Fabry disease (FD) is an X-linked inherited lysosomal storage disease caused by a-galactosidase A (GLA) deficiency. Progressive intracellular accumulation of globotriaosylceramide (Gb3) is considered to be pathogenically responsible for the phenotype variability of FD that causes cardiovascular dysfunction; however, molecular mechanisms underlying the impairment of FD-associated cardiovascular tissues remain unclear. In this study, we reprogrammed human induced pluripotent stem cells (hiPSCs) from peripheral blood cells of patients with FD (FD-iPSCs); subsequently differentiated them into vascular endothelial-like cells (FD-ECs) expressing CD31, VE-cadherin, and vWF; and investigated their ability to form vascular tube-like structures. FD-ECs recapitulated the FD pathophysiological phenotype exhibiting intracellular Gb3 accumulation under a transmission electron microscope. Moreover, compared with healthy control iPSC-derived endothelial cells (NC-ECs), reactive oxygen species (ROS) production considerably increased in FD-ECs. Microarray analysis was performed to explore the possible mechanism underlying Gb3 accumulation-induced ROS production in FD-ECs. Our results revealed that superoxide dismutase 2 (SOD2), a mitochondrial antioxidant, was significantly downregulated in FD-ECs. Compared with NC-ECs, AMPK activity was significantly enhanced in FD-ECs. Furthermore, to investigate the role of Gb3 in these effects, human umbilical vein endothelial cells (HUVECs) were treated with Gb3. After Gb3 treatment, we observed that SOD2 expression was suppressed and AMPK activity was enhanced in a dose-dependent manner. Collectively, our results indicate that excess accumulation of Gb3 suppressed SOD2 expression, increased ROS production, enhanced AMPK activation, and finally caused vascular endothelial dysfunction. Our findings suggest that dysregulated mitochondrial ROS may be a potential target for treating FD.

RationaleA high incidence of GLA IVS4+919 G>A mutation in patients with Fabry disease of the later-onset cardiac phenotype, has been reported in Taiwan. However, suitable biomarkers or potential therapeutic surrogates for Fabry cardiomyopathy (FC) in such patients under enzyme replacement treatment (ERT) remain unknown.ObjectiveUsing FC patients carrying IVS4+919 G>A mutation, we constructed an induced pluripotent stem cell (iPSC)-based disease model to investigate the pathogenetic biomarkers and potential therapeutic targets in ERT-treated FC.Results and methodsThe iPSC-differentiated cardiomyocytes derived from FC-patients (FC-iPSC-CMs) carried IVS4+919 G>A mutation recapitulating FC characteristics, including low α-galactosidase A enzyme activity, cellular hypertrophy, and massive globotriaosylceramide accumulation. Microarray analysis revealed that interleukin-18 (IL-18), a pleiotropic cytokine involved in various myocardial diseases, was the most highly upregulated marker in FC-iPSC-CMs. Meanwhile, IL-18 levels were found to be significantly elevated in the culture media of FC-iPSC-CMs and patients’ sera. Notably, the serum IL-18 levels were highly paralleled with the progression of left ventricular hypertrophy in Fabry patients receiving ERT. Finally, using FC-iPSC-CMs as in vitro FC model, neutralization of IL-18 with specific antibodies combined with ERT synergistically reduced the secretion of IL-18 and the progression of cardiomyocyte hypertrophy in FC-iPSC-CMs.ConclusionOur data demonstrated that cardiac IL-18 and circulating IL-18 are involved in the pathogenesis of FC and LVH. IL-18 may be a novel marker for evaluating ERT efficacy, and targeting IL-18 might be a potential adjunctive therapy combined with ERT for the treatment of advanced cardiomyopathy in FC patients with IVS4+919 G>A mutation.

