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.

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.

Focal Anticoagulation by Somatic Gene Transfer: Towards Preventing Cardioembolic Stroke.

Cardioembolic stroke (CS) has emerged as a leading cause of ischaemic stroke (IS); distinguished by thrombi embolising to the brain from cardiac origins; most often from the left atrial appendage (LAA). Contemporary therapeutic options are largely dependent on systemic anticoagulation as a blanket preventative strategy, yet this does not represent a nuanced or personalised solution. Contraindications to systemic anticoagulation create significant unmedicated and high-risk cohorts, leaving these patients at risk of significant morbidity and mortality. Atrial appendage occlusion devices are increasingly used to mitigate stroke risk from thrombi emerging from the LAA in patients ineligible for oral anticoagulants (OACs). Their use, however, is not without risk or significant cost, and does not address the underlying aetiology of thrombosis and CS. Viral vector-based gene therapy has emerged as a novel strategy to target a spectrum of haemostatic disorders, achieving success through the adeno-associated virus (AAV) based therapy of haemophilia. Yet, thrombotic disorders, such as CS, have had limited exploration within the realm of AAV gene therapy approaches-presenting a gap in the literature and an opportunity for further research. Gene therapy has the potential to directly address the cause of CS by localised targeting of the molecular remodelling that serves to promote thrombosis.

In Search of Adeno-Associated Virus Vectors With Enhanced Cardiac Tropism for Gene Therapy.

Globally, adeno-associated virus (AAV) vectors have been increasingly used for clinical gene therapy trials. In Australia, AAV-based gene therapy is available for hereditary diseases such as retinal dystrophy or spinal muscular atrophy 1 (SMA1). Many preclinical studies have used AAV vectors for gene therapy in models of cardiac disease with outcomes of varying translational potential. However, major barriers to effective and safe therapeutic gene delivery to the human heart remain to be overcome. These include tropism, efficient gene transfer, mitigating off-target gene delivery and avoidance of the host immune response. Developing such an enhanced AAV vector for cardiac gene therapy is of great interest to the field of advanced cardiac therapeutics. In this review, we provide an overview of the approaches currently being employed in the search for cardiac cell-specific AAV capsids, ranging from natural AAVs selected as a result of infection and latency in the heart, to the use of cutting-edge molecular techniques to engineer and select AAVs specific for cardiac cells with the use of high-throughput methods.

Psychological, functional and social outcomes in adolescent and young adult cancer survivors over time: A systematic review of longitudinal studies.

OBJECTIVE: Most adolescents and young adults (AYA) can expect to survive a cancer diagnosis and treatment, but all will be left with the potential of long-term negative effects that can impact their ability to reach their full potential in life. Understanding aspects of psychological, functional, and social health and well-being outcomes, is pivotal for optimising long-term well-being. METHODS: We completed a systematic review of longitudinal studies reporting outcomes after anti-cancer treatment for Adolescents and Young Adults diagnosed between the age of 12-29 years according to established systematic review processes. The protocol was registered with PROSPERO (ID: CRD 42020203116). RESULTS: Thirteen reports from 10 studies met eligibility criteria representing 17,645 individuals (50.3% female, mean age at diagnosis 22 years, and 26 years at last, follow up). Eleven reports were from eight quantitative studies that relied on self-report surveys and two were qualitative studies. Psychological outcomes were reported to improve over time, as were functional health outcomes, although reported health behaviours were inconsistent between studies. Neurocognitive deficits were reported to affect the ability to return to work and impacts on fertility and sexuality were sustained over time. CONCLUSIONS: While some outcomes for AYA are reported to improve over time, particularly for physical functioning, and anxiety and depression, the long-term impact of cancer on many important domains remains largely unknown. Specifically, the evidence to understand what changes occur over time, and when, remains underdeveloped.

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.

Screening for atrial fibrillation and other arrhythmias in primary care.

