Interventional Radiology Obesity Therapeutics: Proceedings from the Society of Interventional Radiology Foundation Research Consensus Panel.

The Society of Interventional Radiology Foundation commissioned a Research Consensus Panel to establish a research agenda on "Obesity Therapeutics" in interventional radiology (IR). The meeting convened a multidisciplinary group of physicians and scientists with expertise in obesity therapeutics. The meeting was intended to review current evidence on obesity therapies, familiarize attendees with the regulatory evaluation process, and identify research deficiencies in IR bariatric interventions, with the goal of prioritizing future high-quality research that would move the field forward. The panelists agreed that a weight loss of >8%-10% from baseline at 6-12 months is a desirable therapeutic endpoint for future IR weight loss therapies. The final consensus on the highest priority research was to design a blinded randomized controlled trial of IR weight loss interventions versus sham control arms, with patients receiving behavioral therapy.

Food and Drug Administration insights on clinical study of weight-loss devices intended for adolescent patients.

BACKGROUND: Medical devices intended for weight loss may provide clinically meaningful benefit to children who are overweight and have obesity; however, no device has been approved by the U.S. Food and Drug Administration (FDA) for use in patients below 18 years of age. Encouragingly, FDA regularly sees new device designs because the field of weight-loss devices is advancing rapidly. As more devices for weight loss are in development, their use in adolescent populations is expected to follow, but supporting data are needed. OBJECTIVES: This report describes efforts that FDA has taken to understand the unmet clinical need, understand how pediatric patients might benefit from a weight-loss device, and provide considerations for how to best design weight-loss device clinical studies considering device-specific patient risk for adolescents. METHODS: We review the recommendations provided to the FDA in 2005 via a Pediatric Advisory Committee meeting and discuss feedback received in 2018 through our Network of Experts programme. RESULTS: FDA encourages weight-loss device manufacturers and academic researchers to collect data through properly controlled trials so that more treatment options can be accessible to pediatric patients. CONCLUSIONS: FDA remains open to considering risk-based clinical study designs incorporating pediatric patients and will continue to take into account the risk to adolescent study participants when determining whether the benefit-risk evidence supports initiation of an adolescent weight-loss device study.

Research: Fluorescence Microscopy-Based Protocol for Detecting Residual Bacteria on Medical Devices.

Standard methods are needed to reliably and efficiently assess bacterial contamination of processed medical devices. This article demonstrates a standard operating procedure (SOP) for fluorescence microscopy-based detection of residual bacteria on medical devices (BAC-VIS). BAC-VIS uses a 4',6-diamidino-2-phenylindole (DAPI) stain with fluorescent microscopy to quickly and cost-effectively detect bacterial contamination of processed medical device parts. The BAC-VIS protocol was optimized and achieved greater than 80% staining efficiency and a signal-to-noise ratio of more than 20 using four representative organisms. The SOP was first validated for use on a buildup biofilm model, accessory channels of contaminated clinically used devices, and inoculated endoscope end caps and O-rings. The buildup biofilm model was used to evaluate BAC-VIS after repeated treatment of adherent bacteria with three common high-level disinfectants: glutaraldehyde, ortho-phthalaldehyde, and peracetic acid. Next, BAC-VIS was used to assess clinically used endoscope parts that cultured positive for Gram-negative bacteria. DAPI-stained cells were found on all culture-positive devices, especially in grooves and imperfections on the surface. Finally, BAC-VIS was used to detect bacteria on inoculated endoscope device components. The results showed potential for BAC-VIS to be a valuable tool for industry and academic/medical researchers for investigations of contaminated medical devices. Results obtained using BAC-VIS can increase understanding of the role of design in cleanability, wear, and prevention of contamination and may lead to improvements in materials and design that could make processed endoscope use safer for patients. Of note, this protocol is not for detecting bacteria on scopes or scope parts that will be put back into clinical use.

US Food and Drug Administration and Off-Label Use of Metal Expandable Biliary Stents within the Peripheral Vasculature-Update.

