Genome-wide association study identifies genetic variants which predict the response of bone mineral density to teriparatide therapy.

OBJECTIVES: Teriparatide (TPTD) is an effective treatment for osteoporosis but the individual response to therapy is variable for reasons that are unclear. This study aimed to determine whether the response to TPTD might be influenced by genetic factors. METHODS: We searched for predictors of the response of bone mineral density (BMD) to TPTD using a two-stage genome-wide association study in 437 patients with osteoporosis from three referral centres. Demographic and clinical data including the response of BMD to treatment at the lumbar spine and hip were extracted from the medical records of each participant. RESULTS: Allelic variation at rs6430612 on chromosome 2, close to the CXCR4 gene was associated with the response of spine BMD to TPTD at a genome wide significant level (p=9.2×10-9 beta=-0.35 (-0.47 to -0.23)). The increase in BMD was almost twice as great in AA homozygotes at rs6430612 as compared with GG homozygotes with intermediate values in heterozygotes. The same variant was also associated with response of femoral neck and total hip BMD (p=0.007). An additional locus on chromosome 19 tagged by rs73056959 was associated with the response of femoral neck BMD to TPTD (p=3.5×10-9, beta=-1.61 (-2.14 to -1.07)). CONCLUSIONS: Genetic factors influence the response to TPTD at the lumbar spine and hip with a magnitude of effect that is clinically relevant. Further studies are required to identify the causal genetic variants and underlying mechanisms as well as to explore how genetic testing for these variants might be implemented in clinical practice.

Probiotics Inhibit Cartilage Damage and Progression of Osteoarthritis in Mice.

Increasing interest has focussed on the possible role of alterations in the microbiome in the pathogenesis of metabolic disease, inflammatory disease, and osteoporosis. Here we examined the role of the microbiome in a preclinical model of osteoarthritis in mice subjected to destabilisation of medical meniscus (DMM). The intestinal microbiome was depleted by broad-spectrum antibiotics from 1 week before birth until the age of 6 weeks when mice were subjected reconstitution of the microbiome with faecal microbial transplant (FMT) followed by the administration of a mixture of probiotic strains Lacticaseibacillus paracasei 8700:2, Lactiplantibacillus plantarum HEAL9 and L. plantarum HEAL19 or vehicle. All mice were subjected to DMM at the age of 8 weeks. The severity of osteoarthritis was evaluated by histological analysis and effects on subchondral bone were investigated by microCT analyses. The combination of FMT and probiotics significantly inhibited cartilage damage at the medial femoral condyle such that the OARSI score was 4.64 ± 0.32 (mean ± sem) in the FMT and probiotic group compared with 6.48 ± 0.53 in the FMT and vehicle group (p = 0.007). MicroCT analysis of epiphyseal bone from the femoral condyle showed that the probiotic group had higher BV/TV, increased Tb.Th, and moderately thicker subchondral bone plates than the control group. There was no difference between groups in joint inflammation or in serum concentrations of inflammatory cytokines and chemokines. We conclude that treatment with probiotics following FMT in mice where the microbiome has been depleted inhibits DMM-induced cartilage damage and impacts on the structure of subchondral bone particularly at the femoral condyle. While further studies are required to elucidate the mechanism of action, our research suggests that these probiotics may represent a novel intervention for the treatment of osteoarthritis.

Gorham-Stout case report: a multi-omic analysis reveals recurrent fusions as new potential drivers of the disease.

BACKGROUND: Gorham-Stout disease is a rare condition characterized by vascular proliferation and the massive destruction of bone tissue. With less than 400 cases in the literature of Gorham-Stout syndrome, we performed a unique study combining whole-genome sequencing and RNA-Seq to probe the genomic features and differentially expressed pathways of a presented case, revealing new possible drivers and biomarkers of the disease. CASE PRESENTATION: We present a case report of a white 45-year-old female patient with marked bone loss of the left humerus associated with vascular proliferation, diagnosed with Gorham-Stout disease. The analysis of whole-genome sequencing showed a dominance of large structural DNA rearrangements. Particularly, rearrangements in chromosomes seven, twelve, and twenty could contribute to the development of the disease, especially a gene fusion involving ATG101 that could affect macroautophagy. The study of RNA-sequencing data from the patient uncovered the PI3K/AKT/mTOR pathway as the most affected signaling cascade in the Gorham-Stout lesional tissue. Furthermore, M2 macrophage infiltration was detected using immunohistochemical staining and confirmed by deconvolution of the RNA-seq expression data. CONCLUSIONS: The way that DNA and RNA aberrations lead to Gorham-Stout disease is poorly understood due to the limited number of studies focusing on this rare disease. Our study provides the first glimpse into this facet of the disease, exposing new possible therapeutic targets and facilitating the clinicopathological diagnosis of Gorham-Stout disease.

