SiO2 stimulates macrophage stress to induce the transformation of lung fibroblasts into myofibroblasts and its relationship with the sphingomyelin metabolic pathway.

Silicosis is a serious occupational disease with the highest incidence in China. However, its pathogenesis has not been fully elucidated. Studies have shown that the sphingomyelin signaling pathway may play an important role in different fibrotic diseases but its role in silicosis-mediated fibrosis is still unclear. In this study, the supernatant of human peripheral blood mononuclear cell line (THP-1)-derived macrophages exposed to silica (SiO2 ) was used to stimulate the transformation of human embryonic lung fibroblast cell line (HFL-1) into myofibroblasts, and the intervention effect of recombinant human acid ceramidase (rAC) was observed. The results showed that SiO2 stimulated the production of reactive oxygen species and malondialdehyde in the supernatant of THP-1-derived macrophages and increased the secretion of TGF-ß1, TNF-α, and IL-8. In addition, we found that the expression levels of α-SMA, FN, Col I, and Col III in HFL-1 cells increased. Meanwhile, the activities of ASMase and ACase and the expression levels of Cer, Sph, and S1P were increased. Intervention by rAC can suppress these changes to different degrees. In conclusion, the present study shows that SiO2 dust poisoning may stimulate HFL-1 cell differentiation into myofibroblasts by inducing oxidative stress in THP-1-derived macrophages, thereby promoting the secretion of a variety of inflammatory factors and activating the sphingolipid signaling pathway in HFL-1 cells. Exogenous rAC can effectively interfere with the stimulation of HFL-1 cells by silica in vitro.

A New Fluorescent Method to Detect Sulfamidase Activity in Blood, Tissue Extracts and Dried Blood Spots

Abstract Mucopolysaccharidosis type IIIA (MPS IIIA) is a lysosomal storage disorder due to the deficient activity of sulfamidase (SGSH). Traditionally, measurement of this enzymatic activity has been performed using a fluorescently (4-MU) labeled glycoside substrate. While this substrate is inexpensive and readily available, the current method requires a 2-step procedure that is performed over 2 days. Here we report a new and simplified procedure using the 4-MU substrate. Major advantages of this assay method over the existing fluorescent method include a single step vs. 2-step procedure, an incubation time of 1 hour, and high sensitivity. The reaction is also run on UPLC equipment, which is available in most research labs and permits separation of the endogenous, autofluorescent material from the 4-MU signal. This assay method was developed using the MPS IIIA mouse model, and was validated using mouse plasma, liver and brain extracts, and dried blood spots. Human MPS IIIA skin fibroblasts and dried blood spots also were used to validate the method.

Adeno-associated viral vector serotype 9-based gene therapy for Niemann-Pick disease type A.

Niemann-Pick disease type A (NPD-A) is a lysosomal storage disorder characterized by neurodegeneration and early death. It is caused by loss-of-function mutations in the gene encoding for acid sphingomyelinase (ASM), which hydrolyzes sphingomyelin into ceramide. Here, we evaluated the safety of cerebellomedullary (CM) cistern injection of adeno-associated viral vector serotype 9 encoding human ASM (AAV9-hASM) in nonhuman primates (NHP). We also evaluated its therapeutic benefit in a mouse model of the disease (ASM-KO mice). We found that CM injection in NHP resulted in widespread transgene expression within brain and spinal cord cells without signs of toxicity. CM injection in the ASM-KO mouse model resulted in hASM expression in cerebrospinal fluid and in different brain areas without triggering an inflammatory response. In contrast, direct cerebellar injection of AAV9-hASM triggered immune response. We also identified a minimally effective therapeutic dose for CM injection of AAV9-hASM in mice. Two months after administration, the treatment prevented motor and memory impairment, sphingomyelin (SM) accumulation, lysosomal enlargement, and neuronal death in ASM-KO mice. ASM activity was also detected in plasma from AAV9-hASM CM-injected ASM-KO mice, along with reduced SM amount and decreased inflammation in the liver. Our results support CM injection for future AAV9-based clinical trials in NPD-A as well as other lysosomal storage brain disorders.

CD40 Enhances Sphingolipids in Orbital Fibroblasts: Potential Role of Sphingosine-1-Phosphate in Inflammatory T-Cell Migration in Graves' Orbitopathy.

