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Isolation and culture of mouse aortic endothelial cells (MAEC). Mouse thoracic aortas were surgically removed from the proximal end to the middle portion and dissected away from connective tissues. The aortic rings (approximately 2 mm) were placed carefully onto collagen-coated 12-well culture dishes. After 2 d in culture, aortic endothelial cells first were released into plating medium and grown. At day 5, tissues were discarded, and the MAEC were cultured continuously to confluence.
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Fabry disease is an X-linked inherited loss of alpha-galactosidase A (alpha-Gal A). Affected patients experience complications that include neuropathy, renal failure, and cardiovascular disease. Although the genetic and biochemical basis of this sphingolipidosis is well studied, the basis for the vascular disease remains poorly understood. In an at...
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... culture and the growth of MAEC, in vitro, were depen- dent on the genotypes of mice used in this study. Although the MAEC isolated from both Gla/0 and Gla/0 mice grew free of aortic rings in plating medium after 2 d culture (Figure 1), by culture day 10, the number of MAEC that were harvested from 10 age-matched Gla/0 mice was 2.0 to 2.3 10 6 , whereas the MAEC yield from the same number of Gla/0 mice was 1.5 to 1.6 10 6 . Under identical culture conditions, a longer culture time (2 to 3 d) was required for Gla/0 MAEC to reach 90% confluence when compared with the culture and growth of Gla/0 MAEC, even though the number of cells seeded was always the same. ...
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... combination of dt-EtDO-P4 and recombinant human -Gal A on the removal of Gb3 was assessed using a single and low dose for a shorter period of incubation ( Figure 10). Cul- tured Gla/0 MAEC were exposed to 0.5 M dt-EtDO-P4 or recombinant human -Gal A (1 g/ml) or a mixture of 0.5 M dt-EtDO-P4 plus 1 g/ml recombinant human -Gal A for 24 h. ...
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... serve as antigens on blood cells, epithelia, and other tissues. Globo- series glycosphingolipids (Figure 11) are also oncofetal antigens and are expressed at the early cleavage stage in embryos and are markers of embryonic mesoderm, extra-embryonic visceral endoderm, and visceral yolk sac cells (21,22). In early embryo- genesis, globoside (Gb4) and two other related stage-specific antigens (Gal-Gb4 and LKE) play significant roles in preim- plantation development (23). ...
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... is a 3-N-acetylglucosaminyltransferase re- lated to globoside synthase (3GalT3) (26,27). The arthro-series glycosphingolipids in drosophila play critical roles in oogene- Figure 10. Synergistic effects of dt-EtDO-P4 and -Gal A on the clearance of Gb3 in Gla/0 MAEC. ...
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... accumulation of Gb3 and strategies designed to lower tissue Gb3 levels need to be viewed in the context of glyco- sphingolipid metabolism in general and globo-series glyco- sphingolipid metabolism in particular ( Figure 11). An interven- tion that lowers Gb3 by catabolism to lactosylceramide or by inhibition of an upstream glycosyltransferase will predictably have secondary effects on the levels of other sphingolipids. ...
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Fabry disease is an X-linked recessive and progressive disease caused by α-galactosidase A (α-GaL A) deficiency. We sought to assess the prevalence of unrecognized Fabry disease in dialysis-dependent patients and the efficacy of serum globotriaosylceramide (GL3) screening.
A total of 480 patients of 1,230 patients among 17 clinics were enrolled. Se...
Fabry disease is a metabolic disorder without a specific treatment, caused by a deficiency of the lysosomal enzyme alpha-galactosidase A (alpha-gal A). Most patients experience debilitating neuropathic pain and premature mortality because of renal failure, cardiovascular disease, or cerebrovascular disease.
To evaluate the safety and efficacy of in...
