The Transcriptome Profile of Retinal Pigment Epithelium and Müller Cell Lines Protected by Risuteganib Against Hydrogen Peroxide Stress.

Purpose: Oxidative stress contributes to the pathogenesis of vision-impairing diseases. In the retina, retinal pigment epithelium (RPE) and Müller cells support neuronal homeostasis, but also contribute to pathological development under stressed conditions. Recent studies found that the investigational drug risuteganib (RSG) has a good safety profile, provided protection in experimental models, and improved visual acuity in patients. The present in vitro study evaluated the effects of RSG in RPE and Müller cell lines stressed with the oxidant hydrogen peroxide (H2O2). Methods: Human RPE (ARPE-19) and Müller (MIO-M1) cell lines were treated with various combinations of RSG and H2O2. Trypan blue assay was used to investigate the effect of compounds on cell viability. Gene expression was measured using RNA sequencing to identify regulated genes and the biological processes and pathways involved. Results: Trypan blue assay found RSG pre-treatment significantly protected against H2O2-induced cell death in ARPE-19 and MIO-M1 cells. Transcriptome analysis found H2O2 regulated genes in several disease-relevant biological processes, including cell adhesion, migration, death, and proliferation; ECM organization; angiogenesis; metabolism; and immune system processes. RSG pre-treatment modulated these gene expression profiles in the opposite direction of H2O2. Pathway analysis found genes in integrin, AP-1, and syndecan signaling pathways were regulated. Expression of selected RSG-regulated genes was validated using qRT-PCR. Conclusions: RSG protected cultured human RPE and Müller cell lines against H2O2-induced cell death and mitigated the associated transcriptome changes in biological processes and pathways relevant to the pathogenesis of retinal diseases. These results demonstrate RSG reduced oxidative stress-induced toxicity in two retinal cell lines with potential relevance to the treatment of human diseases.

Tungsten disulfide nanotubes enhance flow-induced crystallization and radio-opacity of polylactide without adversely affecting in vitro toxicity.

Treatment of vascular disease, from peripheral ischemia to coronary heart disease (CHD), is poised for transformation with the introduction of transient implants designed to "scaffold" regeneration of blood vessels and ultimately leave nothing behind. Improved materials could expand the use of these devices. Here, we examine one of the leading polymers for bioresorbable scaffolds (BRS), polylactide (PLA), as the matrix of nanocomposites with tungsten disulfide (WS2) nanotubes (WSNT), which may provide mechanical reinforcement and enhance radio-opacity. We evaluate in vitro cytotoxicity using vascular cells, flow-induced crystallization and radio-opacity of PLA-WSNT nanocomposites at low WSNT concentration. A small amount of WSNT (0.1 wt%) can effectively promote oriented crystallization of PLA without compromising molecular weight. And radio-opacity improves significantly: as little as 0.5 to 1 wt% WSNT doubles the radio-opacity of PLA-WSNT relative to PLA at 17 keV. The results suggest that a single component, WSNT, has the potential to increase the strength of BRS to enable thinner devices and increase radio-opacity to improve intraoperative visualization. The in vitro toxicity results indicate that PLA-WSNT nanocomposites are worthy of investigation in vivo. Although substantial further preclinical studies are needed, PLA-WSNT nanocomposites may provide a complement of material properties that may improve BRS and expand the range of lesions that can be treated using transient implants. STATEMENT OF SIGNIFICANCE: Bioresorbable Scaffolds (BRSs) support regeneration of arteries without permanent mechanical constraint. Poly-L-lactide (PLLA) is the structural material of the first approved BRS for coronary heart disease (ABSORB BVS), withdrawn due to adverse events in years 1-3. Here, we examine tungsten disulfide (WS2) nanotubes (WSNT) in PLA to address two contributors to early complications: (1) reinforce PLLA (enable thinner BRS), and (2) increase radiopacity (provide intraoperative visibility). For BRS, it is significant that WSNT disperse, remain dispersed, reduce friction and improve mechanical properties without additional chemicals or surface modifications. Like WS2 nanospheres, bare WSNT and PLA-WSNT nanocomposites show low cytotoxicity in vitro. PLA-WSNT show enhanced flow-induced crystallization relative to PLA, motivating future study of the processing behavior and strength of these materials.

Differential effects of risuteganib and bevacizumab on AMD cybrid cells.