Targeted Inactivation of Rin3 Increases Trabecular Bone Mass by Reducing Bone Resorption and Favouring Bone Formation.

Common genetic variants at the RIN3 locus on chromosome 14q32 predispose to Paget's disease of bone (PDB) but the mechanisms by which they do so are unknown. Here, we analysed the skeletal phenotype of female mice with targeted inactivation of the mouse Rin3 gene (Rin3-/-) as compared with wild-type littermates. The Rin3-/- mice had higher trabecular bone volume (BV/TV%) compared with wild type. Mean ± standard deviation values at the distal femur at 8 weeks were 9.0 ± 2.5 vs. 7.0 ± 1.5 (p = 0.002) and at 52 weeks were 15.8 ± 9.5 vs. 8.5 ± 4.2 (p = 0.002). No differences were observed in femoral cortical bone parameters with the exception of marrow diameter which was significantly smaller in 52-week-old Rin3-/- mice compared to wild type: (0.43 mm ± 0.1 vs. 0.57 mm ± 0.2 (p = 0.001). Bone histomorphometry showed a lower osteoclast surface / bone surface (Oc.S/BS%) at 8 weeks in Rin3-/- mice compared to wild type (24.1 ± 4.7 vs. 29.7 ± 6.6; p = 0.025) but there were no significant differences in markers of bone formation at this time. At 52 weeks, Oc.S/BS did not differ between genotypes but single labelled perimeter (SL.Pm/B.Pm (%)) was significantly higher in Rin3-/- mice (24.4 ± 6.4 vs. 16.5 ± 3.8, p = 0.003). We conclude that Rin3 negatively regulates trabecular bone mass in mice by inhibiting osteoclastic bone resorption and favouring bone formation. Our observations also suggest that the variants that predispose to PDB in humans probably do so by causing a gain-in-function of RIN3.

Chondrocytes from osteoarthritic cartilage of obese patients show altered adiponectin receptors expression and response to adiponectin.

Obesity and osteoarthritis (OA) are well-known comorbidities and their precise molecular interactions are still unidentified. Adiponectin, a major adipokine, known to have an anti-inflammatory effect in atherosclerosis or Type 2 Diabetes Mellitus (T2DM), has also been postulated to be pro-inflammatory in OA. This dual role of adiponectin is still not explained. The precise mechanism by which adiponectin affects cartilage and chondrocytes remains to be elucidated. In the present observational study chondrocytes from 30 patients with OA (18 females and 12 males) undergoing total knee replacement (TKR) were isolated. Expression of adiponectin receptors 1 and 2 (ADIPOR1 and ADIPOR2) was examined both at gene and protein levels in chondrocytes. The difference in adiponectin receptor expression between lean and obese patients with OA and the role of adiponectin in regulating pro-inflammatory genes (MCP-1, IL-6, and VCAM-1, MMP-1, MMP-2, and TIMP-1) has been investigated. We found that ADIPOR1 represented the most abundant adiponectin receptor in primary OA chondrocytes. ADIPOR1 and ADIPOR2 genes and ADIPOR1 protein were differently expressed in OA chondrocytes obtained from obese compared with lean patients with OA. Adiponectin induced gene expression of MCP-1, IL-6, and MMP-1 in all OA patients' chondrocytes. In contrast, VCAM-1 and MMP-2 were differently regulated by adiponectin depending on the patient's body mass index. This study suggests that adiponectin and ADIPOR1 may have important roles in the pathogenesis of cartilage degeneration in OA of obese subjects.

Analysis of Transcriptional Regulation in Bone Cells.

Transcription is a process by which the rate of RNA synthesis is regulated. Here we describe the techniques for carrying out promoter-reporter assays, electrophoretic mobility shift assays, chromosome conformation capture (3C) assays, chromatin immunoprecipitation assays, and CRISPR-Cas9 assay, five commonly used methods for studying and altering gene transcription.

The Potential of Intrinsically Magnetic Mesenchymal Stem Cells for Tissue Engineering.