Purpose: Graves' orbitopathy (GO) is an autoimmune orbital disorder associated with Graves' disease caused by thyrotropin receptor autoantibodies. Orbital fibroblasts (OFs) and CD40 play a key role in disease pathogenesis. The bioactive lipid sphingosine-1-phosphate (S1P) has been implicated in promoting adipogenesis, fibrosis, and inflammation in OFs. We investigated the role of CD40 signaling in inducing S1P activity in orbital inflammation. Methods: OFs and T cells were derived from GO patients and healthy control (Ctl) persons. S1P abundance in orbital tissues was evaluated by immunofluorescence. OFs were stimulated with CD40 ligand and S1P levels were determined by ELISA. Further, activities of acid sphingomyelinase (ASM), acid ceramidase, and sphingosine kinase were measured by ultraperformance liquid chromatography. Sphingosine and ceramide contents were analyzed by mass spectrometry. Finally, the role for S1P in T-cell attraction was investigated by T-cell migration assays. Results: GO orbital tissue showed elevated amounts of S1P as compared to control samples. Stimulation of CD40 induced S1P expression in GO-derived OFs, while Ctl-OFs remained unaffected. A significant increase of ASM and sphingosine kinase activities, as well as lipid formation, was observed in GO-derived OFs. Migration assay of T cells in the presence of SphK inhibitor revealed that S1P released by GO-OFs attracted T cells for migration. Conclusions: The results demonstrated that CD40 ligand stimulates GO fibroblast to produce S1P, which is a driving force for T-cell migration. The results support the use of S1P receptor signaling modulators in GO management.

The Role of S1P and the Related Signaling Pathway in the Development of Tissue Fibrosis.

Tissue fibrosis, including pulmonary fibrosis, hepatic fibrosis, and cardiac fibrosis, is an important stage in the development of many diseases. It can lead to structural damage and dysfunction and even severe carcinogenesis or death. There is currently no effective method for the treatment of fibrosis. At present, the molecular mechanism of tissue fibrosis has not yet been fully elucidated, but many studies have demonstrated that it is involved in conveying the complex messages between fibroblasts and various cytokines. Sphingosine 1-phosphate (S1P) is a naturally bioactive sphingolipid. S1P and the related signaling pathways are important intracellular metabolic pathways involved in many life activities, including cell proliferation, differentiation, apoptosis, and cellular signal transduction. Increasing evidence suggests that S1P and its signaling pathways play an important role in the development of tissue fibrosis; however, the mechanisms of these effects have not yet been fully elucidated, and even the role of S1P and its signaling pathways are still controversial. This article focuses on the role of S1P and the related signaling pathways in the development of fibrosis of lung, liver, heart, and other tissues, with emphasis on the application of inhibitors of some of molecules in the pathway in clinical treatment of fibrosis diseases.

Acid Ceramidase Deficiency is characterized by a unique plasma cytokine and ceramide profile that is altered by therapy.

Acid Ceramidase Deficiency (Farber disease, FD) is an ultra-rare Lysosomal Storage Disorder that is poorly understood and often misdiagnosed as Juvenile Idiopathic Arthritis (JIA). Hallmarks of FD are accumulation of ceramides, widespread macrophage infiltration, splenomegaly, and lymphocytosis. The cytokines involved in this abnormal hematopoietic state are unknown. There are dozens of ceramide species and derivatives, but the specific ones that accumulate in FD have not been investigated. We used a multiplex assay to analyze cytokines and mass spectrometry to analyze ceramides in plasma from patients and mice with FD, controls, Farber patients treated by hematopoietic stem cell transplantation (HSCT), JIA patients, and patients with Gaucher disease. KC, MIP-1α, and MCP-1 were sequentially upregulated in plasma from FD mice. MCP-1, IL-10, IL-6, IL-12, and VEGF levels were elevated in plasma from Farber patients but not in control or JIA patients. C16-Ceramide (C16-Cer) and dhC16-Cer were upregulated in plasma from FD mice. a-OH-C18-Cer, dhC12-Cer, dhC24:1-Cer, and C22:1-Cer-1P accumulated in plasma from patients with FD. Most cytokines and only a-OH-C18-Cer returned to baseline levels in HSCT-treated Farber patients. Sphingosines were not altered. Chitotriosidase activity was also relatively low. A unique cytokine and ceramide profile was seen in the plasma of Farber patients that was not observed in plasma from HSCT-treated Farber patients, JIA patients, or Gaucher patients. The cytokine profile can potentially be used to prevent misdiagnosis of Farber as JIA and to monitor the response to treatment. Further understanding of why these signaling molecules and lipids are elevated can lead to better understanding of the etiology and pathophysiology of FD and inform development of future treatments.

