Overexpression of multidrug resistance-associated protein 1 protects against cardiotoxicity by augmenting the doxorubicin efflux from cardiomyocytes.

Doxorubicin is a commonly used anti-cancer drug used in treating a variety of malignancies. However, a major adverse effect is cardiotoxicity, which is dose dependent and can be either acute or chronic. Doxorubicin causes injury by DNA damage, the formation of free reactive oxygen radicals and induction of apoptosis. Our aim is to induce expression of the multidrug resistance-associated protein 1 (MRP1) in cardiomyocytes derived from human iPS cells (hiPSC-CM), to determine whether this will allow cells to effectively remove doxorubicin and confer cardioprotection. We generated a lentivirus vector encoding MRP1 (LV.MRP1) and validated its function in HEK293T cells and stem cell-derived cardiomyocytes (hiPSC-CM) by quantitative PCR and western blot analysis. The activity of the overexpressed MRP1 was also tested, by quantifying the amount of fluorescent dye exported from the cell by the transporter. We demonstrated reduced dye sequestration in cells overexpressing MRP1. Finally, we demonstrated that hiPSC-CM transduced with LV.MRP1 were protected against doxorubicin injury. In conclusion, we have shown that we can successfully overexpress MRP1 protein in hiPSC-CM, with functional transporter activity leading to protection against doxorubicin-induced toxicity.

Ghrelin mediated cardioprotection using in vitro models of oxidative stress.

Ghrelin is commonly known as the 'hunger hormone' due to its role in stimulating food intake in humans. However, the roles of ghrelin extend beyond regulating hunger. Our aim was to investigate the ability of ghrelin to protect against hydrogen peroxide (H2O2), a reactive oxygen species commonly associated with cardiac injury. An in vitro model of oxidative stress was developed using H2O2 injured H9c2 cells. Despite lentiviral ghrelin overexpression, H9c2 cell viability and mitochondrial function were not protected following H2O2 injury. We found that H9c2 cells lack expression of the preproghrelin cleavage enzyme prohormone convertase 1 (encoded by PCSK1), required to convert ghrelin to its active form. In contrast, we found that primary rat cardiomyocytes do express PCSK1 and were protected from H2O2 injury by lentiviral ghrelin overexpression. In conclusion, we have shown that ghrelin expression can protect primary rat cardiomyocytes against H2O2, though this effect was not observed in other cell types tested.

Development of new adeno-associated virus capsid variants for targeted gene delivery to human cardiomyocytes.

Recombinant adeno-associated viruses (rAAVs) have emerged as one of the most promising gene therapy vectors that have been successfully used in pre-clinical models of heart disease. However, this has not translated well to humans due to species differences in rAAV transduction efficiency. As a result, the search for human cardiotropic capsids is a major contemporary challenge. We used a capsid-shuffled rAAV library to perform directed evolution in human iPSC-derived cardiomyocytes (hiPSC-CMs). Five candidates emerged, with four presenting high sequence identity to AAV6, while a fifth divergent variant was related to AAV3b. Functional analysis of the variants was performed in vitro using hiPSC-CMs, cardiac organoids, human cardiac slices, non-human primate and porcine cardiac slices, as well as mouse heart and liver in vivo. We showed that cell entry was not the best predictor of transgene expression efficiency. The novel variant rAAV.KK04 was the best-performing vector in human-based screening platforms, exceeding the benchmark rAAV6. None of the novel capsids demonstrate a significant transduction of liver in vivo. The range of experimental models used revealed the value of testing for tropism differences under the conditions of human specificity, bona fide, myocardium and cell type of interest.

Promoter Optimization Circumvents Bcl-2 Transgene-Mediated Suppression of Lentiviral Vector Production.

