDs, Crohn's disease and ulcerative colitis. This streamlined informatics approach for cytometry data analysis leverages publicly available software, can be applied to manually or computationally gated cytometry data, is suitable for any clinical or pre-clinical setting, and embraces ultra-high content flow and mass cytometry as a discovery engine.

Identification of Circulating MicroRNA Signatures in Crohn's Disease Using the Nanostring nCounter Technology.

BACKGROUND: Current clinical indices, such as Harvey-Bradshaw index, are often inadequate for the assessment of disease activity in Crohn's disease (CD). Alternative methods including imaging modalities and laboratory markers, such as C-reactive protein (CRP), are routinely applied to assess disease activity. However, laboratory markers poorly reflect the actual disease activity. Consequently, novel biomarkers represent a clinical necessity for CD patient management. We hypothesized that circulating serum-derived microRNAs may be used as diagnosis and disease activity monitoring tools of CD patients. METHODS: To test this hypothesis, we performed microRNA expression profiling through Nanostring nCounter technology in blood serum samples of CD patients and healthy control subjects. Harvey-Bradshaw index score was used to capture clinical disease activity; CRP was measured as part of standard clinical practice. The expression profile of circulating microRNAs and the levels of CRP correlated with Harvey-Bradshaw index. RESULTS: We identified a signature of 10 circulating microRNAs that are differentially expressed in CD patients compared with healthy control subjects. Two of these microRNAs (hsa-miR-1286 and hsa-miR-1273d) correlated with CD disease activity and exhibited higher correlation values compared with CRP. Further analysis revealed distinct microRNA signatures between CD patients with ileal and colonic involvement. CONCLUSIONS: Circulating microRNAs show superior value as diagnostic and disease activity markers in comparison to traditional methods. Circulating microRNAs could improve CD patient management, if applied in combination with current state-of-the-art diagnostic and disease activity assessment modalities.

Optimization of human mesenchymal stem cell manufacturing: the effects of animal/xeno-free media.

Due to their immunosuppressive properties, mesenchymal stem cells (MSC) have been evaluated for the treatment of immunological diseases. However, the animal-derived growth supplements utilized for MSC manufacturing may lead to clinical complications. Characterization of alternative media formulations is imperative for MSC therapeutic application. Human BMMSC and AdMSC were expanded in media supplemented with either human platelet lysates (HPL), serum-free media/xeno-free FDA-approved culture medium (SFM/XF), or fetal bovine serum (FBS) and the effects on their properties were investigated. The immunophenotype of resting and IFN-γ primed BMMSC and AdMSC remained unaltered in all media. Both HPL and SFM/XF increased the proliferation of BMMSC and AdMSC. Expansion of BMMSC and AdMSC in HPL increased their differentiation, compared to SFM/XF and FBS. Resting BMMSC and AdMSC, expanded in FBS or SFM/XF, demonstrated potent immunosuppressive properties in both non-primed and IFN-γ primed conditions, whereas HPL-expanded MSC exhibited diminished immunosuppressive properties. Finally, IFN-γ primed BMMSC and AdMSC expanded in SFM/XF and HPL expressed attenuated levels of IDO-1 compared to FBS. Herein, we provide strong evidence supporting the use of the FDA-approved SFM/XF medium, in contrast to the HPL medium, for the expansion of MSC towards therapeutic applications.

Assessment of Circulating MicroRNAs for the Diagnosis and Disease Activity Evaluation in Patients with Ulcerative Colitis by Using the Nanostring Technology.

BACKGROUND: Clinical decision and patient care management in inflammatory bowel diseases is largely based on the assessment of clinical symptoms, while the biomarkers currently in use poorly reflect the actual disease activity. Therefore, the identification of novel biomarkers will serve an unmet clinical need for IBD screening and patient management. We examined the utility of circulating microRNAs for diagnosis and disease activity monitoring in patients with ulcerative colitis (UC). METHODS: Blood serum microRNAs were isolated from patients with UC with active and inactive disease and healthy donors. High-throughput microRNA profiling was performed using the Nanostring technology platform. Clinical disease activity was captured by calculating the partial Mayo score. C-reactive protein was measured in patients with UC as part of their clinical monitoring. The profiles of circulating microRNAs and C-reactive protein were correlated with clinical disease indices. RESULTS: We have identified a signature of 12 circulating microRNAs that differentiate patients with UC from control subjects. Moreover, 6 of these microRNAs significantly correlated with UC disease activity. Importantly, a set of 4 microRNAs (hsa-miR-4454, hsa-miR-223-3p, hsa-miR-23a-3p, and hsa-miR-320e), which correlated with UC disease activity were found to have higher sensitivity and specificity values than C-reactive protein. CONCLUSIONS: Circulating microRNAs provide a novel diagnostic and prognostic marker for patients with UC. The use of an FDA-approved platform could accelerate the application of microRNA screening in a gastrointenstinal clinical setting. When used in combination with current diagnostic and disease activity assessment modalities, microRNAs could improve both IBD screening and care management.