The molecular medicine of acid ceramidase.

Acid ceramidase (N-acylsphingosine deacylase, EC 3.5.1.23; AC) is the lipid hydrolase responsible for the degradation of ceramide into sphingosine and free fatty acids within lysosomes. The enzymatic activity was first identified over four decades ago and is deficient in two rare inherited disorders, Farber lipogranulomatosis (Farber disease) and spinal muscular atrophy with myoclonic epilepsy (SMA-PME). Importantly, AC not only hydrolyzes ceramide into sphingosine within acidic compartments, but also can synthesize ceramide from sphingosine at neutral pH, suggesting that the enzyme may have diverse functions depending on its subcellular location and the local pH. Within cells, AC exists in a complex with other lipid hydrolases and requires a polypeptide cofactor (saposin D) for full hydrolytic activity. Recent studies also have shown that AC is overexpressed in several human cancers, and that inhibition of this enzyme may be a useful cancer drug target. Aberrant AC activity has also been described in several other common diseases. The cDNA and gene (ASAH1) encoding AC have been isolated, several mouse models of AC deficiency have been constructed, and the recombinant enzyme is currently being manufactured for the treatment of Farber disease and SMA-PME. Current information concerning the biology of this enzyme and its role in human disease is reviewed within.

Pathological roles of the VEGF/SphK pathway in Niemann-Pick type C neurons.

Sphingosine is a major storage compound in Niemann-Pick type C disease (NP-C), although the pathological role(s) of this accumulation have not been fully characterized. Here we found that sphingosine kinase (SphK) activity is reduced in NP-C patient fibroblasts and NP-C mouse Purkinje neurons (PNs) due to defective vascular endothelial growth factor (VEGF) levels. Sphingosine accumulation due to inactivation of VEGF/SphK pathway led to PNs loss via inhibition of autophagosome-lysosome fusion in NP-C mice. VEGF activates SphK by binding to VEGFR2, resulting in decreased sphingosine storage as well as improved PNs survival and clinical outcomes in NP-C cells and mice. We also show that induced pluripotent stem cell (iPSC)-derived human NP-C neurons are generated and the abnormalities caused by VEGF/SphK inactivity in these cells are corrected by replenishment of VEGF. Overall, these results reveal a pathogenic mechanism in NP-C neurons where defective SphK activity is due to impaired VEGF levels.

Recombinant human acid sphingomyelinase as an adjuvant to sorafenib treatment of experimental liver cancer.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most common form of liver cancer and the third leading cause of cancer death worldwide. The only approved systemic treatment for unresectable HCC is the oral kinase inhibitor, sorafenib. Recombinant human acid sphingomyelinase (rhASM), which hydrolyzes sphingomyelin to ceramide, is an orphan drug under development for the treatment of Type B Niemann-Pick disease (NPD). Due to the hepatotropic nature of rhASM and its ability to generate pro-apoptotic ceramide, this study evaluated the use of rhASM as an adjuvant treatment with sorafenib in experimental models of HCC. METHODOLOGY/PRINCIPAL FINDINGS: In vitro, rhASM/sorafenib treatment reduced the viability of Huh7 liver cancer cells more than sorafenib. In vivo, using a subcutaneous Huh7 tumor model, mouse survival was increased and proliferation in the tumors decreased to a similar extent in both sorafenib and rhASM/sorafenib treatment groups. However, combined rhASM/sorafenib treatment significantly lowered tumor volume, increased tumor necrosis, and decreased tumor blood vessel density compared to sorafenib. These results were obtained despite poor delivery of rhASM to the tumors. A second (orthotopic) model of Huh7 tumors also was established, but modest ASM activity was similarly detected in these tumors compared to healthy mouse livers. Importantly, no chronic liver toxicity or weight loss was observed from rhASM therapy in either model. CONCLUSIONS/SIGNIFICANCE: The rhASM/sorafenib combination exhibited a synergistic effect on reducing the tumor volume and blood vessel density in Huh7 xenografts, despite modest activity of rhASM in these tumors. No significant increases in survival were observed from the rhASM/sorafenib treatment. The poor delivery of rhASM to Huh7 tumors may be due, at least in part, to low expression of mannose receptors. The safety and efficacy of this approach, together with the novel findings regarding enzyme targeting, merits further investigation.