Fabry disease is a lysosomal storage disorder caused by a deficiency of the lysosomal enzyme alpha-galactosidase A (alpha-gal A). This enzymatic defect results in the accumulation of the glycosphingolipid globotriaosylceramide (Gb(3); also referred to as ceramidetrihexoside) throughout the body. To investigate the effects of purified alpha-gal A, 1...
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... A previous in vitro study on Fabry disease using mouse aortic endothelial cells from α-Gal A-deficient mice showed that rhα-GLA treatment for 48 hr reduced Gb3 at a dose from 0.1-60 µg/ml 20 . A previous study also demonstrated that a longer incubation time was associated with a lower required dose of the enzyme. ...
Fabry disease is an X-linked lysosomal storage disease caused by a mutation in the galactosidase alpha (GLA) gene. Despite advances in therapeutic technologies, the lack of humanized experimental models of Fabry disease has limited the development of new therapies to cure the disease. Herein, we modeled Fabry disease using human inducible pluripotent stem cell (iPSC)-derived kidney organoids and the CRISPR–Cas9 genome-editing system. GLA-mutant human kidney organoids revealed deformed podocytes and tubular cells with accumulation of globotriaosylceramide (Gb3). Ultrastructural analysis showed abundant electron-dense granular deposits and electron-dense lamellate lipid-like deposits that formed concentric bodies (zebra bodies) in the cytoplasm of podocytes and tubules. The oxidative stress level was increased in GLA-mutant kidney organoids, and the increase was accompanied by apoptosis. Enzyme replacement treatment (ERT) with recombinant human α-Gal A decreased the Gb3 accumulation and oxidative stress, which resulted in amelioration of the deformed cellular structure of the GLA-mutant kidney organoids. Transcription profile analyses showed decreased glutathione (GSH) metabolism in GLA-mutant kidney organoids. GSH replacement treatment decreased oxidative stress and attenuated the structural deformity of the GLA-mutant kidney organoids. GSH treatment also increased the expression of podocyte and tubular markers and decreased apoptosis. In conclusion, GLA-mutant kidney organoids derived from human iPSCs are valuable tools for studying the mechanisms and developing novel therapeutic alternatives for Fabry disease.
... An in vitro model of FD was developed from α-Gal A KO mouse, in particular, primary cultures of aortic endothelial cells were generated, characterized and maintained in culture. Further, these primary cultures were used to compare the effects of recombinant α-Gal A and a strong glucosylceramide synthase inhibitor on Gb3 metabolism (ethylenedioxyphenyl-P4) 73 . ...
... Advantages: GL-3 accumulates in the lysosomes of these cells, inducing alteration close to cardiac tissue of FD patients; Disadvantages: Difficulties in studying other clinical features (i.e renal failure or neuronal dysfunction) 73 . ...
Fabry (or Anderson‐Fabry) is a rare pan‐ethnic disease affecting males and females. Fabry is an X‐linked lysosomal storage disease, affecting glycosphingolipid metabolism, that is caused by mutations of the GLA gene that codes for α‐galactosidase A. Fabry disease (FD) can be classified into a severe, classical phenotype, most often seen in men with no residual enzyme activity, that usually appear before 18 years and a usually milder, nonclassical (later‐onset) phenotype that usually appear above 18 years. Affected patients show multifactorial complications, including renal failure, cardiovascular problems, and neuropathy. In this review, we briefly report the clinical trials so far performed with the available therapies, and then we focus on the in vitro and the in vivo experimental models of the disease, to highlight the relevance in improving the existing therapeutics and understand the mechanism of this rare disorder. Current available in vivo and in vitro models can assist in better comprehension of the pathogenesis and underlying mechanisms of FD, thus the existing therapeutic approaches can be optimized, and new options can be developed.