BACKGROUND: Atrial fibrillation (AF) and other arrhythmias are prevalent and often encountered by general practitioners (GPs). In response to the growing prevalence and to assist practitioners in the diagnosis and management of AF, the Cardiac Society of Australia & New Zealand and Heart Foundation of Australia published the first Australian AF Guidelines in 2018. We aimed to examine (a) the proportion of GPs who performed any form of AF screening and identify the methods they applied, (b) GPs' awareness of the AF Guidelines and approaches to arrhythmia screening, (c) the roles of conventional 12-lead ECG and mobile health devices, and (d) GPs' confidence in ECG interpretation and need for training. METHODS: A cross-sectional online survey titled "GPs Screen their patients for Atrial Fibrillation and othEr aRrhythmia (GPSAFER)" was conducted from October 2018 to March 2019. The participants were recruited via various GP networks across Australia. Ethics approval was granted by The University of Sydney. RESULTS: A total of 463 surveys were completed. Many GPs (394/463, 85.1%, 95% CI 81.5-88.2%) performed some forms of AF screening and applied at least one AF screening method, most frequently pulse palpation (389/463, 84.0%). Some (299/463, 64.6%) GPs considered assessing their patients for other arrhythmias (237/299, 79.3% for complete heart block and 236/299, 78.9% for long-QT). Most GPs (424/463, 91.6%) were not using mobile ECG devices in their practice but some (147/463, 31.7%) were contemplating it. One third (175/463, 37.8%) of GPs were aware of the Australian AF Guidelines; those aware were more likely to perform AF screening (98.9% vs 76.7%, p <  0.001). Factors significantly and positively associated with AF screening were "awareness of the AF Guidelines" (p <  0.001), "number of years working in general practice" (p <  0.001), and "confidence in ECG interpretation of AF" (p = 0.003). Most GPs reported that they were very or extremely confident in interpreting AF (381/463, 82.3%) and complete heart block (266/463, 57.5%). Many GPs (349/463, 75.4%) would like to receive online ECG interpretation training. CONCLUSIONS: Assessment of arrhythmias is common in general practice and GPs are open to further training in ECG interpretation and using mobile ECG devices to aid their clinical practice. Increasing awareness of AF Guidelines and improving confidence in ECG interpretation may increase AF screening.

The transcription factor Klf5 is essential for intrahepatic biliary epithelial tissue remodeling after cholestatic liver injury.

Under various conditions of liver injury, the intrahepatic biliary epithelium undergoes dynamic tissue expansion and remodeling, a process known as ductular reaction. Mouse models defective in inducing such a tissue-remodeling process are more susceptible to liver injury, suggesting a crucial role of this process in liver regeneration. However, the molecular mechanisms regulating the biliary epithelial cell (BEC) dynamics in the ductular reaction remain largely unclear. Here, we demonstrate that the transcription factor Krüppel-like factor 5 (Klf5) is highly enriched in mouse liver BECs and plays a key role in regulating the ductular reaction, specifically under cholestatic injury conditions. Although mice lacking Klf5 in the entire liver epithelium, including both hepatocytes and BECs (Klf5-LKO (liver epithelial-specific knockout) mice), did not exhibit any apparent phenotype in the hepatobiliary system under normal conditions, they exhibited significant defects in biliary epithelial tissue remodeling upon 3,5-diethoxycarbonyl-1,4-dihydrocollidine-induced cholangitis, concomitantly with exacerbated cholestasis and reduced survival rate. In contrast, mice lacking Klf5 solely in hepatocytes did not exhibit any such phenotypes, confirming Klf5's specific role in BECs. RNA-sequencing analyses of BECs isolated from the Klf5-LKO mouse livers revealed that the Klf5 deficiency primarily affected expression of cell cycle-related genes. Moreover, immunostaining analysis with the proliferation marker Ki67 disclosed that the Klf5-LKO mice had significantly reduced BEC proliferation levels upon injury. These results indicate that Klf5 plays a critical role in the ductular reaction and biliary epithelial tissue expansion and remodeling by inducing BEC proliferation and thereby contributing to liver regeneration.

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.

The Role of Text Messaging in Cardiovascular Risk Factor Optimization.