In the past, some medical device manufacturers were obtaining marketing clearance for metallic stents indicated for biliary use; however, these devices were being promoted and used in the peripheral vasculature, creating a patient safety problem. The US Food and Drug Administration (FDA) acted in 2006-2008 to help decrease off-label use of metal expandable biliary stents. This communication describes the early and continued efforts of the FDA to address safety concerns relating to off-label use of metal expandable biliary stents and the status of this issue. An analysis of Medical Device Reporting (MDR) data from January 1, 2005, through December 31, 2018, was conducted to determine the percentage of MDR reports associated with off-label use. The percentage was approximately 90% in 2001-2006 and decreased to < 40% a decade later. In reports associated with off-label use, these devices are still associated with death and serious injury; however, the percentage of injury MDR reports associated with off-label device use has trended down since 2007. Whereas 92%-95% of reported serious injuries were with off-label placement in 2005-2007, 43%-79% of injuries were with off-label placement in 2008-2018. Collaborative efforts among the FDA, manufacturers, and physicians appear to have made progress in addressing this issue.

Loss of miR-17~92 results in dysregulation of Cftr in nephron progenitors.

We have previously demonstrated that loss of miR-17~92 in nephron progenitors in a mouse model results in renal hypodysplasia and chronic kidney disease. Clinically, decreased congenital nephron endowment because of renal hypodysplasia is associated with an increased risk of hypertension and chronic kidney disease, and this is at least partly dependent on the self-renewal of nephron progenitors. Here, we present evidence for a novel molecular mechanism regulating the self-renewal of nephron progenitors and congenital nephron endowment by the highly conserved miR-17~92 cluster. Whole transcriptome sequencing revealed that nephron progenitors lacking this cluster demonstrated increased Cftr expression. We showed that one member of the cluster, miR-19b, is sufficient to repress Cftr expression in vitro and that perturbation of Cftr activity in nephron progenitors results in impaired proliferation. Together, these data suggest that miR-19b regulates Cftr expression in nephron progenitors, with this interaction playing a role in appropriate nephron progenitor self-renewal during kidney development to generate normal nephron endowment.

The Regulatory Perspectives on Endoscopic Devices for Obesity.

The recent increase in US Food and Drug Administration-approved weight-loss devices has diversified obesity treatment options. The regulatory pathways for endoscopically placed weight-loss devices and considerations for clinical trials are discussed, including the benefit-risk paradigm intended to aid in weight-loss-device trial development. Also discussed is the benefit-risk analysis of recently approved endoscopic devices. A strategic priority of the FDA Center for Devices and Radiological Health is to increase the use of patient input in decision making. Thus, we consider how endoscopic weight-loss devices with profiles similar to those that have been approved may be viewed in a patient preference study.

MicroRNA changes, activation of progenitor cells and severity of liver injury in mice induced by choline and folate deficiency.

Dietary deficiency in methyl-group donors and cofactors induces liver injury that resembles many pathophysiological and histopathological features of human nonalcoholic fatty liver disease (NAFLD), including an altered expression of microRNAs (miRNAs). We evaluated the consequences of a choline- and folate-deficient (CFD) diet on the expression of miRNAs in the livers of male A/J and WSB/EiJ mice. The results demonstrate that NAFLD-like liver injury induced by the CFD diet in A/J and WSB/EiJ mice was associated with marked alterations in hepatic miRNAome profiles, with the magnitude of miRNA expression changes being greater in WSB/EiJ mice, the strain characterized by the greatest severity of liver injury. Specifically, WSB/EiJ mice exhibited more prominent changes in the expression of common miRNAs as compared to A/J mice and distinct miRNA alterations, including the overexpression of miR-134, miR-409-3p, miR-410 and miR-495 miRNAs that were accompanied by an activation of hepatic progenitor cells and fibrogenesis. This in vivo finding was further confirmed by in vitro experiments showing an overexpression of these miRNAs in undifferentiated progenitor hepatic HepaRG cells compared to in fully differentiated HepaRG cells. Additionally, a marked elevation of miR-134, miR-409-3p, miR-410 and miR-495 was found in plasma of WSB/EiJ mice fed the CFD diet, while none of the miRNAs was changed in plasma of A/J mice. These findings suggest that miRNAs may be crucial regulators responsible for the progression of NAFLD and may be useful as noninvasive diagnostic indicators of the severity and progression of NAFLD.