Cisplatin (CDDP) is an important anti-cancer drug commonly used in various human cancers, including nasopharyngeal carcinoma (NPC). How to overcome the drug resistance of CDDP provides opportunities to improve clinical outcomes of NPC. O 6 -methylguanine-DNA methyltransferase (MGMT) has been well-characterized to be a therapeutic determinant of O 6 -alkylguanine alkylating drugs. However, the underlying mechanism and clinical relevance between MGMT and CDDP remain poorly defined in NPC. In this study, we showed that MGMT-proficient cells were highly resistant to the cytotoxic effects of CDDP as compared to MGMT-deficient cells. Further studies showed that the platinum level of DNA after CDDP exposure was significantly lower in MGMT-proficient cells than in MGMT-deficient cells. Host cell reactivation assay revealed that MGMT protected NPC cells from CDDP-induced DNA damage by enhancing DNA repair capacity. Importantly, we demonstrated for the first time that MGMT protein directly bound to CDDP-induced DNA damages. Subsequently, CDDP-bound MGMT protein became ubiquitinated and was degraded through ubiquitin-mediated proteasome system. We further analyzed the relationship between MGMT expression and clinical survivals in a cohort of 83 NPC patients. NPC patients who received CDDP-based concurrent chemoradiotherapy (CCRT), with high MGMT expression level, exhibited shorter progression-free survival (PFS; p 5 0.022) and overall survival (OS; p 5 0.015), than patients with low MGMT expression level. Furthermore, high MGMT expression level remained to be an independent prognostic factor for worse PFS (p 5 0.01, hazard ratio 2.23) and OS (p 5 0.018, hazard ratio 2.14). Our findings suggest that MGMT protein is important to determine the efficacy of CDDP in NPC.

The CRISPR/Cas9 Genome-editing system has revealed promising potential for generating gene mutation, deletion, and correction in human cells. Application of this powerful tool in Fabry disease (FD), however, still needs to be explored. Enzyme replacement therapy (ERT), a regular administration of recombinant human α Gal A (rhα-GLA), is a currently available and effective treatment to clear the accumulated Gb3 in FD patients. However, the short half-life of rhα-GLA in human body limits its application. Moreover, lack of an appropriate in vitro disease model restricted the high-throughput screening of drugs for improving ERT efficacy. Therefore, it is worth establishing a large-expanded in vitro FD model for screening potential candidates, which can enhance and prolong ERT potency. Using CRISPR/Cas9-mediated gene knockout of GLA in HEK-293T cells, we generated GLA-null cells to investigate rhα-GLA cellular pharmacokinetics. The half-life of administrated rhα-GLA was around 24 h in GLA-null cells; co-administration of proteasome inhibitor MG132 and rhα-GLA significantly restored the GLA enzyme activity by two-fold compared with rhα-GLA alone. Furthermore, co-treatment of rhα-GLA/MG132 in patient-derived fibroblasts increased Gb3 clearance by 30%, compared with rhα-GLA treatment alone. Collectively, the CRISPR/Cas9-mediated GLA-knockout HEK-293T cells provide an in vitro FD model for evaluating the intracellular pharmacokinetics of the rhα-GLA as well as for screening candidates to prolong rhα-GLA potency. Using this model, we demonstrated that MG132 prolongs rhα-GLA half-life and enhanced Gb3 clearance, shedding light on the direction of enhancing ERT efficacy in FD treatment.

Cellular accumulation of mono(2-ethylhexyl)phthalate (MEHP) has been recently demonstrated to disturb fat cell energy metabolism; however, the underlying mechanism remained unclear. The study aimed to determine how MEHP influenced fat cell transcriptome and how the changes might contribute to bioenergetics. Because of the pivotal role of PPARγ in energy metabolism of fat cells, comparative microarray analysis of gene expression in 3T3-L1 adipocytes treated with both MEHP and rosiglitazone was performed. Pathway enrichment analysis and gene ontology (GO) enrichment analysis revealed that both treatments caused up-regulation of genes involved in PPAR signaling/energy metabolism-related pathways and down-regulation of genes related to adipokine/inflammation signals. MEHP/rosiglitazone-treated adipocytes exhibited increased levels of lipolysis, glucose uptake, and glycolysis; the gene expression profiles provided molecular basis for the functional changes. Moreover, MEHP was shown to induce nuclear translocation and activation of PPARγ. The similarity in gene expression and functional changes in response to MEHP and rosiglitazone suggested that MEHP influenced bioenergetics and adipokine network mainly via PPARγ. Importantly, adipokine levels in C57BL/6J mice with di(2-ethylhexyl)phthalate (DEHP) treatments provided in vivo evidence for microarray results. On the basis of correlation between gene expression and functional assays, possible involvements of genes in bioenergetics of MEHP-treated adipocytes were proposed.