PURPOSE OF REVIEW: Many cases of CVD may be avoidable through lowering behavioural risk factors such as smoking and physical inactivity. Mobile health (mHealth) provides a novel opportunity to deliver cardiovascular prevention programs in a format that is potentially scalable. Here, we provide an overview of text messaging-based mHealth interventions in cardiovascular prevention. RECENT FINDINGS: Text messaging-based interventions appear effective on a range of behavioural risk factors and can effect change on multiple risk factors-e.g. smoking, weight, blood pressure-simultaneously. For many texting studies, there are challenges in interpretation as many texting interventions are part of larger complex interventions making it difficult to determine the benefits of the separate components. Whilst there is evidence for text messaging improving cardiovascular risk factor levels in the short-term, future studies are needed to examine the durability of these effects and whether they can be translated to improvements in clinical care and outcomes.

Outdoor areas of Australian residential aged care facilities do not facilitate appropriate sun exposure.

OBJECTIVE: The aim of the present study was to assess whether the outdoor areas of residential aged care facilities used for a sunlight intervention trial had the design features that encouraged participants' use of these spaces. METHODS: The design principles recommended in the 'Vitamin D and the Built Environment in Victoria' guidelines were used to assess the outdoor spaces of residential aged care facilities that were used in a randomised controlled trial (RCT) of sunlight exposure. Attendance rates in the sunlight RCT were analysed in relation to global impression scores of the facilities using one-way analysis of variance. RESULTS: Thirty-six outdoor areas of 31 facilities were assessed. The facilities met the guidelines for sun exposure, and were generally safe and accessible. However, many lacked privacy, security and aesthetic appeal. Most of the outdoor spaces were not used for regularly scheduled activities. Attendance rates were higher in those facilities with the highest global impression scores compared with those with the lowest scores (F(2,367) = 3.262, P = 0.039). CONCLUSIONS: The physical environment of the outdoor areas of residential aged care facilities was associated with their use for sunlight exposure. Suitably designed or modified spaces have the potential to encourage their greater use, and residential aged care facilities should also plan regular activities in those areas. These measures can facilitate safe sun exposure, as well as physical activity and social interaction in older people.

Adeno-associated virus-mediated rescue of neonatal lethality in argininosuccinate synthetase-deficient mice.

Viral vectors based on adeno-associated virus (AAV) are showing exciting promise in gene therapy trials targeting the adult liver. A major challenge in extending this promise to the pediatric liver is the loss of episomal vector genomes that accompanies hepatocellular proliferation during liver growth. Hence maintenance of sufficient transgene expression will be critical for success in infants and children. We therefore set out to explore the therapeutic efficacy and durability of liver-targeted gene transfer in the challenging context of a neonatal lethal urea cycle defect, using the argininosuccinate synthetase deficient mouse. Lethal neonatal hyperammonemia was prevented by prenatal and early postnatal vector delivery; however, hyperammonemia subsequently recurred limiting survival to no more than 33 days despite vector readministration. Antivector antibodies acquired in milk from vector-exposed dams were subsequently shown to be blocking vector readministration, and were avoided by crossfostering vector-treated pups to vector-naive dams. In the absence of passively acquired antivector antibodies, vector redelivery proved efficacious with mice surviving to adulthood without recurrence of significant hyperammonemia. These data demonstrate the potential of AAV vectors in the developing liver, showing that vector readministration can be used to counter growth-associated loss of transgene expression provided the challenge of antivector humoral immunity is addressed.

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.

Beyond Medical Care: How Different National Models of Care Impact the Experience of Adolescent and Young Adult Cancer Patients.