Lentiviral vectors are a robust gene delivery tool for inducing transgene expression in a variety of cells. They are well suited to facilitate the testing of therapeutic candidate genes in vitro, due to relative ease of packaging and ability to transduce dividing and non-dividing cells. Our goal was to identify a gene that could be delivered to the heart to protect against cancer-therapy-induced cardiotoxicity. We sought to generate a lentivirus construct with a ubiquitous CMV promoter driving expression of B-cell lymphocyte/leukemia 2 gene (Bcl-2), a potent anti-apoptotic gene. Contrary to our aim, overexpression of Bcl-2 induced cell death in the producer HEK293T cells, resulting in failure to produce usable vector titre. This was circumvented by exchanging the CMV promoter to the cardiac-specific NCX1 promoter, leading to the successful production of a lentiviral vector which could induce cardioprotective expression of Bcl-2. In conclusion, reduced expression of Bcl-2 driven by a weaker promoter improved vector yield, and led to the production of functional cardioprotective Bcl-2 in primary cardiomyocytes.

Recombinant Adeno-Associated Viral Vector-Mediated Gene Transfer of hTBX18 Generates Pacemaker Cells from Ventricular Cardiomyocytes.

Sinoatrial node dysfunction can manifest as bradycardia, leading to symptoms of syncope and sudden cardiac death. Electronic pacemakers are the current standard of care but are limited due to a lack of biological chronotropic control, cost of revision surgeries, and risk of lead- and device-related complications. We therefore aimed to develop a biological alternative to electronic devices by using a clinically relevant gene therapy vector to demonstrate conversion of cardiomyocytes into sinoatrial node-like cells in an in vitro context. Neonatal rat ventricular myocytes were transduced with recombinant adeno-associated virus vector 6 encoding either hTBX18 or green fluorescent protein and maintained for 3 weeks. At the endpoint, qPCR, Western blot analysis and immunocytochemistry were used to assess for reprogramming into pacemaker cells. Cell morphology and Arclight action potentials were imaged via confocal microscopy. Compared to GFP, hTBX18-transduced cells showed that hTBX18, HCN4 and Cx45 were upregulated. Cx43 was significantly downregulated, while sarcomeric α-actinin remained unchanged. Cardiomyocytes transduced with hTBX18 acquired the tapering morphology of native pacemaker cells, as compared to the block-like, striated appearance of ventricular cardiomyocytes. Analysis of the action potentials showed phase 4 depolarization and a significant decrease in the APD50 of the hTBX18-transduced cells. We have demonstrated that rAAV-hTBX18 gene transfer to ventricular myocytes results in morphological, molecular, physiological, and functional changes, recapitulating the pacemaker phenotype in an in vitro setting. The generation of these induced pacemaker-like cells using a clinically relevant vector opens new prospects for biological pacemaker development.

Performance of Cardiotropic rAAV Vectors Is Dependent on Production Method.

Gene therapy is making significant impact on a modest, yet growing, number of human diseases. Justifiably, the preferred viral vector for clinical use is that based on recombinant adeno-associated virus (rAAV). There is a need to scale up rAAV vector production with the transition from pre-clinical models to human application. Standard production methods based on the adherent cell type (HEK293) are limited in scalability and other methods, such as those based on the baculovirus and non-adherent insect cell (Sf9) system, have been pursued as an alternative to increase rAAV production. In this study, we compare these two production methods for cardiotropic rAAVs. Transduction efficiency for both production methods was assessed in primary cardiomyocytes, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), and in mice following systemic delivery. We found that the rAAV produced by the traditional HEK293 method out-performed vector produced using the baculovirus/Sf9 system in vitro and in vivo. This finding provides a potential caveat for vector function when using the baculovirus/Sf9 production system and underscores the need for thorough assessment of vector performance when using diverse rAAV production methods.

Analysis of recombinant adeno-associated viral vector shedding in sheep following intracoronary delivery.