MicroRNA Responses to the Genotoxic Carcinogens Aflatoxin B1 and Benzo[a]pyrene in Human HepaRG Cells.

Recent advances in toxicogenomics present an opportunity to develop new in vitro testing methodologies to identify human carcinogens. We have investigated microRNA expression responses to the treatment of human liver HepaRG cells with the human genotoxic carcinogens aflatoxin B1 (AFB1) and benzo[a]pyrene (B[a]P), and the structurally similar compounds aflatoxin B2 (AFB2) and benzo[e]pyrene (B[e]P) that exhibit minimal carcinogenic potential. We demonstrate that treatment of HepaRG cells with AFB1 or B[a]P resulted in specific changes in the expression of miRNAs as compared with their non-carcinogenic analogues, particularly in a marked over-expression of miR-410. An additional novel finding is the dose- and time-dependent inhibition of miR-122 in AFB1-treated HepaRG cells. Mechanistically, the AFB1-induced down-regulation of miR-122 was attributed to inhibition of the HNF4A/miR-122 regulatory pathway. These results demonstrate that HepaRG cells can be used to investigate miRNA responses to xenobiotic exposure, and illustrate the existence of early non-genotoxic events, in addition to a well-established genotoxic mode of action changes, in the mechanism of AFB1 and B[a]P carcinogenicity.

Differentially expressed MicroRNAs provide mechanistic insight into fibrosis-associated liver carcinogenesis in mice.

Hepatocellular carcinoma (HCC) is one of the most prevalent human cancers, with a rising incidence worldwide. The molecular mechanisms associated with the development of HCC are complex and include multiple interconnected molecular alterations with mounting evidence indicating an important role of microRNAs (miRNAs) in the pathogenesis of HCC. In humans, the development of HCC is commonly associated with liver cirrhosis. To study fibrosis-associated liver carcinogenesis, we used a mouse model designed to emulate the development of HCC in cirrhotic liver. Specifically, we were interested in evaluating the role of miRNAs in the molecular pathogenesis of liver carcinogenesis in male B6C3F1/J mice treated with N-nitrosodiethylamine (DEN) or carbon tetrachloride (CCl4 ) alone or a combination of DEN and CCl4 and characterized by a differential tumor incidence that increased in the following order: DEN

Renal stromal miRNAs are required for normal nephrogenesis and glomerular mesangial survival.

MicroRNAs are small noncoding RNAs that post-transcriptionally regulate mRNA levels. While previous studies have demonstrated that miRNAs are indispensable in the nephron progenitor and ureteric bud lineage, little is understood about stromal miRNAs during kidney development. The renal stroma (marked by expression of FoxD1) gives rise to the renal interstitium, a subset of peritubular capillaries, and multiple supportive vascular cell types including pericytes and the glomerular mesangium. In this study, we generated FoxD1(GC);Dicer(fl/fl) transgenic mice that lack miRNA biogenesis in the FoxD1 lineage. Loss of Dicer activity resulted in multifaceted renal anomalies including perturbed nephrogenesis, expansion of nephron progenitors, decreased renin-expressing cells, fewer smooth muscle afferent arterioles, and progressive mesangial cell loss in mature glomeruli. Although the initial lineage specification of FoxD1(+) stroma was not perturbed, both the glomerular mesangium and renal interstitium exhibited ectopic apoptosis, which was associated with increased expression of Bcl2l11 (Bim) and p53 effector genes (Bax, Trp53inp1, Jun, Cdkn1a, Mmp2, and Arid3a). Using a combination of high-throughput miRNA profiling of the FoxD1(+)-derived cells and mRNA profiling of differentially expressed transcripts in FoxD1(GC);Dicer(fl/fl) kidneys, at least 72 miRNA:mRNA target interactions were identified to be suppressive of the apoptotic program. Together, the results support an indispensable role for stromal miRNAs in the regulation of apoptosis during kidney development.