... The loss of GLA in EV-GLA samples could be attributed to the presence of free GLA co-precipitated with the EV-GLA during the isolation procedure ( Figure S) Overall, cell internalization and crude enzymatic activity assays suggested that EVs are suitable delivery systems for lysosomal enzymes. For further confirmation that EVs could be therapeutically effective, in vitro efficacy assays were conducted in MAEC derived from Fabry KO mice, a very handful in vitro model of the Fabry disease (Shu et al., 2005). Indeed, due to the lack of endogenous alpha-galactosidase A activity, these cells accumulate high levels of Gb3, the predominant storage product in Fabry patients and the main responsible of the clinical manifestations of the disease . ...
... Cell culture conditions: HEK293T cells (CRL-3216, ATCC) were routinely maintained in RPMI medium supplemented with 10% foetal bovine serum (Life Technologies, Paisley, UK), 2 mM L-Glutamine (Life Technologies), 1 X Non-essential aminoacids (Life Technologies), 1X antimycotic-antibiotic (Life Technologies) solution at 37 • C and 5% CO 2 . Mouse endothelial aortic cells (MAEC) were isolated from descending aorta of GLA deficient mice (GlatmKul1) at the ICTS NANBIOSIS (U20) following procedures previously established procedures (Giannotti et al., 2016;Nazarenus et al., 2015;Shu et al., 2005). Endothelial origin of isolated cells was confirmed by CD105 staining (12-1051, eBioscience) and flow cytometry (FacScalibur, Becton Dickinson). ...
In the present study the use of extracellular vesicles (EVs) as vehicles for therapeutic enzymes in lysosomal storage disorders was explored. EVs were isolated from mammalian cells overexpressing alpha‐galactosidase A (GLA) or N‐sulfoglucosamine sulfohydrolase (SGSH) enzymes, defective in Fabry and Sanfilippo A diseases, respectively. Direct purification of EVs from cell supernatants was found to be a simple and efficient method to obtain highly active GLA and SGSH proteins, even after EV lyophilization. Likewise, EVs carrying GLA (EV‐GLA) were rapidly uptaken and reached the lysosomes in cellular models of Fabry disease, restoring lysosomal functionality much more efficiently than the recombinant enzyme in clinical use. In vivo, EVs were well tolerated and distributed among all main organs, including the brain. DiR‐labelled EVs were localized in brain parenchyma 1 h after intra‐arterial (internal carotid artery) or intravenous (tail vein) administrations. Moreover, a single intravenous administration of EV‐GLA was able to reduce globotriaosylceramide (Gb3) substrate levels in clinically relevant tissues, such kidneys and brain. Overall, our results demonstrate that EVs from cells overexpressing lysosomal enzymes act as natural protein delivery systems, improving the activity and the efficacy of the recombinant proteins and facilitating their access to organs neglected by conventional enzyme replacement therapies.
... We still decided to identify STxB + profiles as Gb3, due to the 3-fold higher affinity of STxB for Gb3 (Gallegos et al., 2012). Furthermore, it was shown that Gb4 was not increased in GLA KO mice, compared to wildtype littermates in an in vitro rodent model of primary aortic endothelial cells (Shu et al., 2005). Together with the complete absence of STxB + accumulations in control skin, iPSC, and neurons, we hypothesized that if Gb4 is present in our cells, its concentration is most likely below the detection threshold of our assay and hence of minor relevance. ...