Acid ceramidase maintains the chondrogenic phenotype of expanded primary chondrocytes and improves the chondrogenic differentiation of bone marrow-derived mesenchymal stem cells.

Acid ceramidase is required to maintain the metabolic balance of several important bioactive lipids, including ceramide, sphingosine and sphingosine-1-phosphate. Here we show that addition of recombinant acid ceramidase (rAC) to primary chondrocyte culture media maintained low levels of ceramide and led to elevated sphingosine by 48 hours. Surprisingly, after three weeks of expansion the chondrogenic phenotype of these cells also was markedly improved, as assessed by a combination of histochemical staining (Alcian Blue and Safranin-O), western blotting (e.g., Sox9, aggrecan, collagen 2A1), and/or qPCR. The same effects were evident in rat, equine and human cells, and were observed in monolayer and 3-D cultures. rAC also reduced the number of apoptotic cells in some culture conditions, contributing to overall improved cell quality. In addition to these effects on primary chondrocytes, when rAC was added to freshly harvested rat, equine or feline bone marrow cultures an ~2-fold enrichment of mesenchymal stem cells (MSCs) was observed by one week. rAC also improved the chondrogenic differentiation of MSCs, as revealed by histochemical and immunostaining. These latter effects were synergistic with TGF-beta1. Based on these results we propose that rAC could be used to improve the outcome of cell-based cartilage repair by maintaining the quality of the expanded cells, and also might be useful in vivo to induce endogenous cartilage repair in combination with other techniques. The results also suggest that short-term changes in sphingolipid metabolism may lead to longer-term effects on the chondrogenic phenotype.

Pentosan polysulfate: a novel therapy for the mucopolysaccharidoses.

BACKGROUND: Pentosan polysulfate (PPS) is an FDA-approved, oral medication with anti-inflammatory and pro-chondrogenic properties. We have previously shown that animal models of the mucopolysaccharidoses (MPS) exhibit significant inflammatory disease, contributing to cartilage degeneration. Enzyme replacement therapy (ERT) only partly reduced inflammation, and anti-TNF-alpha antibody therapy significantly enhanced clinical and pathological outcomes. Here we describe the use of PPS for the treatment of MPS type VI rats. METHODOLOGY/PRINCIPAL FINDINGS: Treatment began during prenatal development and at 1 and 6 months of age. All animals were treated until they were 9 months old. Significant reductions in the serum and tissue levels of several inflammatory markers (e.g., TNF-alpha, MIP-1alpha and RANTES/CCL5) were observed, as was reduced expression of inflammatory markers in cultured articular chondrocytes. ADAMTS-5/aggrecanase-2 levels also were reduced in chondrocytes, consistent with an elevation of serum tissue inhibitor of metalloproteinase 1. Marked improvements in motility and grooming behavior occurred, along with a reduction in eye and nasal secretions and a lessening of the tracheal deformities. MicroCT and radiographic analyses further revealed that the treated MPS skulls were longer and thinner, and that the teeth malocclusions, misalignments and mineral densities were improved. MicroCT analysis of the femurs and vertebrae revealed improvements in trabecular bone mineral densities, number and spacing in a subset of treated MPS animals. Biomechanical assessments of PPS-treated spines showed partially restored torsional behaviors, suggesting increased spinal stability. No improvements were observed in cortical bone or femur length. The positive changes in the PPS-treated MPS VI rats occurred despite glycosaminoglycan accumulation in their tissues. CONCLUSIONS: Based on these findings we conclude that PPS could be a simple and effective therapy for MPS that might provide significant clinical benefits alone and in combination with other therapies.

Identification of cystatin SA as a novel inhibitor of acid ceramidase.