The magnetization of mesenchymal stem cells (MSC) has the potential to aid tissue engineering approaches by allowing tracking, targeting, and local retention of cells at the site of tissue damage. Commonly used methods for magnetizing cells include optimizing uptake and retention of superparamagnetic iron oxide nanoparticles (SPIONs). These appear to have minimal detrimental effects on the use of MSC function as assessed by in vitro assays. The cellular content of magnetic nanoparticles (MNPs) will, however, decrease with cell proliferation and the longer-term effects on MSC function are not entirely clear. An alternative approach to magnetizing MSCs involves genetic modification by transfection with one or more genes derived from Magnetospirillum magneticum AMB-1, a magnetotactic bacterium that synthesizes single-magnetic domain crystals which are incorporated into magnetosomes. MSCs with either or mms6 and mmsF genes are followed by bio-assimilated synthesis of intracytoplasmic magnetic nanoparticles which can be imaged by magnetic resonance (MR) and which have no deleterious effects on MSC proliferation, migration, or differentiation. The stable transfection of magnetosome-associated genes in MSCs promotes assimilation of magnetic nanoparticle synthesis into mammalian cells with the potential to allow MR-based cell tracking and, through external or internal magnetic targeting approaches, enhanced site-specific retention of cells for tissue engineering.

TIA1 variant drives myodegeneration in multisystem proteinopathy with SQSTM1 mutations.

Multisystem proteinopathy (MSP) involves disturbances of stress granule (SG) dynamics and autophagic protein degradation that underlie the pathogenesis of a spectrum of degenerative diseases that affect muscle, brain, and bone. Specifically, identical mutations in the autophagic adaptor SQSTM1 can cause varied penetrance of 4 distinct phenotypes: amyotrophic lateral sclerosis (ALS), frontotemporal dementia, Paget's disease of the bone, and distal myopathy. It has been hypothesized that clinical pleiotropy relates to additional genetic determinants, but thus far, evidence has been lacking. Here, we provide evidence that a TIA1 (p.N357S) variant dictates a myodegenerative phenotype when inherited, along with a pathogenic SQSTM1 mutation. Experimentally, the TIA1-N357S variant significantly enhances liquid-liquid-phase separation in vitro and impairs SG dynamics in living cells. Depletion of SQSTM1 or the introduction of a mutant version of SQSTM1 similarly impairs SG dynamics. TIA1-N357S-persistent SGs have increased association with SQSTM1, accumulation of ubiquitin conjugates, and additional aggregated proteins. Synergistic expression of the TIA1-N357S variant and a SQSTM1-A390X mutation in myoblasts leads to impaired SG clearance and myotoxicity relative to control myoblasts. These findings demonstrate a pathogenic connection between SG homeostasis and ubiquitin-mediated autophagic degradation that drives the penetrance of an MSP phenotype.

Regional Differences Between Perisynovial and Infrapatellar Adipose Tissue Depots and Their Response to Class II and Class III Obesity in Patients With Osteoarthritis.

OBJECTIVE: Obesity is associated with an increased risk of developing osteoarthritis (OA), which is postulated to be secondary to adipose tissue-dependent inflammation. Periarticular adipose tissue depots are present in synovial joints, but the association of this tissue with OA has not been extensively explored. The aim of this study was to investigate differences in local adipose tissue depots in knees with OA and characterize the changes related to class II and class III obesity in patients with end-stage knee OA. METHODS: Synovium and the infrapatellar fat pad (IPFP) were collected during total knee replacement from 69 patients with end-stage OA. Histologic changes, changes in gene and protein expression of adiponectin, peroxisome proliferator-activated receptor γ (PPARγ), and Toll-like receptor 4 (TLR-4), and immune cell infiltration into the adipose tissue were investigated. RESULTS: IPFP and synovium adipose tissue depots differed significantly and were influenced by the patient's body mass index. Compared to adipocytes from the IPFP and synovium of lean patients, adipocytes from the IPFP of obese patients were significantly larger and the synovium of obese patients displayed marked fibrosis, increased macrophage infiltration, and higher levels of TLR4 gene expression. The adipose-related markers PPARγ in the IPFP and adiponectin and PPARγ in the synovium were expressed at lower levels in obese patients compared to lean patients. Furthermore, there were increased numbers of CD45+ hematopoietic cells, CD45+CD14+ total macrophages, and CD14+CD206+ M2-type macrophages in both the IPFP and synovial tissue of obese patients. CONCLUSION: These differences suggest that IPFP and synovium may contain 2 different white adipose tissue depots and support the theory of inflammation-induced OA in patients with class II or III obesity. These findings warrant further investigation as a potentially reversible, or at least suppressible, cause of OA in obese patients.