Fabry disease (FD) is an X-linked lysosomal storage disorder caused by deficiency of the α-galactosidase A (GLA), leading to intracellular accumulations of globotriaosylceramide (Gb3). Acral burning pain, which can be triggered by heat, fever or physical activity is an early hallmark of FD and greatly reduces patients’ quality of life. The pathophysiology of FD pain is unknown and research is hindered by the limited in vivo availability of suitable human biomaterial. To overcome this obstacle, we generated induced pluripotent stem cells (iPSC) from one female and two male patients with a differing pain phenotype, and developed a refined differentiation protocol for sensory neurons to increase reliability and survival of these neurons, serving as an in vitro disease model. Neurons were characterized for the correct neuronal subtype using immunocytochemistry, gene expression analysis, and for their functionality using electrophysiological measurements. iPSC and sensory neurons from the male patients showed Gb3 accumulations mimicking the disease phenotype, whereas no Gb3 depositions were detected in sensory neurons derived from the female cell line, likely caused by a skewed X-chromosomal inactivation in favor of healthy GLA. Using super-resolution imaging techniques we showed that Gb3 is localized in neuronal lysosomes of male patients and in a first experiment using dSTORM microscopy we were able to visualize the neuronal membrane in great detail. To test our disease model, we treated the neurons with enzyme replacement therapy (ERT) and analyzed its effect on the cellular Gb3 load, which was reduced in the male FD-lines, compared to non-treated cells. We also identified time-dependent differences of Gb3 accumulations, of which some seemed to be resistant to ERT. We also used confocal Ca2+ imaging to investigate spontaneous neuronal network activity, but analysis of the dataset proofed to be difficult, nonetheless showing a high potential for further investigations. We revealed that neurons from a patient with pain pain are more easily excitable, compared to cells from a patient without pain and a healthy control. We provide evidence for the potential of patient-specific iPSC to generate a neuronal in vitro disease model, showing the typical molecular FD phenotype, responding to treatment, and pointing towards underlying electrophysiological mechanisms causing different pain phenotypes. Our sensory neurons are suitable for state-of-the-art microscopy techniques, opening new possibilities for an in-depth analysis of cellular changes, caused by pathological Gb3 accumulations. Taken together, our system can easily be used to investigate the effect of the different mutations of GLA on a functional and a molecular level in affected neurons.
... Shu et al. 26 Human telomerase reverse transcriptase introduced in FD hemizygote endothelial cells. ...
... 21 This is particularly interesting to understand the underlying disease mechanism and provide a cellular and molecular platform for developing novel treatment strategies. 22 In vitro cell models (Table 1), initially, used cells obtained from FD patients especially of target organs in FD: endothelial cells [23][24][25][26][27]29 kidney cells [48][49][50][52][53][54] to determine glycosphingolipids accumulation and a-Gal A activity, and obtain lines for further research. Fibroblast 32,34 and bone marrow cells 30,31 were subjected to different techniques to incorporate an a-Gal A for enzyme expression, as well as for the analysis of different mutations present in FD patients. ...
Fabry disease (FD) is a progressive, X-linked inherited disorder of glycosphingolipid metabolism due to deficient or absent lysosomal α-galactosidase A (α-Gal A) activity which results in progressive accumulation of globotriaosylceramide (Gb3) and related metabolites. One prominent feature of Fabry disease is neuropathic pain. Accumulation of Gb3 has been documented in dorsal root ganglia (DRG) as well as other neurons, and has lately been associated with the mechanism of pain though the pathophysiology is still unclear. Small fiber (SF) neuropathy in FD differs from other entities in several aspects related to the perception of pain, alteration of fibers as well as drug therapies used in the practice with patients, with therapies far from satisfying. In order to develop better treatments, more information on the underlying mechanisms of pain is needed. Research in neuropathy has gained momentum from the development of preclinical models where different aspects of pain can be modelled and further analyzed. This review aims at describing the different in vitro and FD animal models that have been used so far, as well as some of the insights gained from their use. We focus especially in recent findings associated with ion channel alterations -that apart from the vascular alterations-, could provide targets for improved therapies in pain.
... Previously, our group used primary cells isolated from mouse aortas of GLA knockout mice (mouse aortic endothelial cells, MAECs), which were useful for characterizing lipid changes and efficacy of GlcCer synthase inhibition for decreasing accumulating Gb3 (Shu, Murphy et al. 2005). However, this model was not sustainable for studying long-term consequences of treating cells with different agents since these cells could only be maintained in cell culture for a limited time (3 to 6 days). ...