Autoproteolytic cleavage of the inactive acid ceramidase (AC) precursor into the active heterodimer exposes a free cysteine residue, leading us to study whether AC could be regulated by one or more members of the cystatin family. Co-expression of the full-length AC and cystatin SA (cysSA) cDNAs led to significant reduction of AC activity in the transfected cells. Expression of cysSA also inhibited endogenous AC activity in cells and increased ceramide. Conversely, cysSA siRNA expression led to elevated AC activity and reduction in ceramide. The effects of cysSA siRNA expression could be reversed by the addition of recombinant cysSA into the culture media. These results were consistent with detection of a physical interaction between AC and cysSA, assessed by co-immunoprecipitation and nickel-nitrilotriacetic acid affinity chromatography, and further supported by co-localization of the endogenous proteins using confocal microscopy. In vitro kinetic analysis of purified, recombinant AC and cysSA confirmed the transfection results and suggested a non-competitive type of inhibition with a K(i) in the low micromolar range. Processing of the AC precursor into the active form was not affected by cysSA expression, suggesting that it likely inhibits AC by allosteric interference. Computer modeling and expression studies identified several potential inhibitory domains in cysSA, including a small "AC-like" domain (identical to the AC cleavage site, TICT). Small peptides, synthesized with combinations of this and a "cystatin-like" domain (QXVXG), exhibited significant AC inhibition as well. Such peptide-based AC inhibitors could potentially be used to regulate AC activity in cancer cells that are known to overexpress this enzyme alone and in combination with conventional anti-cancer drugs.

Acid sphingomyelinase deficiency attenuates bleomycin-induced lung inflammation and fibrosis in mice.

BACKGROUND/AIMS: The sphingomyelin/ceramide signaling pathway is an important component of many cellular processes implicated in the pathogenesis of lung disease. Acid sphingomyelinase (ASM) is a key mediator of this pathway, but its specific role in pulmonary fibrosis has not been previously investigated. Here we used the bleomycin model of pulmonary fibrosis to investigate fibrotic responses in normal and ASM knockout (ASM(-/-)) mice, and in NIH3T3 fibroblasts with and without ASM siRNA treatment. METHODS: Mice and cells with and without ASM activity were treated with bleomycin, and the effects on lung inflammation, formation of collagen producing myofibroblasts, and apoptosis were assessed. RESULTS: The development of bleomycin-induced inflammation and fibrosis in wildtype mice correlated with the rapid activation of ASM, and was markedly attenuated in the absence of ASM activity. Along with the elevated ASM activity, there also was an elevation of acid ceramidase (AC) activity, which was sustained for up to 14 days post-bleomycin treatment. Studies in NIH3T3 fibroblasts confirmed these findings, and revealed a direct effect of ASM/AC activation on the formation of myofibroblasts. Cell studies also showed that a downstream effect of bleomycin treatment was the production of sphingosine-1-phosphate. CONCLUSIONS: These data demonstrate that the sphingomyelin/ceramide signaling pathway is involved in the pathogenesis of bleomycin-induced pulmonary fibrosis, and suggest that inhibition of ASM may potentially slow the fibrotic process in the lung.

Bone marrow-derived mesenchymal stem cells prevent the loss of Niemann-Pick type C mouse Purkinje neurons by correcting sphingolipid metabolism and increasing sphingosine-1-phosphate.

Niemann-Pick type C (NP-C) disease exhibits neuronal sphingolipid storage and cerebellar Purkinje neuron (PN) loss. Although it is clear that PNs are compromised in this disorder, it remains to be defined how neuronal lipid storage causes the PN loss. Our previous studies have shown that bone marrow-derived mesenchymal stem cells (BM-MSCs) transplantation prevent PN loss in NP-C mice. The aim of the present study was therefore to examine the neuroprotective mechanism of BM-MSCs on PNs. We found that NP-C PNs exhibit abnormal sphingolipid metabolism and defective lysosomal calcium store compared to wild-type mice PNs. BM-MSCs promote the survival of NP-C PNs by correction of the altered calcium homeostasis, restoration of the sphingolipid imbalance, as evidenced by increased sphingosine-1-phosphate levels and decreased sphingosine, and ultimately, inhibition of apoptosis pathways. These effects suggest that BM-MSCs modulate sphingolipid metabolism of endogenous NP-C PNs, resulting in their survival and improved clinical outcome in mice.

Involvement of the Toll-like receptor 4 pathway and use of TNF-alpha antagonists for treatment of the mucopolysaccharidoses.