PURPOSE: Intravitreal injections of anti-vascular endothelial growth factor (VEGF) treatments are currently used to treat wet age-related macular degeneration (AMD), diabetic retinopathy, and macular edema. Chronic, repetitive treatments with anti-VEGF may have unintended consequences beyond the inhibition of angiogenesis. Most recently, clinical trials have been conducted with risuteganib (RSG, Luminate®), which is anti-angiogenic and has neuroprotective and anti-inflammatory properties. Mitochondrial damage and dysfunction play a major role in development of AMD. Transmitochondrial cybrids are cell lines established by fusing human retinal pigment epithelial (RPE) cells that are Rho0 (lacking mtDNA) with platelets isolated from AMD subjects or age-matched normal subjects. Cybrid cell lines have identical nuclei but mitochondria from different subjects, enabling investigation of the functional consequences of damaged AMD mitochondria. The present study compares the responses of AMD cybrids treated with bevacizumab (Bmab, Avastin®) versus risuteganib (RSG, Luminate®). METHODS: Cybrids were created by fusing mtDNA depleted ARPE-19 cells with platelets from AMD or age-matched normal patients. AMD (n = 5) and normal (n = 3) cybrids were treated for 48 h with or without 1x clinical dose of 1.25 mg/50 µl (25,000 µg/ml) of Bmab or 1.0 mg/50 µl (20,000 µg/ml) of RSG. Cultures were analyzed for levels of cleaved caspase 3/7 and NucLight Rapid Red staining (IncuCyte® Live Cell Imager), mitochondrial membrane potential (ΔΨm, JC1 assay) or reactive oxygen species (ROS, H2DCFDA assay). Expression levels of genes related to the following pathways were analyzed with qRT-PCR: Apoptosis (BAX, BCL2L13, CASP-3, -7, -9); angiogenesis (VEGFA, HIF1α, PDGF); integrins (ITGB-1, -3, -5, ITGA-3, -5, -V); mitochondrial biogenesis (PGC1α, POLG); oxidative stress (SOD2, GPX3, NOX4); inflammation (IL-6, -18, -1ß, IFN-ß1); and signaling (P3KCA, PI3KR1). Statistical analyses were performed using GraphPad Prism software. RESULTS: The untreated AMD cybrids had significantly higher levels of cleaved caspase 3/7 compared to the untreated normal cybrids. The Bmab-treated AMD cybrids showed elevated levels of cleaved caspase 3/7 compared to untreated AMD or RSG-treated AMD cybrids. The Bmab-treated cybrids had lower ΔΨm compared to untreated AMD or RSG-treated AMD cybrids. The ROS levels were not changed with Bmab or RSG treatment. Results showed that Bmab-treated cybrids had higher expression levels of inflammatory (IL-6, IL1-ß), oxidative stress (NOX4) and angiogenesis (VEGFA) genes compared to untreated AMD, while RSG-treated cybrids had lower expression levels of apoptosis (BAX), angiogenesis (VEGFA) and integrin (ITGB1) genes. CONCLUSIONS: These data suggest that the mechanism(s) of action of RSG, an integrin regulator, and Bmab, a recombinant monoclonal antibody, affect the AMD RPE cybrid cells differently, with the former having more anti-apoptosis properties, which may be desirable in treating degenerative ocular diseases.

Risuteganib Protects against Hydroquinone-induced Injury in Human RPE Cells.

Purpose: Cigarette smoking has been implicated in the pathogenesis of AMD. Integrin dysfunctions have been associated with AMD. Herein, we investigate the effect of risuteganib (RSG), an integrin regulator, on RPE cell injury induced by hydroquinone (HQ), an important oxidant in cigarette smoke. Methods: Cultured human RPE cells were treated with HQ in the presence or absence of RSG. Cell death, mitochondrial respiration, reactive oxygen species production, and mitochondrial membrane potential were measured by flow cytometry, XFe24 analyzer, and fluorescence plate reader, respectively. Whole transcriptome analysis and gene expression were analyzed by Illumina RNA sequencing and quantitative PCR, respectively. F-actin aggregation was visualized with phalloidin. Levels of heme oxygenase-1, P38, and heat shock protein 27 proteins were measured by Western blot. Results: HQ induced necrosis and apoptosis, decreased mitochondrial bioenergetics, increased reactive oxygen species levels, decreased mitochondrial membrane potential, increased F-actin aggregates, and induced phosphorylation of P38 and heat shock protein 27. HQ, but not RSG alone, induced substantial transcriptome changes that were regulated by RSG cotreatment. RSG cotreatment significantly protected against HQ-induced necrosis and apoptosis, prevented HQ-reduced mitochondrial bioenergetics, decreased HQ-induced reactive oxygen species production, improved HQ-disrupted mitochondrial membrane potential, reduced F-actin aggregates, decreased phosphorylation of P38 and heat shock protein 27, and further upregulated HQ-induced heme oxygenase-1 protein levels. Conclusions: RSG has no detectable adverse effects on healthy RPE cells, whereas RSG cotreatment protects against HQ-induced injury, mitochondrial dysfunction, and actin reorganization, suggesting a potential role for RSG therapy to treat retinal diseases such as AMD.