... Substrate deprivation is an alternative approach to reducing accumulation of globo-series GSLs, where a small molecule inhibitor of glucosylceramide (GlcCer) synthase prevents the de novo synthesis of downstream GSLs, including LacCer, Gb3, and Gb4 (Abe, Arend et al. 2000, Abe, Gregory et al. 2000. A previous in vitro experiment performed with MAECs from Gla-knockout mice reported that treating cells with recombinant GLA or a GlcCer synthase inhibitor reduced the accumulation of Gb3 (Shu, Murphy et al. 2005). Similarly, treating CRISPR/Cas9 GLA-deficient EA.hy926 cells with exogenous GLA or the GlcCer inhibitor, eliglustat, decreased the accumulation of globoseries GSLs (figs. ...
Fabry disease, a rare, X-linked lysosomal storage disease, arises from deficiency of the lysosomal hydrolase, α-galactosidase A (GLA) which disrupts the catabolism of globo- series glycosphingolipids (GSLs). One potential link between GLA deficiency and vascular dysfunction may be changes in endothelial nitric oxide synthase (eNOS) function. GLA-deficient EA.hy926 cells were obtained by siRNA knockdown of GLA expression and by mutation of GLA with CRISPR/Cas9 gene editing to investigate the effects of GLA deficiency on eNOS. As previously observed with siRNA knockdown of GLA, globotriaosylceramide (Gb3) accumulated in EA.hy926 cells. In contrast, Gb3 did not accumulate in CRISPR/Cas9 gene edited GLA-deficient cells, but instead, globotetraosylceramide (Gb4). However, in both the siRNA and CRISPR/Cas9 models globotriaosylsphingosine (lyso-Gb3) was elevated. As was previously observed with siRNA knockdown of GLA expression, CRISPR/Cas9 GLA-deficient cells had lower eNOS activity. Restoring GLA activity in GLA-deficient cells with exogenous GLA treatment improved eNOS activity. In contrast, treating cells with the glucosylceramide synthase inhibitor, eliglustat, decreased NOS activity. These results suggest that eNOS uncoupling is due to GLA deficiency, and not necessarily due to elevated Gb3 per se. It was observed that lyso-Gb3 inhibits eNOS activity.
... Previously it was shown that two days of recombinant human GLA (α-Gal A) treatment lowered Gb3 in Gla-null mouse aortic endothelial cells 37 . When eNOS activity was examined in a subsequent study, the reduction of Gb3 by the potent glucosylceramide synthase inhibitor, eliglustat, failed to restore the decreased eNOS activity significantly in these cells 31 . ...
Fabry disease results from loss of activity of the lysosomal enzyme α-galactosidase A (GLA), leading to the accumulation of globoseries glycosphingolipids in vascular endothelial cells. Thrombosis and stroke are life-threatening complications of Fabry disease; however, the mechanism of the vasculopathy remains unclear. We explored the relationship between GLA deficiency and endothelial cell von Willebrand factor (VWF) secretion in in vivo and in vitro models of Fabry disease. Plasma VWF was significantly higher at two months and increased with age in Gla-null compared to wild-type mice. Disruption of GLA in a human endothelial cell line by siRNA and CRISPR/Cas9 resulted in a 3-fold and 5-fold increase in VWF secretion, respectively. The increase in VWF levels was associated with decreased endothelial nitric oxide synthase (eNOS) activity in both in vitro models. Pharmacological approaches that increase nitric oxide bioavailability or decrease reactive oxygen species completely normalized the elevated VWF secretion in GLA deficient cells. In contrast, the abnormality was not readily reversed by recombinant human GLA or by inhibition of glycosphingolipid synthesis with eliglustat. These results suggest that GLA deficiency promotes VWF secretion through eNOS dysregulation, which may contribute to the vasculopathy of Fabry disease.