The role for microRNAs in drug toxicity and in safety assessment.

INTRODUCTION: Adverse drug reactions present significant challenges that impact pharmaceutical development and are major burdens to public health services worldwide. In response to this need, the field of toxicology is rapidly expanding to identify key pathways involved in drug toxicity. AREAS COVERED: MicroRNAs (miRNAs) are a class of small evolutionary conserved endogenous non-coding RNAs that regulate the translation of protein-coding genes. A wide range of toxicants alter miRNA levels in target organs and these altered miRNAs can also be detected in easily accessible biological fluids. This, combined with an early miRNA response to toxic insults and miRNA stability, substantiates the potential for these small molecules to be useful biomarkers for drug safety assessment. EXPERT OPINION: miRNAs are early indicators and useful tools to detect drug-induced toxicity. Incorporation of miRNA profiling into the drug safety testing process will complement currently used techniques and may substantially enhance drug safety. With the increasing interests in translational research, the field of miRNA biomarker research will continue to expand and become an important part of the investigation of human drug toxicity.

Suppression of CYP2C9 by microRNA hsa-miR-128-3p in human liver cells and association with hepatocellular carcinoma.

Published studies have identified genetic variants, somatic mutations, and changes in gene expression profiles that are associated with hepatocellular carcinoma (HCC), particularly involving genes that encode drug metabolizing enzymes (DMEs). CYP2C9, one of the most abundant and important DMEs, is involved in the metabolism of many carcinogens and drugs and is down-regulated in HCC. To investigate the molecular mechanisms that control CYP2C9 expression, we applied integrative approaches including in silico, in vitro, and in vivo analyses to elucidate the role of microRNA hsa-miR-128-3p in the regulation of CYP2C9 expression and translation. RNA electrophoresis mobility shift assays demonstrated a direct interaction between hsa-miR-128-3p and its cognate target, the CYP2C9 transcript. Furthermore, the expression of a luciferase reporter gene containing the 3'-UTR of CYP2C9 and the endogenous expression of CYP2C9 were suppressed by transfection of hsa-miR-128-3p. Importantly, chemically-induced up- or down-regulation of hsa-miR-128-3p correlated inversely with the expression of CYP2C9. Finally, an association analysis revealed that the expression of hsa-miR-128-3p is inversely correlated with the expression of CYP2C9 in HCC tumor tissues. Altogether, the study helped to elucidate the mechanism of CYP2C9 regulation by hsa-miR-128-3p, and the inverse association in HCC.

miRNA-based buffering of the cobblestone-lissencephaly-associated extracellular matrix receptor dystroglycan via its alternative 3'-UTR.

Many proteins are expressed dynamically during different stages of cellular life and the accuracy of protein amounts is critical for cell endurance. Therefore, cells should have a perceptive system that notifies about fluctuations in the amounts of certain components and an executive system that efficiently restores their precise levels. At least one mechanism that evolution has employed for this task is regulation of 3'-UTR length for microRNA targeting. Here we show that in Drosophila the microRNA complex miR-310s acts as an executive mechanism to buffer levels of the muscular dystrophy-associated extracellular matrix receptor dystroglycan via its alternative 3'-UTR. miR-310s gene expression fluctuates depending on dystroglycan amounts and nitric oxide signalling, which perceives dystroglycan levels and regulates microRNA gene expression. Aberrant levels of dystroglycan or deficiencies in miR-310s and nitric oxide signalling result in cobblestone brain appearance, resembling human lissencephaly type II phenotype.

Noncoding RNA response to xenobiotic exposure: an indicator of toxicity and carcinogenicity.

