Genetic basis and hematologic manifestations of sitosterolemia in a group of Turkish patients.

BACKGROUND: Sitosterolemia is a rare lipid disorder caused by mutations in adenosine triphosphate-binding cassette genes (ABCG) 5 and 8. OBJECTIVE: To evaluate the phenotypic/genotypic features of sitosterolemia in a group of Turkish patients. METHODS: Seven probands with unexplained hematologic abnormalities and their 13 relatives were enrolled. Sterol levels were measured by gas chromatography and genetic studies were performed using Sanger sequencing. Individuals were diagnosed with sitosterolemia if they were found to have frankly elevated sitosterol level >15 µg/mL and/or pathogenic variants of the ABCG5/ABCG8. RESULTS: The seven probands and their six relatives  were diagnosed with frank sitosterolemia, and all these patients had hematologic abnormalities. The remaining seven relatives were asymptomatic heterozygous carriers. Three novel variants in the ABCG5 gene (c.161G>A, c.1375C>T, IVS10-1G>T), one novel variant in the ABCG8 gene (c.1762G>C) and one known variant in the ABCG5 gene (c.1336 C>T) were identified. No variant was identified in one case. The mean sitosterol level was significantly higher and mean platelet count was significantly lower in patients with homozygous variants compared to heterozygous variants (p<0.05, for all). Diets low in plant sterols were recommended for 13 symptomatic cases. Four homozygotes received ezetimibe, and their splenomegaly, anemia, and thrombocytopenia completely resolved except one. CONCLUSION: The five pathogenic variants identified in this study indicate the genetic heterogeneity of sitosterolemia in Turkish population. Patients with unexplained hematologic abnormalities (specifically macrothrombocytopenia) should have their sterol level measured as initial testing. Ezetimibe can be a good choice for sitosterolemia.

Improvement in the sensitivity of newborn screening for Fabry disease among females through the use of a high-throughput and cost-effective method, DNA mass spectrometry.

Many female carriers of Fabry disease are likely to develop severe morbidity and mortality. However, by our own estimation, around 80% of female newborns are missed by our current enzyme-based screening approach. Our team's aim was to develop an improved cost-effective screening method that is able to detect Fabry disease among female newborns. In Taiwan, based on a database of 916,000 newborns, ~98% of Fabry patients carry mutations out of a pool of only 21 pathogenic mutations. An Agena iPLEX platform was designed to detect these 21 pathogenic mutations using only a single-assay panel. A total of 54,791 female infants were screened and 136 female newborns with the IVS4 + 919G > A mutation and one female newborn with the c.656T > C mutation were identified. Using the current enzyme-based newborn screening approach as baseline, around 83% of female newborns are being missed. Through a family study of the IVS4 female newborns, 30 IVS4 adult family members were found to have left ventricular hypertrophy. Ten patients received endomyocardial biopsy and all were found to have significant globotriaosylceramide (Gb3) accumulation in their cardiomyocytes. All of these individuals now receive enzyme replacement therapy. We have demonstrated that the Agena iPLEX assay is a powerful tool for detecting females with Fabry disease. Furthermore, through this screening, we also have been able to identify many disease-onset adult family members who were originally undiagnosed for Fabry disease. This screening helps them to receive treatment in time before severe and irreversible cardiac damage has occurred.

Arecoline activates latent transforming growth factor ß1 via mitochondrial reactive oxygen species in buccal fibroblasts: Suppression by epigallocatechin-3-gallate.

