A Degradable Sustained-Release Drug Delivery System for Bleb-Forming Glaucoma Surgery.

Fibrosis of the filtering bleb is one of the main causes of failure after bleb-forming glaucoma surgery. Intraoperative application of mitomycin C (MMC) is the current gold standard to reduce the fibrotic response. However, MMC is cytotoxic and one-time application is often insufficient. A sustained-release drug delivery system (DDS), loaded with MMC, may be less cytotoxic and equally or more effective. Two degradable (polycaprolactone (PCL) and polylactic-co-glycolic acid (PLGA)) MMC-loaded DDSs are developed. Release kinetics are first assessed in vitro followed by rabbit implants in conjunction with the PRESERFLO MicroShunt. As a control, the MicroShunt is implanted with adjunctive use of a MMC solution. Rabbits are euthanized at postoperative day (POD) 28 and 90. The PLGA and PCL DDSs release (on average) 99% and 75% of MMC, respectively. All groups show functioning blebs until POD 90. Rabbits implanted with a DDS show more inflammation with avascular thin-walled blebs when compared to the control. However, collagen is more loosely arranged. The PLGA DDS shows less inflammation, less foreign body response (FBR), and more complete degradation at POD 90 when compared to the PCL DDS. Further optimization with regard to dosage is required to reduce side effects to the conjunctiva.

Wound Healing Response After Bleb-Forming Glaucoma Surgery With a SIBS Microshunt in Rabbits.

Purpose: The PreserFlo MicroShunt is an innovative implant for the surgical treatment of glaucoma. Although usually effective, surgeries can still fail due to fibrosis. This study was conducted to gain insight into the histological aspects of the fibrotic response and find potential targets to reduce postoperative fibrosis. Methods: Fifteen New Zealand White rabbits were implanted with a microshunt and followed up for 40 days. Animals were euthanized at postoperative days (PODs) 1, 5, and 40 to collect eyes for histological evaluation. Bleb formation and ocular health were assessed by slit-lamp (SL) biomicroscopy and optical coherence tomography (OCT). Intraocular pressure (IOP) was measured using rebound tonometry. Results: Blebs failed after approximately 2 weeks based on bleb survival and IOP measurements. No severe complications were observed with OCT and SL. Histology revealed a wide variety of cells, in the bleb and around the microshunt, including polymorphonuclear leucocytes (PMNs), myofibroblasts, and foreign body giant cells, at different PODs. Conclusions: Implantation of a poly(styrene-b-isobutylene-b-styrene) microshunt in rabbits resulted in the occurrence of a wide variety of cells during the wound-healing response. Future research should further elucidate the potential of these (earlier often overlooked) cells to target the fibrotic response in vivo-for example, by developing novel antifibrotic drugs, methods for sustained delivery of medications, or augmenting material properties. Translational Relevance: Current antifibrotic therapies aim to inhibit myofibroblasts; however, a wide variety of cells are involved in the fibrotic response. Future research focusing on these cells could offer novel methods for reducing the fibrotic response after glaucoma surgery.

Repeatability, reproducibility, and agreement of three tonometers for measuring intraocular pressure in rabbits.

The aim of this study was to evaluate repeatability, reproducibility, and agreement of three commonly used tonometers in animal research (TonoLab, TonoVet, and TonoPEN AVIA) in a cohort of 24 rabbits. Additionally, the impact of sedation on IOP was investigated in 21 New Zealand White rabbits with the TonoVet tonometer. Repeatability was determined using the coefficient of variation (CoV) for two observers. For the TonoLab (6.55%) and TonoVet (6.38%) the CoV was lower than for the TonoPEN AVIA (10.88%). The reproducibility was highest for the TonoVet (0.2 ± 3.3 mmHg), followed by the TonoLab (0 ± 12.89 mmHg) and lowest for the TonoPEN AVIA (- 1.48 ± 10.3 mmHg). The TonoLab and TonoVet showed the highest agreement (r = 0.85, R2 = 0.73). After sedation, a significant IOP reduction (often > 25%) was observed. Our results show that among the three tonometers tested, the TonoVet tonometer is best for use in rabbits while the TonoLab should be avoided. The impact of sedation on IOP was substantial and should be taken into account during experimentation.

History, presence, and future of mitomycin C in glaucoma filtration surgery.

PURPOSE OF REVIEW: Mitomycin C (MMC) is an alkylating agent with extraordinary ability to crosslink DNA, preventing DNA synthesis. By this virtue, MMC is an important antitumor drug. In addition, MMC has become the gold standard medication for glaucoma filtration surgery (GFS). This eye surgery creates a passage for drainage of aqueous humor (AqH) out of the eye into the sub-Tenon's space with the aim of lowering the intraocular pressure. A major cause of failure of this operation is fibrosis and scarring in the sub-Tenon's space, which will restrict AqH outflow. Intraoperative application of MMC during GFS has increased GFS success rate, presumably mainly by reducing fibrosis after GFS. However, still 10% of glaucoma surgeries fail within the first year. RECENT FINDINGS: In this review, we evaluate risks and benefits of MMC as an adjuvant for GFS. In addition, we discuss possible improvements of its use by adjusting dose and method of administration. SUMMARY: One way of improving GFS outcome is to prolong MMC delivery by using a drug delivery system.