Biosynthesis of magnetic nanoparticles by human mesenchymal stem cells following transfection with the magnetotactic bacterial gene mms6.

The use of stem cells to support tissue repair is facilitated by loading of the therapeutic cells with magnetic nanoparticles (MNPs) enabling magnetic tracking and targeting. Current methods for magnetizing cells use artificial MNPs and have disadvantages of variable uptake, cellular cytotoxicity and loss of nanoparticles on cell division. Here we demonstrate a transgenic approach to magnetize human mesenchymal stem cells (MSCs). MSCs are genetically modified by transfection with the mms6 gene derived from Magnetospirillum magneticum AMB-1, a magnetotactic bacterium that synthesises single-magnetic domain crystals which are incorporated into magnetosomes. Following transfection of MSCs with the mms6 gene there is bio-assimilated synthesis of intracytoplasmic magnetic nanoparticles which can be imaged by MR and which have no deleterious effects on cell proliferation, migration or differentiation. The assimilation of magnetic nanoparticle synthesis into mammalian cells creates a real and compelling, cytocompatible, alternative to exogenous administration of MNPs.

Functional roles of CSPG4/NG2 in chondrosarcoma.

CSPG4/NG2 is a multifunctional transmembrane protein with limited distribution in adult tissues including articular cartilage. The purpose of this study was to investigate the possible roles of CSPG4/NG2 in chondrosarcomas and to establish whether this molecule may have potential for targeted therapy. Stable knock-down of CSPG4/NG2 in the JJ012 chondrosarcoma cell line by shRNA resulted in decreased cell proliferation and migration as well as a decrease in gene expression of the MMP (matrix metalloproteinase) 3 protease and ADAMTS4 (aggrecanase). Chondrosarcoma cells in which CSPG4/NG2 was knocked down were more sensitive to doxorubicin than wild-type cells. The results indicate that CSPG4/NG2 has roles in regulating chondrosarcoma cell function in relation to growth, spread and resistance to chemotherapy and that anti-CSPG4/NG2 therapies may have potential in the treatment of surgically unresectable chondrosarcoma.

Optineurin Negatively Regulates Osteoclast Differentiation by Modulating NF-κB and Interferon Signaling: Implications for Paget's Disease.

Paget's disease of bone (PDB) is a common disease characterized by osteoclast activation that leads to various skeletal complications. Susceptibility to PDB is mediated by a common variant at the optineurin (OPTN) locus, which is associated with reduced levels of mRNA. However, it is unclear how this leads to the development of PDB. Here, we show that OPTN acts as a negative regulator of osteoclast differentiation in vitro and that mice with a loss-of-function mutation in Optn have increased osteoclast activity and bone turnover. Osteoclasts derived from Optn mutant mice have an increase in NF-κB activation and a reduction in interferon beta expression in response to RANKL when compared to wild-type mice. These studies identify OPTN as a regulator of bone resorption and are consistent with a model whereby genetically determined reductions in OPTN expression predispose to PDB by enhancing osteoclast differentiation.

MCP-1-induced protein attenuates endotoxin-induced myocardial dysfunction by suppressing cardiac NF-кB activation via inhibition of IкB kinase activation.

Myocardial contractile dysfunction is a major consequence of septic shock, which is mainly mediated by nuclear factor-kappa B (NF-кB)-dependent production of inflammatory mediators in the heart. A novel zinc-finger protein, MCP-1-induced protein (MCPIP), is thought to have NF-кB inhibitory activity in certain cell cultures, but its pathophysiological consequence in vivo remains undefined. This study aims to clarify whether the anti-inflammatory potency of MCPIP contribute to amelioration of septic myocardial inflammation and dysfunction in vivo. Transgenic mice (TG) with cardiac-specific expression of MCPIP and their littermate wild-type (WT) controls were challenged with Escherichia coli LPS (10mg/kg ip) and myocardial function was assessed 18 h later using echocardiography. LPS administration markedly deteriorated myocardial contractile function evidenced by reduction of the percentage of left ventricular fractional shortening, which was significantly attenuated by myocardial expression of MCPIP. MCPIP TG mice exhibited a markedly reduced myocardial inflammatory cytokines, less of iNOS expression and peroxynitrite formation, decreased caspase-3/7 activities and apoptotic cell death compared with LPS-treated WT mice. Activation of cardiac NF-кB observed in LPS-challenged WT mice was suppressed by the presence of MCPIP, as evidenced by decreased phosphorylation of IкB kinase (IKKα/ß), reduced degradation of the cytosolic IкBα, and decreased nuclear translocation of NF-кB p65 subunit and its target DNA-binding activity. These results suggest that MCPIP has therapeutic values to protect heart from inflammatory pathologies, possibly through inhibition of IкB kinase complex, leading to blockade of NF-кB activation, and subsequently, attenuation of the proinflammatory state and nitrosative stress in the myocardium.