MicroRNA214 Is Associated With Progression of Ulcerative Colitis, and Inhibition Reduces Development of Colitis and Colitis-Associated Cancer in Mice.

BACKGROUND & AIMS: Persistent activation of the inflammatory response contributes to the development of inflammatory bowel diseases, which increase the risk of colorectal cancer. We aimed to identify microRNAs that regulate inflammation during the development of ulcerative colitis (UC) and progression to colitis-associated colon cancer (CAC). METHODS: We performed a quantitative polymerase chain reaction analysis to measure microRNAs in 401 colon specimens from patients with UC, Crohn's disease, irritable bowel syndrome, sporadic colorectal cancer, or CAC, as well as subjects without these disorders (controls); levels were correlated with clinical features and disease activity of patients. Colitis was induced in mice by administration of dextran sodium sulfate (DSS), and carcinogenesis was induced by addition of azoxymethane; some mice also were given an inhibitor of microRNA214 (miR214). RESULTS: A high-throughput functional screen of the human microRNAome found that miR214 regulated the activity of nuclear factor-κB. Higher levels of miR214 were detected in colon tissues from patients with active UC or CAC than from patients with other disorders or controls and correlated with disease progression. Bioinformatic and genome-wide profile analyses showed that miR214 activates an inflammatory response and is amplified through a feedback loop circuit mediated by phosphatase and tensin homolog (PTEN) and PDZ and LIM domain 2 (PDLIM2). Interleukin-6 induced signal transducer and activator of transcription 3 (STAT3)-mediated transcription of miR214. A miR214 chemical inhibitor blocked this circuit and reduced the severity of DSS-induced colitis in mice, as well as the number and size of tumors that formed in mice given azoxymethane and DSS. In fresh colonic biopsy specimens from patients with active UC, the miR214 inhibitor reduced inflammation by increasing levels of PDLIM2 and PTEN. CONCLUSIONS: Interleukin-6 up-regulates STAT3-mediated transcription of miR214 in colon tissues, which reduces levels of PDLIM2 and PTEN, increases phosphorylation of AKT, and activates nuclear factor-κB. The activity of this circuit correlates with disease activity in patients with UC and progression to colorectal cancer.

A microRNA signature in pediatric ulcerative colitis: deregulation of the miR-4284/CXCL5 pathway in the intestinal epithelium.

BACKGROUND: Twenty to 25% of the patients with inflammatory bowel disease (IBD) present the disease before the age of 18 to 20, with worse extent and severity, compared with adult-onset IBD. We sought to identify the differential expression of microRNAs in pediatric ulcerative colitis (UC) and their association with different clinical phenotypes. METHODS: MicroRNA expression analysis was performed in colonic tissues derived from pediatric patients with UC and controls without IBD. MiR-4284 levels were verified by real-time quantitative polymerase chain reaction in 2 additional cohorts of pediatric patients with UC. Bioinformatics analysis was performed to predict the targets of miR-4284. In vitro experiments using luciferase reporter assays and real-time polymerase chain reaction evaluated the direct effect of miR-4284 on CXCL5 mRNA. In vivo experiments were performed in 2 mouse models of experimental colitis. RESULTS: A 24-microRNA signature was identified in colonic tissues derived from pediatric patients with UC. The most downregulated microRNA in the tissue of pediatric patients UC, relative to non-IBD controls, was miR-4284. In situ hybridization revealed that miR-4284 is present in colonic epithelial cells, and its levels correlate with the disease activity. Furthermore, we found that miR-4284 regulates CXCL5 mRNA expression through binding to its 3'UTR. CXCL5 had increased mRNA levels in colonic tissue from pediatric patients with UC and correlated with disease activity. Furthermore, we found an inverse correlation between miR-4284 and CXCL5 levels in the colonic pediatric UC tissues and in 2 mouse models of experimental colitis. CONCLUSIONS: Our data reveal a novel microRNA pediatric UC signature and provide evidence that miR-4284 directly regulates CXCL5 and correlates with the disease activity.