... A model of Fabry disease using primary cultures of mouse aortic endothelial cells was established. 39 When these cells were compared with those from wild-type mice, Gb3 accumulation was observed in plasma membrane fractions and in lipid raft fractions as well. 40 The increase in Gb3 was associated with a decrease in the molar ratio of cholesterol, suggesting that Gla deficiency fundamentally could alter the membrane composition and structure within the lipid raft domains. ...
Sphingolipids, including ceramides, glycosphingolipids, sphingomyelin, and sphingosine-1-phosphate, have been recognized as important molecules that regulate critical cellular functions. Although originally studied in the context of lysosomal storage diseases, the roles of these compounds in more common disorders involving metabolism, vascular disease, and aberrant growth has been the focus of recent studies, including in disorders that affect the kidneys. These efforts have led to new insights into Fabry disease, a classic disorder of lysosomal function that results in renal failure as well as in more common renal diseases including diabetic nephropathy and polycystic kidney disease. Pathways for glycosphingolipid synthesis can be targeted with orally available small-molecule inhibitors, creating new opportunities for the treatment of both rare and common kidney diseases.
... following procedures previously described. 11,38 Endothelial origin of isolated cells was 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 IC50 values were calculated using GraphPad Prism 5 software. ...
Lysosomal storage disorders are currently treated by enzyme replacement therapy (ERT) through the direct administration of the unprotected recombinant protein to the patients. Herein we present an ionically crosslinked polyelectrolyte complex (PEC) composed of trimethyl chitosan (TMC) and α-galactosidase A (GLA), the defective enzyme in Fabry disease, with the capability of directly targeting endothelial cells by incorporating peptide ligands containing the RGD sequence. We assessed the physicochemical properties, cytotoxicity and hemocompatibility of RGD-targeted and un-targeted PECs, the uptake by endothelial cells and the intracellular activity of PECs in cell culture models of Fabry disease. Moreover, we also explored the effect of different freeze-drying procedures in the overall activity of the PECs. Our results indicate that the use of integrin-binding RGD moiety within the PEC increases their uptake and the efficacy of the GLA enzyme, while the freeze-drying allows keeping intact the activity of the therapeutic protein. Overall, these results highlight the potential of TMC-based PECs as a highly versatile and feasible drug delivery system for improving the ERT of lysosomal storage disorders.
... In Vitro Cell Assays: Primary cultures of MAEC of GLA deficient mice (Gla tmKul1 ) were isolated at the In vivo Experimentation Platform from CIBER-BBN and Vall d'Hebron Institute of Research (VHIR) following procedures previously described. [47] Endothelial origin of isolated cells was confirmed by CD105 staining. For activity assays, cells in passages 2-5 were seeded in 24 well plates and maintained at 37 °C and 5% of CO 2 . ...
Lysosomal storage disorders (LSD) are caused by lysosomal dysfunction usually as a consequence of deficiency of a single enzyme required for the metabolism of macromolecules, such as lipids, glycoproteins, and mucopolysaccharides. For instance, the lack of α-galactosidase A (GLA) activity in Fabry disease patients causes the accumulation of glycosphingolipids in the vasculature leading to multiple organ pathology. Enzyme replacement therapy, which is the most common treatment of LSD, exhibits several drawbacks mainly related to the instability and low efficacy of the exogenously administered therapeutic enzyme. In this work, the unprecedented increased enzymatic activity and intracellular penetration achieved by the association of a human recombinant GLA to nanoliposomes functionalized with Arginine-Glycine-Aspartic acid (RGD) peptides is reported. Moreover, these new GLA loaded nanoliposomes lead to a higher efficacy in the reduction of the GLA substrate named globotriasylceramide in a cellular model of Fabry disease, than that achieved by the same concentration of the free enzyme. The preparation of these new liposomal formulations by DELOS-SUSP, based on the depressurization of a CO2 -expanded liquid organic solution, shows the great potential of this CO2 -based methodology for the one-step production of protein-nanoliposome conjugates as bioactive nanomaterials with therapeutic interest.