MCP-1 (monocyte chemotactic protein-1)-induced protein, a recently identified zinc finger protein, induces adipogenesis in 3T3-L1 pre-adipocytes without peroxisome proliferator-activated receptor gamma.

Adipogenesis is a key differentiation process relevant to obesity and associated diseases such as type 2 diabetes. This process involves temporally regulated genes controlled by a set of transcription factors, CCAAT/enhancer-binding proteins (C/EBP) beta, C/EBPdelta, and C/EBPalpha and peroxisome proliferator-activated receptor gamma (PPARgamma). Currently, PPARgamma is universally accepted as the master regulator that is necessary and sufficient to induce adipogenesis as no known factor can induce adipogenesis without PPARgamma. We present evidence that a novel zinc finger protein, MCP-1-induced protein (MCPIP), can induce adipogenesis without PPARgamma. Classical adipogenesis-inducing medium induces MCP-1 production and expression of MCPIP in 3T3-L1 cells before the induction of the C/EBP family of transcription factors and PPARgamma. Knockdown of MCPIP prevents their expression and adipogenesis as measured by expression of adipocyte markers and lipid droplet accumulation. Treatment of 3T3-L1 cells with MCP-1 or forced expression of MCPIP induces expression of C/EBPbeta, C/EBPdelta, C/EBPalpha, and PPARgamma and adipogenesis without any other inducer. Forced expression of MCPIP induces expression of the C/EBP family of transcription factors and adipogenesis in PPARgamma(-/-) mouse embryonic fibroblasts. Thus, MCPIP is a newly identified protein that can induce adipogenesis without PPARgamma.

Cardiac-targeted expression of soluble fas attenuates doxorubicin-induced cardiotoxicity in mice.

Doxorubicin (Dox) is known to cause cardiomyopathy and congestive heart failure upon chronic administration. The mechanisms underlying these toxicities remain uncertain but have been attributed, at least in part, by induction of cardiac cell apoptosis. Fas ligation with its cognate ligand (FasL) induces apoptosis and activates cellular inflammatory responses associated with tissue injury. We determined whether interruption of Fas/FasL interaction by cardiac-targeted expression of soluble Fas (sFas), a competitive inhibitor of FasL, would protect against Dox chronic cardiotoxicity in mice. Wild-type (WT) and sFas transgenic mice were administrated intravenously with 4 mg/kg Dox or with an equivalent volume of saline twice a week for a total of 10 injections. There were 25% mortality in WT mice, but no death was observed in sFas mice during the period of Dox treatment. Echocardiographic evaluation revealed a significant decrease in left ventricle fractional shortening after Dox treatment in WT mice but not in sFas mice. WT mice treated with Dox developed extensive myocardial cytoplasmic vacuolization, apoptosis, and interstitial fibrosis, which were much less or absent in sFas mice. The increased inducible nitric oxide synthase expression, nitric oxide production, superoxide generation, and peroxynitrite formation after Dox treatment in WT mice were attenuated by sFas expression. sFas expression also attenuated Dox-mediated induction of proinflammatory cytokines, tumor necrosis factor-alpha, interleukin (IL)-1beta, and IL-6 in the myocardium. These observations indicate that FasL is an important mediator in Dox-associated cardiotoxicity by generating reactive oxygen and nitrogen species.

Monocyte chemotactic protein (MCP)-1 promotes angiogenesis via a novel transcription factor, MCP-1-induced protein (MCPIP).