Enzyme replacement therapy is currently available for three of the mucopolysaccharidoses (MPSs) but has limited effects on the skeletal lesions. We investigated the involvement of the Toll-like receptor 4 (TLR4) signaling pathway in the pathogenesis of MPS bone and joint disease, and the use of the anti-TNF-alpha drug, Remicade (Centocor, Inc.), for treatment. TLR4 KO (TLR4(lps-/-)) mice were interbred with MPS VII mice to produce double-KO (DKO) animals. The DKO mice had longer and thinner faces and longer femora as revealed by micro-computed tomography analysis compared with MPS VII mice. Histological analyses also revealed more organized and thinner growth plates. The serum levels of TNF-alpha were normalized in the DKO animals, and the levels of phosphorylated STAT1 and STAT3 in articular chondrocytes were corrected. These findings led us to evaluate the effects of Remicade in MPS VI rats. When initiated at 1 month of age, i.v. treatment prevented the elevation of TNF-alpha, receptor activator of NF-kappaB, and other inflammatory molecules not only in the blood but in articular chondrocytes and fibroblast-like synoviocytes (FLSs). Treatment of 6-month-old animals also reduced the levels of these molecules to normal. The number of apoptotic articular chondrocytes in MPS VI rats was similarly reduced, with less infiltration of synovial tissue into the underlying bone. These studies revealed the important role of TLR4 signaling in MPS bone and joint disease and suggest that targeting TNF-alpha may have positive therapeutic effects.

Characterization of common SMPD1 mutations causing types A and B Niemann-Pick disease and generation of mutation-specific mouse models.

Herein we describe detailed characterization of four common mutations (L302P, H421Y, R496L and DeltaR608) within the acid sphingomyelinase (ASM) gene causing types A and B Niemann-Pick disease (NPD). In vitro and in situ enzyme assays revealed marked deficiencies of ASM activity in NPD cell lines homoallelic for each mutation, although Western blotting and fluorescent microscopy showed that the mutant ASM polypeptides were expressed at normal levels and trafficked to lysosomes. Co-immunoprecipitation of the polypeptides with the ER chaperone, BiP, confirmed these findings, as did in vitro expression of the mutant cDNAs in reticulocyte lysates. We further developed a computer assisted, three-dimensional model of human ASM based on homologies to known proteins, and used this model to map each NPD mutation in relation to putative substrate binding, hydrolysis and zinc-binding domains. Lastly, we generated transgenic mice expressing the R496L and DeltaR608 mutations on the complete ASM knock-out background (ASMKO), and established breeding colonies for the future evaluation of enzyme enhancement therapies. Analysis of these mice demonstrated that the mutant ASM transgenes were expressed at high levels in the brain, and in the case of the DeltaR608 mutation, produced residual ASM activity that was significantly above the ASMKO background.

Autoproteolytic cleavage and activation of human acid ceramidase.

Herein we report the mechanism of human acid ceramidase (AC; N-acylsphingosine deacylase) cleavage and activation. A highly purified, recombinant human AC precursor underwent self-cleavage into alpha and beta subunits, similar to other members of the N-terminal nucleophile hydrolase superfamily. This reaction proceeded with first order kinetics, characteristic of self-cleavage. AC self-cleavage occurred most rapidly at acidic pH, but also at neutral pH. Site-directed mutagenesis and expression studies demonstrated that Cys-143 was an essential nucleophile that was required at the cleavage site. Other amino acids participating in AC cleavage included Arg-159 and Asp-162. Mutations at these three amino acids prevented AC cleavage and activity, the latter assessed using BODIPY-conjugated ceramide. We propose the following mechanism for AC self-cleavage and activation. Asp-162 likely forms a hydrogen bond with Cys-143, initiating a conformational change that allows Arg-159 to act as a proton acceptor. This, in turn, facilitates an intermediate thioether bond between Cys-143 and Ile-142, the site of AC cleavage. Hydrolysis of this bond is catalyzed by water. Treatment of recombinant AC with the cysteine protease inhibitor, methyl methanethiosulfonate, inhibited both cleavage and enzymatic activity, further indicating that cysteine-mediated self-cleavage is required for ceramide hydrolysis.

Mechanism of glycosaminoglycan-mediated bone and joint disease: implications for the mucopolysaccharidoses and other connective tissue diseases.