Patient experience is positively associated with clinical effectiveness, quality care, and patient safety. This study examines the experience of care of adolescents and young adult (AYA) cancer patients from Australia and the United States, allowing a comparison of patient experiences in the context of different national models of cancer care delivery. Participants (n = 190) were aged 15-29 years and received cancer treatment from 2014 to 2019. Australians (n = 118) were recruited nationally by health care professionals. U.S. participants (n = 72) were recruited nationally via social media. The survey included demographic and disease variables, and questions regarding medical treatment, information and support provision, care coordination, and satisfaction across the treatment pathway. Sensitivity analyses examined the possible contribution of age and gender. Most patients from both countries were satisfied or very satisfied with their medical treatment (chemotherapy, radiotherapy, and surgery). There were significant differences between countries in the provision of fertility preservation services, age-appropriate communication, and psychosocial support. Our findings suggest when a national system of oversight with both state and federal funding is implemented, as is the case in Australia but not in the United States, significantly more AYAs with cancer receive age-appropriate information and support services, and improved access to specialist services such as fertility care. A national approach with government funding and centralized accountability appears to be associated with substantial benefits for the well-being of AYAs undergoing cancer treatment.

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.

Gene therapy for metabolic disorders: an overview with a focus on urea cycle disorders.

Many metabolic diseases are compelling candidates for gene therapy, and are the subject of vigorous pre-clinical research. Successful phenotype correction in mouse models is now commonplace and research effort is increasingly being directed towards addressing the translational challenges inherent in human clinical trials. This paper places current efforts to develop gene therapy approaches to metabolic disease in historical context and describes contemporary research in the authors' laboratory on urea cycle defects, particularly ornithine transcarbamylase deficiency, in a manner that is illustrative of the general state of the field.

Induction and prevention of severe hyperammonemia in the spfash mouse model of ornithine transcarbamylase deficiency using shRNA and rAAV-mediated gene delivery.

Urea cycle defects presenting early in life with hyperammonemia remain difficult to treat and commonly necessitate liver transplantation. Gene therapy has the potential to prevent hyperammonemic episodes while awaiting liver transplantation, and possibly also to avert the need for transplantation altogether. Ornithine transcarbamylase (OTC) deficiency, the most prevalent urea cycle disorder, provides an ideal model for the development of liver-targeted gene therapy. While we and others have successfully cured the spf(ash) mouse model of OTC deficiency using adeno-associated virus (AAV) vectors, a major limitation of this model is the presence of residual OTC enzymatic activity which confers a mild phenotype without clinically significant hyperammonemia. To better model severe disease we devised a strategy involving AAV2/8-mediated delivery of a short hairpin RNA (shRNA) to specifically knockdown residual endogenous OTC messenger RNA (mRNA). This strategy proved highly successful with vector-treated mice developing severe hyperammonemia and associated neurological impairment. Using this system, we showed that the dose of an AAV rescue construct encoding the murine OTC (mOTC) cDNA required to prevent hyperammonemia is fivefold lower than that required to control orotic aciduria. This result is favorable for clinical translation as it indicates that the threshold for therapeutic benefit is likely to be lower than indicated by earlier studies.

Hospital data reporting on postpartum hemorrhage: under-estimates recurrence and over-estimates the contribution of uterine atony.

This study aimed to explore whether recording of a prior adverse pregnancy outcome (postpartum hemorrhage) in a medical record increases the likelihood that recurrence of the same event is reported in hospital data. Using a sample of 588 pregnancies [2 consecutive pregnancies for 294 randomly selected women with at least one postpartum hemorrhage (PPH)], we compared 'coded' recurrence rates in hospital data with those obtained from medical record audit. 'Coded' recurrence in a second pregnancy was also compared for women with or without a documented history of prior PPH. We found a 'coded' recurrence rate of 18.5% and an 'audited' recurrence rate of 28.4%. The 'coded' rate of recurrence among women who had a documented history of PPH was 27.4% compared to 19.1% when the previous PPH was not noted in the second pregnancy medical record. Medical record reporting of uterine atony as the cause for postpartum hemorrhages in first and second births was 37.9 and 34.0% while 'coded' hospital data reporting attributed 79.8 and 73.9% respectively to atony. Our study results indicate that a history of postpartum hemorrhage may be a stronger risk factor for subsequent PPH than previously demonstrated. A recorded history of PPH was associated with an increased likelihood of reporting a subsequent PPH, and in such cases recurrence rates approximate true recurrence. The contribution of uterine atony as a cause of postpartum hemorrhage is over-estimated using hospital data.

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.