Differences between mouse and human hearts pose a significant limitation to the value of small animal models when predicting vector behavior following recombinant adeno-associated viral (rAAV) vector-mediated cardiac gene therapy. Hence, sheep have been adopted as a preclinical animal, as they better model the anatomy and cardiac physiological processes of humans. There is, however, no comprehensive data on the shedding profile of rAAV in sheep following intracoronary delivery, so as to understand biosafety risks in future preclinical and clinical applications. In this study, sheep received intracoronary delivery of rAAV serotypes 2/6 (2 × 1012 vg), 2/8, and 2/9 (1 × 1013 vg) at doses previously administered in preclinical and clinical trials. This was followed by assessment over 96 h to examine vector shedding in urine, feces, nasal mucus, and saliva samples. Vector genomes were detected via real-time quantitative PCR in urine and feces up to 48 and 72 h post vector delivery, respectively. Of these results, functional vector particles were only detected via a highly sensitive infectious replication assay in feces samples up to 48 h following vector delivery. We conclude that rAAV-mediated gene transfer into sheep hearts results in low-grade shedding of non-functional vector particles for all excreta samples, except in the case of feces, where functional vector particles are present up to 48 h following vector delivery. These results may be used to inform containment and decontamination guidelines for large animal dealings, and to understand the biosafety risks associated with future preclinical and clinical uses of rAAV.

Human Connexin40 Mutations Slow Conduction and Increase Propensity for Atrial Fibrillation.

BACKGROUND: Patch clamping studies using non-cardiomyocytes revealed that the human connexin40 mutations P88S, G38D, and A96S are associated with reduced gap junction conductances compared to wild type connexin40 (wtCx40). Their effects within myocytes however are unclear. We aimed to characterise P88S, G38D, and A96S after expression in rat hearts and primary cardiomyocyte cultures. METHODS: Adult Sprague-Dawley rat atria were transduced with a lentivector containing a transgene encoding wtCx40, P88S, G38D, A96S, or eGFP (n=6 per transgene). Electrophysiology studies (EPS) were performed just prior to and 7 days after surgery. Left atria were assessed for connexin expression, mRNA levels, inflammation and fibrosis. Primary cardiomyocyte cultures were also transduced with the abovementioned vectors (n=6 per transgene) and monolayer conduction velocities (CV) and protein expression were assessed at 96hours. RESULTS: At day 7 EPS, P wave and induced atrial fibrillation (AF) durations were significantly longer in the mutant groups when compared to wtCx40 controls (p<0.05). There were no significant differences in inflammation, fibrosis, or heart to body weight ratios. Monolayer CV's were reduced in the A96S group compared to the wtCx40 group. While similar to wtCx40 controls, P88S velocities were reduced compared to eGFP controls. G38D monolayers possessed spontaneous fibrillatory activity and could not be paced. Immunofluorescence revealed that P88S and G38D reduced native connexin43 myocyte coupling while A96S appeared to co-localise with connexin43 in gap junctions. Connexin43 mRNA levels were similar between groups. CONCLUSIONS: The A96S, G38D, and P88S Cx40 mutations slow conduction and increased the propensity for inducible AF.

Vitamin D Receptor Ablation and Vitamin D Deficiency Result in Reduced Grip Strength, Altered Muscle Fibers, and Increased Myostatin in Mice.

Vitamin D deficiency is associated with muscle weakness, pain, and atrophy. Serum vitamin D predicts muscle strength and age-related muscle changes. However, precise mechanisms by which vitamin D affects skeletal muscle are unclear. To address this question, this study characterizes the muscle phenotype and gene expression of mice with deletion of vitamin D receptor (VDRKO) or diet-induced vitamin D deficiency. VDRKO and vitamin D-deficient mice had significantly weaker grip strength than their controls. Weakness progressed with age and duration of vitamin D deficiency, respectively. Histological assessment showed that VDRKO mice had muscle fibers that were significantly smaller in size and displayed hyper-nuclearity. Real-time PCR also indicated muscle developmental changes in VDRKO mice with dysregulation of myogenic regulatory factors (MRFs) and increased myostatin in quadriceps muscle (>2-fold). Vitamin D-deficient mice also showed increases in myostatin and the atrophy marker E3-ubiqutin ligase MuRF1. As a potential explanation for grip strength weakness, both groups of mice had down-regulation of genes encoding calcium-handling and sarco-endoplasmic reticulum calcium transport ATPase (Serca) channels. This is the first report of reduced strength, morphological, and gene expression changes in VDRKO and vitamin D-deficient mice where confounding by calcium, magnesium, and phosphate have been excluded by direct testing. Although suggested in earlier in vitro work, this study is the first to report an in vivo association between vitamin D, myostatin, and the regulation of muscle mass. These findings support a direct role for vitamin D in muscle function and corroborate earlier work on the presence of VDR in this tissue.