We have previously shown that glycosaminoglycan (GAG) storage in animal models of the mucopolysaccharidoses (MPS) leads to inflammation and apoptosis within cartilage. We have now extended these findings to synovial tissue and further explored the mechanism underlying GAG-mediated disease. Analysis of MPS rats, cats, and/or dogs revealed that MPS synovial fibroblasts and fluid displayed elevated expression of numerous inflammatory molecules, including several proteins important for lipopolysaccharide signaling (eg, Toll-like receptor 4 and lipoprotein-binding protein). The expression of tumor necrosis factor, in particular, was elevated up to 50-fold, leading to up-regulation of the osteoclast survival factor, receptor activator of nuclear factor-kappaB ligand, and the appearance of multinucleated osteoclast-like cells in the MPS bone marrow. Treatment of normal synovial fibroblasts with GAGs also led to production of the prosurvival lipid sphingosine-1-phosphate, resulting in enhanced cell proliferation, consistent with the hyperplastic synovial tissue observed in MPS patients. In contrast, GAG treatment of normal chondrocytes led to production of the proapoptotic lipid ceramide, confirming the enhanced cell death we had previously observed in MPS cartilage. These findings have important implications for the pathogenesis and treatment of MPS and have further defined the mechanism of GAG-stimulated disease.

Bone marrow transplantation for feline mucopolysaccharidosis I.

Severe mucopolysaccharidosis type I (MPS I) is a fatal neuropathic lysosomal storage disorder with significant skeletal involvement. Treatment involves bone marrow transplantation (BMT), and although effective, is suboptimal, due to treatment sequelae and residual disease. Improved approaches will need to be tested in animal models and compared to BMT. Herein we report on bone marrow transplantation to treat feline mucopolysaccharidosis I (MPS I). Five MPS I stably engrafted kittens, transplanted with unfractionated bone marrow (6.3x10(7)-1.1x10(9) nucleated bone marrow cells per kilogram) were monitored for 13-37 months post-engraftment. The tissue total glycosaminoglycan (GAG) content was reduced to normal levels in liver, spleen, kidney, heart muscle, lung, and thyroid. Aorta GAG content was between normal and affected levels. Treated cats had a significant decrease in the brain GAG levels relative to untreated MPS I cats and a paradoxical decrease relative to normal cats. The alpha-l-iduronidase (IDUA) activity in the livers and spleens of transplanted MPS I cats approached heterozygote levels. In kidney cortex, aorta, heart muscle, and cerebrum, there were decreases in GAG without significant increases in detectable IDUA activity. Treated animals had improved mobility and decreased radiographic signs of disease. However, significant pathology remained, especially in the cervical spine. Corneal clouding appeared improved in some animals. Immunohistochemical and biochemical analysis documented decreased central nervous system ganglioside storage. This large animal MPS I study will serve as a benchmark of future therapies designed to improve on BMT.

A lipid analogue that inhibits sphingomyelin hydrolysis and synthesis, increases ceramide, and leads to cell death.

We report the synthesis and characterization of a novel thiourea derivative of sphingomyelin (AD2765). In vitro assays using pure enzyme and/or cell extracts revealed that this compound inhibited the hydrolysis of BODIPY-conjugated or 14C-labeled sphingomyelin by acid sphingomyelinase and Mg2+-dependent neutral sphingomyelinase. Studies in normal human skin fibroblasts further revealed that AD2765 was taken up by cells and inhibited the hydrolysis of BODIPY-conjugated sphingomyelin in situ. In situ and in vitro studies also showed that this compound inhibited the synthesis of sphingomyelin from BODIPY-conjugated ceramide. The specificity of AD2765 for enzymes involved in sphingomyelin metabolism was demonstrated by the fact that it had no effect on the hydrolysis of BODIPY-conjugated ceramide by acid ceramidase or on the synthesis of BODIPY-conjugated glucosylceramide from BODIPY-conjugated ceramide. The overall effect of AD2765 on sphingomyelin metabolism was concentration-dependent, and treatment of normal human skin fibroblasts or cancer cells with this compound at concentrations > 10 microM led to an increase in cellular ceramide and cell death. Thus, AD2765 might be used to manipulate sphingomyelin metabolism in various ways, potentially to reduce substrate accumulation in cells from types A and B Niemann-Pick disease patients, and/or to affect the growth of human cancer cells.