Bioavailability and Bioaccumulation of 6:2 Fluorotelomer Sulfonate, 6:2 Chlorinated Polyfluoroalkyl Ether Sulfonates, and Perfluorophosphinates in a Soil-Plant System.

As emerging alternatives of legacy perfluoroalkyl substances, 6:2 fluorotelomer sulfonate (6:2 FTS), 6:2 chlorinated polyfluoroalkyl ether sulfonates (6:2 Cl-PFESA), and perfluorophosphinates (C6/C6 and C8/C8 PFPiAs) are supposed to be partitioned to soil and highly persistent in the environment. The uptake of novel per- and polyfluoroalkyl substances (PFASs) by plants represents a potential pathway for their transfer in the food chain. In this study, the bioavailability of these four novel PFASs in soil and the bioaccumulation characteristics in greenhouse-grown wheat (Triticum aestivum L.), maize (Zea mays L.), soybean (Glycine max L. Merrill), and pumpkin (Cucurbita maxima L.) were investigated. The results indicated that these novel PFASs with higher hydrophobicity were more easily sequestrated in soil, and the fractions extracted by methanol could well describe their bioavailability, which could be stimulated by low-molecular-weight organic acids at rhizospheric concentrations. A negative relationship was found between root soil concentration factors (RSCFs) and hydrophobicity (log Kow) of the target PFASs. This correlation was also found in the translocation factors (TF) from roots to shoots. Furthermore, the uptake and transfer of the target PFASs were regulated by the protein contents in plant roots and shoots.

Loss of Pten synergizes with c-Met to promote hepatocellular carcinoma development via mTORC2 pathway.

Hepatocellular carcinoma (HCC) is a deadly malignancy with limited treatment options. Activation of the AKT/mTOR cascade is one of the most frequent events along hepatocarcinogenesis. mTOR is a serine/threonine kinase and presents in two distinct complexes: mTORC1 and mTORC2. While mTORC1 has been extensively studied in HCC, the functional contribution of mTORC2 during hepatocarcinogenesis has not been well characterized, especially in vivo. Pten expression is one of the major mechanisms leading to the aberrant activation of the AKT/mTOR signaling. Here, we show that concomitant downregulation of Pten and upregulation of c-Met occurs in a subset of human HCC, mainly characterized by poor prognosis. Using CRISPR-based gene editing in combination with hydrodynamic injection, Pten was deleted in a subset of mouse hepatocytes (sgPten). We found that loss of Pten synergizes with overexpression of c-Met to promote HCC development in mice (sgPten/c-Met). At the molecular level, sgPten/c-Met liver tumor tissues display increased AKT and mTOR signaling. Using Rictor conditional knockout mice, we demonstrate that sgPten/c-Met-driven HCC development strictly depends on an intact mTORC2 complex. Our findings therefore support the critical role of mTORC2 in hepatocarcinogenesis. sgPten/c-Met mouse model represents a novel valuable system that can be used for the development of targeted therapy against this deadly malignancy.

Tankyrase inhibitors suppress hepatocellular carcinoma cell growth via modulating the Hippo cascade.

Previous data indicate that Tankyrase inhibitors exert anti-growth functions in many cancer cell lines due to their ability to inactivate the YAP protooncogene. In the present manuscript, we investigated the effect of Tankyrase inhibitors on the growth of hepatocellular carcinoma (HCC) cell lines and the molecular mechanisms involved. For this purpose, we performed cell proliferation assay by colony-forming ability in seven human HCC cells subjected to XAV-939 and G007-LK Tankyrase inhibitors. Noticeably, the two Tankyrase inhibitors suppressed the HCC cell growth in a dose-dependent manner. Furthermore, we found that Tankyrase inhibitors synergized with MEK and AKT inhibitors to suppress HCC cell proliferation. At the molecular level, Tankyrase inhibitors significantly decreased YAP protein levels, reduced the expression of YAP target genes, and inhibited YAP/TEAD luciferase reporter activity. In addition, Tankyrase inhibitors administration was accompanied by upregulation of Angiomotin-like 1 (AMOTL1) and Angiomotin-like 2 (AMOTL2) proteins, two major negative regulators of YAP. Altogether, the present data indicate that XAV-939 and G007-LK Tankyrase inhibitors could suppress proliferation of hepatocellular carcinoma cells and downregulate YAP/TAZ by stabilizing AMOTL1 and AMOTL2 proteins, thus representing new potential anticancer drugs against hepatocellular carcinoma.