Integrated 'omics analysis reveals new drug-induced mitochondrial perturbations in human hepatocytes.

We performed a multiple 'omics study by integrating data on epigenomic, transcriptomic, and proteomic perturbations associated with mitochondrial dysfunction in primary human hepatocytes caused by the liver toxicant valproic acid (VPA), to deeper understand downstream events following epigenetic alterations in the mitochondrial genome. Furthermore, we investigated persistence of cross-omics changes after terminating drug treatment. Upon transient methylation changes of mitochondrial genes during VPA-treatment, increasing complexities of gene-interaction networks across time were demonstrated, which normalized during washout. Furthermore, co-expression between genes and their corresponding proteins increased across time. Additionally, in relation to persistently decreased ATP production, we observed decreased expression of mitochondrial complex I and III-V genes. Persistent transcripts and proteins were related to citric acid cycle and ß-oxidation. In particular, we identified a potential novel mitochondrial-nuclear signaling axis, MT-CO2-FN1-MYC-CPT1. In summary, this cross-omics study revealed dynamic responses of the mitochondrial epigenome to an impulse toxicant challenge resulting in persistent mitochondrial dysfunctioning. Moreover, this approach allowed for discriminating between the toxic effect of VPA and adaptation.

A cross-omics approach to investigate temporal gene expression regulation by 5-hydroxymethylcytosine via TBH-derived oxidative stress showed involvement of different regulatory kinases.

Regulation of DNA methylation plays a crucial role in biological processes and carcinogenesis. The formation of 5-hydroxymethylcytosine (5hmC) by oxidation of 5-methylcytosine (5mC) has been proposed as an intermediate of active demethylation. However, whether and how active demethylation is regulated by oxidative stress-related processes is not well understood. Here we investigated whether free oxygen radicals are capable of directly forming 5hmC and how this enhanced whole genome gene expression. We applied LC-MS/MS technology for the analysis of 5mC, 5hmC, 5-formylcytosine (5fC) and 5-hydroxymethyluracyl (5hmU) in HepG2 cells exposed to hydroxyl- and methyl radicals, formed by tert-butyl hydroperoxide (TBH) at multiple time points. We observed that TBH is able to induce a significant increase in 5hmC. A detailed evaluation of the hydroxymethylome using a combination of 5hmC-immunoprecipitation and microarrays resulted in the identification of highly dynamic modifications that appear to increase during prolonged oxidant exposure. Analyses of temporal gene expression changes in combination with network analysis revealed different subnetworks containing differentially expressed genes (DEGs) with differentially hydroxyl-methylated regions (DhMRs) in different regulatory kinases enriched with serine-threonine kinases. These serine-threonine kinases compromises MAPK14, RPSK6KA1, RIPK1, and PLK3 and were all previously identified as key-regulators in hepatocarcinogenesis and subject of study for chemotherapeutic interventions.

Nuclear and Mitochondrial DNA Methylation Patterns Induced by Valproic Acid in Human Hepatocytes.

Valproic acid (VPA) is one of the most widely prescribed antiepileptic drugs in the world. Despite its pharmacological importance, it may cause liver toxicity and steatosis through mitochondrial dysfunction. The aim of this study is to further investigate VPA-induced mechanisms of steatosis by analyzing changes in patterns of methylation in nuclear DNA (nDNA) and mitochondrial DNA (mtDNA). Therefore, primary human hepatocytes (PHHs) were exposed to an incubation concentration of VPA that was shown to cause steatosis without inducing overt cytotoxicity. VPA was administered daily for 5 days, and this was followed by a 3 day washout (WO). Methylated DNA regions (DMRs) were identified by using the methylated DNA immunoprecipitation-sequencing (MeDIP-seq) method. The nDNA DMRs after VPA treatment could indeed be classified into oxidative stress- and steatosis-related pathways. In particular, networks of the steatosis-related gene EP300 provided novel insight into the mechanisms of toxicity induced by VPA treatment. Furthermore, we suggest that VPA induces a crosstalk between nDNA hypermethylation and mtDNA hypomethylation that plays a role in oxidative stress and steatosis development. Although most VPA-induced methylation patterns appeared reversible upon terminating VPA treatment, 31 nDNA DMRs (including 5 zinc finger protein genes) remained persistent after the WO period. Overall, we have shown that MeDIP-seq analysis is highly informative in disclosing novel mechanisms of VPA-induced toxicity in PHHs. Our results thus provide a prototype for the novel generation of interesting methylation biomarkers for repeated dose liver toxicity in vitro.