Hepatocyte-specific deletion of ARNT (aryl hydrocarbon Receptor Nuclear Translocator) results in altered fibrotic gene expression in the thioacetamide model of liver injury.

BACKGROUND & AIMS: Recent studies have shown that increased expression of liver hypoxia inducible factor 2-α (HIF-2α) leads to liver inflammation and a pro-fibrotic gene expression signature. Aryl hydrocarbon Receptor Nuclear Translocator (ARNT) is required for HIF-2α transcriptional activity and has previously been shown to regulate hepatic metabolism in mice. In these studies we examined the role of hepatocyte ARNT in the thioacetamide (TAA)-induced model of liver fibrosis. METHODS: Hepatocyte-specific ARNT-null (LARNT) mice were created using an albumin promoter-driven Cre recombinase. LARNT and floxed control (FC) littermates were placed on chow diet and received twice weekly intraperitoneal injections of 0.15mg/g body weight of TAA for 13 weeks. RESULTS: TAA treated LARNT and FC mice had a similar pattern of fibrosis. Quantification of Sirius red histology staining and hydroxyproline content revealed mixed results in terms of collagen deposition in LARNT livers. There was no significant difference in hepatocyte apoptosis or proliferation, as assessed by cleaved Caspase-3 and Ki67 respectively. LARNT mice had decreased macrophage accumulation, and decreased liver mRNA expression of Col1A1, Col1A2, Col5A1, Tgfß1, Tgfß2, Timp1 and Timp2. CONCLUSIONS: Deletion of hepatocyte ARNT leads to altered expression of collagen associated mRNA and reduced macrophage infiltration in the TAA-induced model of liver fibrosis. It appears that hepatocyte ARNT is not a requirement for initiation of liver fibrogenesis, but does regulate pro-fibrotic gene expression and macrophage accumulation.

Rare Mediastinal Hydatid Cyst.

Hydatid disease is a parasitic disease caused by the larval form of Echinococcus granulosus. Most common sites are liver, lungs, and brain. The disease is rarely present in the mediastinum. We report the rare instance of a 52-year-old female who presented with hydatid disease in the uncommon location of posterior mediastinum.

Chilaiditi's syndrome.

Chilaidit's syndrome is a rare condition characterised by the interposition of the colon between the liver and the right hemidiaphragm. We present a case of 20-year-old male who reported with breathlessness and epigastric pain, and he was diagnosed radiologically to have Chilaiditi's syndrome.

A vitamin D receptor/SMAD genomic circuit gates hepatic fibrotic response.

Liver fibrosis is a reversible wound-healing response involving TGFß1/SMAD activation of hepatic stellate cells (HSCs). It results from excessive deposition of extracellular matrix components and can lead to impairment of liver function. Here, we show that vitamin D receptor (VDR) ligands inhibit HSC activation by TGFß1 and abrogate liver fibrosis, whereas Vdr knockout mice spontaneously develop hepatic fibrosis. Mechanistically, we show that TGFß1 signaling causes a redistribution of genome-wide VDR-binding sites (VDR cistrome) in HSCs and facilitates VDR binding at SMAD3 profibrotic target genes via TGFß1-dependent chromatin remodeling. In the presence of VDR ligands, VDR binding to the coregulated genes reduces SMAD3 occupancy at these sites, inhibiting fibrosis. These results reveal an intersecting VDR/SMAD genomic circuit that regulates hepatic fibrogenesis and define a role for VDR as an endocrine checkpoint to modulate the wound-healing response in liver. Furthermore, the findings suggest VDR ligands as a potential therapy for liver fibrosis.

Metformin-mediated Bambi expression in hepatic stellate cells induces prosurvival Wnt/ß-catenin signaling.