INTRODUCTION: Human exposure to certain environmental and occupational chemicals is one of the major risk factors for noncommunicable diseases, including cancer. Therefore, it is desirable to take advantage of subtle exposure-related adverse cellular events for early disease detection and to identify potential dangers caused by new and currently under-evaluated drugs and chemicals. Nongenotoxic events due to carcinogen/toxicant exposure are a general hallmark of sustained cellular stress leading to tumorigenesis. These processes are globally regulated via noncoding RNAs (ncRNAs). Tumorigenesis-associated genotoxic and nongenotoxic events lead to the altered expression of ncRNAs and may provide a mechanistic link between chemical exposure and tumorigenesis. Current advances in toxicogenomics are beginning to provide valuable insight into gene-chemical interactions at the transcriptome level. AREAS COVERED: In this review, we summarize recent information about the impact of xenobiotics on ncRNAs. Evidence highlighted in this review suggests a critical role of ncRNAs in response to carcinogen/toxicant exposure. EXPERT OPINION: Benefits for the use of ncRNAs in carcinogenicity assessment include remarkable tissue specificity, early appearance, low baseline variability, and their presence and stability in biological fluids, which suggests that the incorporation of ncRNAs in the evaluation of cancer risk assessment may enhance substantially the efficiency of toxicity and carcinogenicity testing.

Measurement of metabolic rate in Drosophila using respirometry.

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

MicroRNA-17~92 is required for nephrogenesis and renal function.

Deletion of all microRNAs (miRNAs) in nephron progenitors leads to premature loss of these cells, but the roles of specific miRNAs in progenitors have not been identified. Deletions in the MIR17HG cluster (miR-17~92 in mice), detected in a subset of patients with Feingold syndrome, represent the first miRNA mutations to be associated with a developmental defect in humans. Although MIR17HG is expressed in the developing kidney, and patients with Feingold syndrome caused by MYCN mutations have renal anomalies, it remains unclear to what extent MIR17HG contributes to renal development and function. To define the role of miR-17~92, we generated mice with a conditional deletion of miR-17~92 in nephron progenitors and their derivatives. The nephron progenitor population was preserved in these mice; however, this deletion impaired progenitor cell proliferation and reduced the number of developing nephrons. Postnatally, mutant mice developed signs of renal disease, including albuminuria by 6 weeks and focal podocyte foot process effacement and glomerulosclerosis at 3 months. Taken together, these data support a role for this miRNA cluster in renal development, specifically in the regulation of nephron development, with subsequent consequences for renal function in adult mice.

MicroRNAs: potential regulators of renal development genes that contribute to CAKUT.

Congenital anomalies of the kidney and urinary tract (CAKUT) are the leading cause of childhood chronic kidney disease (CKD). While mutations in several renal development genes have been identified as causes for CAKUT, most cases have not yet been linked to known mutations. Furthermore, the genotype-phenotype correlation is variable, suggesting that there might be additional factors that have an impact on the severity of CAKUT. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at the post-transcriptional level, and are involved in many developmental processes. Although little is known about the function of specific miRNAs in kidney development, several have recently been shown to regulate the expression of, and/or are regulated by, crucial renal development genes present in other organ systems. In this review, we discuss how miRNA regulation of common developmental signaling pathways may be applicable to renal development. We focus on genes that are known to contribute to CAKUT in humans, for which miRNA interactions in other contexts have been identified, with miRNAs that are present in the kidney. We hypothesize that miRNA-mediated processes might play a role in kidney development through similar mechanisms, and speculate that genotypic variations in these small RNAs or their targets could be associated with CAKUT.

Dg-Dys-Syn1 signaling in Drosophila regulates the microRNA profile.