Integrative "-Omics" Analysis in Primary Human Hepatocytes Unravels Persistent Mechanisms of Cyclosporine A-Induced Cholestasis.

Cyclosporine A (CsA) is an undecapeptide with strong immunosuppressant activities and is used a lot after organ transplantation. Furthermore, it may induce cholestasis in the liver. In general, the drug-induced cholestasis (DIC) pathway includes genes involved in the uptake, synthesis, conjugation, and secretion of bile acids. However, whether CsA-induced changes in the cholestasis pathway in vitro are persistent for repeated dose toxicity has not yet been investigated. To explore this, primary human hepatocytes (PHH) were exposed to a subcytotoxic dose of 30 µM CsA daily for 3 and 5 days. To investigate the persistence of induced changes upon terminating CsA exposure after 5 days, a subset of PHH was subjected to a washout period (WO-period) of 3 days. Multiple -omics analyses, comprising whole genome analysis of DNA methylation, gene expression, and microRNA expression, were performed. The CsA-treatment resulted after 3 and 5 days, respectively, in 476 and 20 differentially methylated genes (DMGs), 1353 and 1481 differentially expressed genes (DEGs), and in 22 and 29 differentially expressed microRNAs (DE-miRs). Cholestasis-related pathways appeared induced during CsA-treatment. Interestingly, 828 persistent DEGs and 6 persistent DE-miRs but no persistent DMGs were found after the WO-period. These persistent DEGs and DE-miRs showed concordance for 22 genes. Furthermore, 29 persistent DEGs changed into the same direction as observed in livers from cholestasis patients. None of those 29 DEGs which among others relate to oxidative stress and lipid metabolism are yet present in the DIC pathway or cholestasis adverse outcome pathway (AOP) thus presenting novel findings. In summary, we have demonstrated for the first time a persistent impact of repeated dose administration of CsA on genes and microRNAs related to DIC in the gold standard human liver in vitro model with PHH.

Dynamic Interplay between the Transcriptome and Methylome in Response to Oxidative and Alkylating Stress.

In recent years, it has been shown that free radicals not only react directly with DNA but also regulate epigenetic processes such as DNA methylation, which may be relevant within the context of, for example, tumorigenesis. However, how these free radicals impact the epigenome remains unclear. We therefore investigated whether methyl and hydroxyl radicals, formed by tert-butyl hydroperoxide (TBH), change temporal DNA methylation patterns and how this interferes with genome-wide gene expression. At three time points, TBH-induced radicals in HepG2 cells were identified by electron spin resonance spectroscopy. Total 5-methylcytosine (5mC) levels were determined by liquid chromatography and tandem mass spectrometry and genome-wide changes in 5mC and gene expression by microarrays. Induced methylome changes rather represent an adaptive response to the oxidative stress-related reactions observed in the transcriptome. More specifically, we found that methyl radicals did not induce DNA methylation directly. An initial oxidative and alkylating stress-related response of the transcriptome during the early phase of TBH treatment was followed by an epigenetic response associated with cell survival signaling. Also, we identified genes of which the expression seems directly regulated by DNA methylation. This work suggests an important role of the methylome in counter-regulating primary oxidative and alkylating stress responses in the transcriptome to restore normal cell function. Altogether, the methylome may play an important role in counter-regulating primary oxidative and alkylating stress responses in the transcriptome presumably to restore normal cell function.

Development of novel tools for the in vitro investigation of drug-induced liver injury.

INTRODUCTION: Due to its complex mechanisms and unpredictable occurrence, drug-induced liver injury (DILI) complicates drug identification and classification. Since species-specific differences in metabolism and pharmacokinetics exist, data obtained from animal studies may not be sufficient to predict DILI in humans. AREAS COVERED: Over the last few decades, numerous in vitro models have been developed to replace animal testing. The advantages and disadvantages of commonly used liver-derived in vitro models (e.g., cell lines, hepatocyte models, liver slices, three-dimensional (3D) hepatospheres, etc.) are discussed. Toxicogenomics-based methodologies (genomics, epigenomics, transcriptomics, proteomics and metabolomics) and next-generation sequencing have also been used to enhance the reliability of DILI prediction. This review presents an overview of the currently used alternative toxicological models and of the most advanced approaches in the field of DILI research. EXPERT OPINION: It seems unlikely that a single in vitro system will be able to mimic the complex interactions in the human liver. Three-dimensional multicellular systems may bridge the gap between conventional 2D models and in vivo clinical studies in humans and provide a reliable basis for hepatic toxicity assay development. Next-generation sequencing technologies, in comparison to microarray-based technologies, may overcome the current limitations and are promising for the development of predictive models in the near future.