Monocyte chemotactic protein-1 (MCP-1) has been recognized as an angiogenic chemokine. The molecular mechanism of MCP-1-mediated angiogenesis remains unknown. We recently identified a novel transcription factor, designated MCP-1-induced protein (MCPIP), in human monocytes after treatment with MCP-1. We investigated whether MCP-1-induced angiogenesis is mediated via MCPIP. Treatment of human umbilical vein endothelial cells (HUVECs) with MCP-1 induced expression of MCPIP and capillary-like tube formation. Knockdown of MCPIP by small interfering RNA (siRNA) suppressed MCP-1-induced angiogenesis-related gene VEGF and HIF-1alpha expression as well as tube formation. Transfection of HUVECs with an MCPIP expression vector induced angiogenesis-related genes and tube formation. Chromatin immunoprecipitation analysis revealed that cadherin (cdh) 12 and cdh19 are in vivo targets of MCPIP. Transfection of HUVECs with MCPIP expression vector activated the expression of cdh12 and cdh19 genes. Knockdown of cdh12 or cdh19 expression markedly inhibited MCPIP-induced capillary-like tube formation. Moreover, knockdown of MCPIP also significantly suppressed MCP-1-induced cdh12 and cdh19 gene expression. Our data strongly suggest that MCP-1-induced angiogenesis is mediated via MCPIP, at least in part through transcriptional activation of cdh12 and cdh19.

A novel CCCH-zinc finger protein family regulates proinflammatory activation of macrophages.

Activated macrophages play an important role in many inflammatory diseases. However, the molecular mechanisms controlling macrophage activation are not completely understood. Here we report that a novel CCCH-zinc finger protein family, MCPIP1, 2, 3, and 4, encoded by four genes, Zc3h12a, Zc3h12b, Zc3h12c, and Zc3h12d, respectively, regulates macrophage activation. Northern blot analysis revealed that the expression of MCPIP1 and MCPIP3 was highly induced in macrophages in response to treatment with lipopolysaccharide (LPS). Although not affecting cell surface marker expression and phagocytotic function, overexpression of MCPIP1 significantly blunted LPS-induced inflammatory cytokine and NO(2)(.) production as well as their gene expression. Conversely, short interfering RNA-mediated reduction in MCPIP1 augmented LPS-induced inflammatory gene expression. Further studies demonstrated that MCPIP1 did not directly affect the mRNA stability of tumor necrosis factor alpha and monocyte chemoattractant protein 1 (MCP-1) but strongly inhibited LPS-induced tumor necrosis factor alpha and inducible nitric-oxide synthase promoter activation. Moreover, we found that forced expression of MCPIP1 significantly inhibited LPS-induced nuclear factor-kappaB activation. These results identify MCP-induced proteins, a novel CCCH-zinc finger protein family, as negative regulators in macrophage activation and may implicate them in host immunity and inflammatory diseases.

Protection against lipopolysaccharide-induced myocardial dysfunction in mice by cardiac-specific expression of soluble Fas.

The mechanisms responsible for myocardial dysfunction in the setting of sepsis remain undefined. Fas ligation with its cognate ligand (FasL) induces apoptosis and activates cellular inflammatory responses associated with tissue injury. We determined whether interruption of Fas/FasL interaction by cardiac-specific expression of soluble Fas (sFas), a competitive inhibitor of FasL, would improve myocardial dysfunction and inflammation in a lipopolysaccharide (LPS)-induced mouse model of sepsis. Wild-type (WT) and sFas transgenic mice were injected intraperitoneally with 10 mg/kg LPS or with an equivalent volume of saline. At 18 h after LPS administration, echocardiographic evaluation revealed a significant decrease in left ventricular fractional shortening in the WT mice, whereas the fractional shortening was preserved in the sFas mice. Activation of nuclear factor-kappa B (NF-kappaB) and the increase in the transcript levels of proinflammatory cytokines, tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and IL-6 resulting from LPS treatment were attenuated in the myocardium of sFas mice. sFas expression also inhibited LPS-induced upregulation of Toll-like receptor 4 (TLR-4) and inducible nitric oxide synthase (iNOS), and formation of peroxynitrite in the myocardium. LPS-induced increase in caspase-3/7 activity and apoptotic cell death were suppressed in sFas mice compared with WT mice. LPS-induced lung injury and increase in lung water content were also significantly reduced in sFas mice. These data indicate that neutralization of FasL by expression of sFas significantly preserves cardiac function and reduces inflammatory responses in the heart, suggesting that Fas/FasL signaling pathway is important in mediating the deleterious effects of LPS on myocardial function.