AMP-activated protein kinase (AMPK) regulates lipid, cholesterol, and glucose metabolism in specialized metabolic tissues, such as muscle, liver, and adipose tissue. Agents that activate AMPK, such as metformin and 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR), have beneficial effects on liver glucose and lipid metabolism. In addition, AMPK activation in proliferating hepatic stellate cells (HSC) induces growth arrest and inhibits hepatic fibrosis. As metformin and AICAR act in different ways to achieve their effects, our aim was to examine the effects of AMPK activation in quiescent HSCs with these two agents on HSC function. We found that phospho-AMPK levels were markedly upregulated by both AICAR and metformin in quiescent HSCs. However, although AICAR treatment induced cell death, cells treated with metformin did not differ from untreated controls. AICAR-mediated HSC cell death was paralleled by loss of expression of the TGF-ß decoy receptor Bambi, whereas metformin increased Bambi expression. Transfection of siRNA-Bambi into HSCs also induced cell death, mimicking the effects of AICAR, whereas overexpression of Bambi partially rescued AICAR-treated cells. As Bambi has previously been shown to promote cell survival through Wnt/ß-catenin signaling, a reporter incorporating binding sites for a downstream target of this pathway was transfected into HSCs and was induced. We conclude that although AICAR and metformin both activate AMPK in quiescent HSCs, AICAR rapidly induced cell death, whereas metformin-treated cells remained viable. The finding that metformin increases Bambi expression and activates Wnt/ß-catenin signaling provides a possible mechanistic explanation for this observation. These results suggest that AICAR and metformin may confer disease-specific therapeutic benefits.

Rhou maintains the epithelial architecture and facilitates differentiation of the foregut endoderm.

Rhou encodes a Cdc42-related atypical Rho GTPase that influences actin organization in cultured cells. In mouse embryos at early-somite to early-organogenesis stages, Rhou is expressed in the columnar endoderm epithelium lining the lateral and ventral wall of the anterior intestinal portal. During foregut development, Rhou is downregulated in regions where the epithelium acquires a multilayered morphology heralding the budding of organ primordia. In embryos generated from Rhou knockdown embryonic stem (ES) cells, the embryonic foregut displays an abnormally flattened shape. The epithelial architecture of the endoderm is disrupted, the cells are depleted of microvilli and the phalloidin-stained F-actin content of their sub-apical cortical domain is reduced. Rhou-deficient cells in ES cell-derived embryos and embryoid bodies are less efficient in endoderm differentiation. Impaired endoderm differentiation of Rhou-deficient ES cells is accompanied by reduced expression of c-Jun/AP-1 target genes, consistent with a role for Rhou in regulating JNK activity. Downregulation of Rhou in individual endoderm cells results in a reduced ability of these cells to occupy the apical territory of the epithelium. Our findings highlight epithelial morphogenesis as a required intermediate step in the differentiation of endoderm progenitors. In vivo, Rhou activity maintains the epithelial architecture of the endoderm progenitors, and its downregulation accompanies the transition of the columnar epithelium in the embryonic foregut to a multilayered cell sheet during organ formation.

Cytokine-induced stable neuronal differentiation of human bone marrow mesenchymal stem cells in a serum/feeder cell-free condition.

The characteristics and multilineage differentiation potential of bone marrow mesenchymal stem cells (BM MSC) remain controversial. This study aimed to characterize human BM MSC isolated by plastic adherent or antibody selection and their neuronal differentiation potential using growth factors or chemical inducing agents. MSC were found to express low levels of neuronal markers: neurofilament-M, beta tubulin III, and neuron specific enolase. Under a serum- and feeder cell-free condition, basic fibroblast growth factor, epidermal growth factor, and platelet-derived growth factor induced neuronal morphology in MSC. In addition to the above markers, these cells expressed neurotransmitters or associated proteins: gamma-aminobutyric acid, tyrosine hydroxylase and serotonin. These changes were maintained for up to 3 months in all bone marrow specimens (N = 6). In contrast, butylated hydroxyanisole and dimethylsulfoxide were unable to induce sustained neuronal differentiation. Our results show that MSC isolated by two different procedures produced identical lineage differentiation with defined growth factors in a serum- and feeder cell-free condition.

New developments in developmental biology.

A report on the 15th International Society of Developmental Biologists Congress, Sydney, Australia, 3-7 September 2005.