BACKGROUND: The Dystrophin Glycoprotein Complex (DGC) is at the center of significant inheritable diseases, such as muscular dystrophies that can be fatal and impair neuronal function in addition to muscle degeneration. Recent evidence has shown that it can control cellular homeostasis and work via Dystrophin signaling to regulate microRNA gene expression which implies that disease phenotypes hide an entourage of regulatory and homeostatic anomalies. Uncovering these hidden processes could shed new light on the importance of proper DGC function for an organism's overall welfare and bring forth new ideas for treatments. RESULTS: To better understand a role for the DGC in these processes, we used the genetically advantageous Drosophila muscular dystrophy model to conduct a whole animal microarray screen. Since we have recently found that dystrophic symptoms can be caused by stress even in wild type animals and are enhanced in mutants, we screened stressed animals for microRNA misregulation as well. We were able to define microRNAs misregulated due to stress and/or dystrophy. Our results support the hypothesis that there is a Dystrophin and Dystroglycan dependent circuitry of processes linking stress response, dystrophic conditions and cellular signaling and that microRNAs play an important role in this network. Verification of a subset of our results was conducted via q-PCR and revealed that miR-956, miR-980 and miR-252 are regulated via a Dystroglycan-Dystrophin-Syntrophin dependent pathway. CONCLUSIONS: The results presented in this study support the hypothesis that there is a Dystrophin and Dystroglycan dependent circuitry of processes that includes regulation of microRNAs. Dystrophin signaling has already been found to occur in mammalian musculature; however, our data reveals that this regulation is evolutionarily conserved and also present in at least neuronal tissues. Our data imply that Dystroglycan-Dystrophin-Syntrophin signaling through control of multiple microRNAs is involved in highly managed regulation of gene expression required to adapt cellular homeostasis that is compromised under stress and dystrophic conditions.

New dystrophin/dystroglycan interactors control neuron behavior in Drosophila eye.

BACKGROUND: The Dystrophin Glycoprotein Complex (DGC) is a large multi-component complex that is well known for its function in muscle tissue. When the main components of the DGC, Dystrophin (Dys) and Dystroglycan (Dg) are affected cognitive impairment and mental retardation in addition to muscle degeneration can occur. Previously we performed an array of genetic screens using a Drosophila model for muscular dystrophy in order to find novel DGC interactors aiming to elucidate the signaling role(s) in which the complex is involved. Since the function of the DGC in the brain and nervous system has not been fully defined, we have here continued to analyze the DGC modifiers' function in the developing Drosophila brain and eye. RESULTS: Given that disruption of Dys and Dg leads to improper photoreceptor axon projections into the lamina and eye neuron elongation defects during development, we have determined the function of previously screened components and their genetic interaction with the DGC in this tissue. Our study first found that mutations in chif, CG34400, Nrk, Lis1, capt and Cam cause improper axon path-finding and loss of SP2353, Grh, Nrk, capt, CG34400, vimar, Lis1 and Cam cause shortened rhabdomere lengths. We determined that Nrk, mbl, capt and Cam genetically interact with Dys and/or Dg in these processes. It is notable that most of the neuronal DGC interacting components encountered are involved in regulation of actin dynamics. CONCLUSIONS: Our data indicate possible DGC involvement in the process of cytoskeletal remodeling in neurons. The identification of new components that interact with the DGC not only helps to dissect the mechanism of axon guidance and eye neuron differentiation but also provides a great opportunity for understanding the signaling mechanisms by which the cell surface receptor Dg communicates via Dys with the actin cytoskeleton.

Stress and muscular dystrophy: a genetic screen for dystroglycan and dystrophin interactors in Drosophila identifies cellular stress response components.

In Drosophila, like in humans, Dystrophin Glycoprotein Complex (DGC) deficiencies cause a life span shortening disease, associated with muscle dysfunction. We performed the first in vivo genetic interaction screen in ageing dystrophic muscles and identified genes that have not been shown before to have a role in the development of muscular dystrophy and interact with dystrophin and/or dystroglycan. Mutations in many of the found interacting genes cause age-dependent morphological and heat-induced physiological defects in muscles, suggesting their importance in the tissue. Majority of them is phylogenetically conserved and implicated in human disorders, mainly tumors and myopathies. Functionally they can be divided into three main categories: proteins involved in communication between muscle and neuron, and interestingly, in mechanical and cellular stress response pathways. Our data show that stress induces muscle degeneration and accelerates age-dependent muscular dystrophy. Dystrophic muscles are already compromised; and as a consequence they are less adaptive and more sensitive to energetic stress and to changes in the ambient temperature. However, only dystroglycan, but not dystrophin deficiency causes extreme myodegeneration induced by energetic stress suggesting that dystroglycan might be a component of the low-energy pathway and act as a transducer of energetic stress in normal and dystrophic muscles.