Neurotensin--regulated miR-133α is involved in proinflammatory signalling in human colonic epithelial cells and in experimental colitis.

OBJECTIVE: Neurotensin (NT) mediates colonic inflammation through its receptor neurotensin receptor 1 (NTR1). NT stimulates miR-133α expression in colonic epithelial cells. We investigated the role of miR-133α in NT-associated colonic inflammation in vitro and in vivo. DESIGN: miR-133α and aftiphilin (AFTPH) levels were measured by quantitative PCR. Antisense (as)-miR-133α was administrated intracolonicaly prior to induction of 2, 4, 6-trinitrobenzene sulfonic acid (TNBS)-induced colitis and dextran sodium sulfate (DSS)-induced colitis. The effect of AFTPH was examined by gene silencing in vitro. RESULTS: NT increased miR-133α levels in NCM-460 overexpressing NTR1 (NCM460-NTR1) and HCT-116 cells. NT-induced p38, ERK1/2, c-Jun, and NF-κB activation, as well as IL-6, IL-8 and IL-1ß messenger RNA (mRNA) expression in NCM-460-NTR1 cells were reduced in miR-133α-silenced cells, while overexpression of miR-133α reversed these effects. MiR-133α levels were increased in TNBS (2 day) and DSS (5 day) colitis, while NTR1 deficient DSS-exposed mice had reduced miR-133α levels, compared to wild-type colitic mice. Intracolonic as-miR-133α attenuated several parameters of colitis as well expression of proinflammatory mediators in the colonic mucosa. In silico search coupled with qPCR identified AFTPH as a downstream target of miR-133α, while NT decreased AFTPH expression in NCM-460-NTR1 colonocytes. Gene silencing of AFTPH enhanced NT-induced proinflammatory responses and AFTPH levels were downregulated in experimental colitis. Levels of miR-133α were significantly upregulated, while AFTPH levels were downregulated in colonic biopsies of patients with ulcerative colitis compared to controls. CONCLUSIONS: NT-associated colitis and inflammatory signalling are regulated by miR-133α-AFTPH interactions. Targeting of miR-133α or AFTPH may represent a novel therapeutic approach in inflammatory bowel disease.

FLT3 activation improves post-myocardial infarction remodeling involving a cytoprotective effect on cardiomyocytes.

OBJECTIVES: The goal of this study was to define the role of FMS-like tyrosine kinase 3 (FLT3) in the heart. BACKGROUND: FLT3 is a prominent target of receptor tyrosine kinase inhibitors (TKIs) used for anticancer therapy. TKIs can cause cardiomyopathy but understanding of the mechanisms is incomplete, partly because the roles of specific TKI target receptors in the heart are still obscure. METHODS: Myocardial infarction was induced in mice by permanent ligation of the left anterior descending coronary artery followed by intramyocardial injection of FLT3 ligand (FL) or vehicle into the infarct border zone. Cardiac morphology and function were assessed by echocardiography and histological analysis 1 week after infarction. In addition, FLT3 expression and regulation, as well as molecular mechanisms of FLT3 action, were examined in cardiomyocytes in vitro. RESULTS: The intramyocardial injection of FL into the infarct border zone decreased infarct size and ameliorated post-myocardial infarction remodeling and function in mice. This beneficial effect was associated with reduced apoptosis, including myocytes in the infarct border zone. Cardiomyocytes expressed functional FLT3, and FLT3 messenger ribonucleic acid and protein were up-regulated under oxidative stress, identifying cardiomyocytes as FL target cells. FLT3 activation with FL protected cardiomyocytes from oxidative stress-induced apoptosis via an Akt-dependent mechanism involving Bcl-2 family protein regulation and inhibition of the mitochondrial death pathway. CONCLUSIONS: FLT3 is a cytoprotective system in the heart and a potential therapeutic target in ischemic cardiac injury. The protective mechanisms uncovered here may be further explored in view of potential cardiotoxic effects of FLT3-targeting anticancer therapy, particularly in patients with ischemic heart disease.