BACKGROUND/PURPOSE: Oral submucous fibrosis (OSF) is a premalignant condition caused by the chewing of areca nut (AN). Transforming growth factor ß (TGFß) plays a central role in the pathogenesis of OSF. Connective tissue growth factor (CTGF or CCN2) and early growth response-1 (Egr-1) are important mediators in the fibrotic response to TGFß in several fibrotic disorders including OSF. Arecoline, a major AN alkaloid, induced the synthesis of CCN2 and Egr-1 in human buccal mucosal fibroblast (BMFs). The aims of this study were to investigate whether arecoline-induced CCN2 and Egr-1 syntheses are mediated through TGFß1 signaling and to inspect the detailed mechanisms involved. METHODS: Western blot and TGFß1 Emax® ImmunoAssay were used to measure the effect of arecoline on the TGFß signaling pathways. 2',7'-dichlorodihydrofluorescein diacetate and MitoSOX™ Red were used to measure the effect of arecoline on the cellular and mitochondrial reactive oxygen species (ROS). RESULTS: Arecoline induced latent TGFß1 activation, Smad2 phosphorylation, and mitochondrial and total cellular ROS in BMFs. TGFß-neutralizing antibody completely inhibited the arecoline-induced synthesis of CCN2 and Egr-1. Mito-TEMPO, a mitochondria-targeted antioxidant, completely suppressed arecoline-induced latent TGFß1 activation and mitochondrial and total cellular ROS. Epigallocatechin-3-gallate (EGCG) dose-dependently inhibited arecoline-induced TGFß1 activation and mitochondrial ROS in BMFs. CONCLUSION: Our results indicated that arecoline-induced mitochondrial ROS plays pivotal roles in the activation of latent TGFß1 leading to the initiation of TGFß1 signaling and subsequent increase in the synthesis of CCN2 and Egr-1. EGCG can be a useful agent in the chemoprevention and treatment of OSF.

Controlled release of lovastatin from poly(lactic-co-glycolic acid) nanoparticles for direct pulp capping in rat teeth.

Statin at appropriate concentrations has been shown to induce odontoblastic differentiation, dentinogenesis, and angiogenesis. However, using a carrier to control statin release might reduce toxicity and enhance its therapeutic effects. The aim of this study was to prepare poly(d,l-lactide-co-glycolide acid) (PLGA) nanoparticles that contain lovastatin for application in direct pulp capping. The PLGA-lovastatin particle size was determined using dynamic light scattering measurements and transmission electron microscopy. In addition, the release of lovastatin was quantified using a UV-Vis spectrophotometer. The cytotoxicity and alkaline phosphatase (ALP) activity of PLGA-lovastatin nanoparticles on human dental pulp cells were investigated. Moreover, a real-time polymerase chain reaction (PCR) assay, Western blot analysis, and an enzyme-linked immunosorbent assay (ELISA) were used to examine the osteogenesis gene and protein expression of dentin sialophosphoprotein (DSPP), dentin matrix acidic phosphoprotein 1 (DMP1), and osteocalcin (OCN). Finally, PLGA-lovastatin nanoparticles and mineral trioxide aggregate (MTA) were compared as direct pulp capping materials in Wistar rat teeth. The results showed that the median diameter of PLGA-lovastatin nanoparticles was 174.8 nm and the cumulative lovastatin release was 92% at the 44th day. PLGA-lovastatin nanoparticles demonstrated considerably a lower cytotoxicity than free lovastatin at 5, 9, and 13 days of culture. For ALP activity, the ALP amount of PLGA-lovastatin (100 µg/mL) was significantly higher than that of the other groups for 9 and 13 days of culture. The real-time PCR assay, Western blot analysis, and ELISA assay showed that PLGA-lovastatin (100 µg/mL) induced the highest mRNA and protein expression of DSPP, DMP1, and OCN in pulp cells. Histological evaluation of the animal studies revealed that MTA was superior to the PLGA-lovastatin in stimulating the formation of tubular dentin in an observation period of 2 weeks. However, in an observation period of 4 weeks, it was evident that the PLGA-lovastatin and MTA were competitive in the formation of tubular reparative dentin and a complete dentinal bridge.

Activation of transforming growth factor-ß1 by thrombin via integrins αvß1, αvß3, and αvß5 in buccal fibroblasts: Suppression by epigallocatechin-3-gallate.

BACKGROUND: Transforming growth factor-beta (TGF-ß) plays a central role in the pathogenesis of oral submucous fibrosis (OSF). Thrombin is a key player in tissue repair, inflammation, and fibrosis after injury. METHODS: Effects of thrombin on activated-TGF-ß1 levels, Smad3 phosphorylation, and connective tissue growth factor (CTGF/CCN2) synthesis in primary human buccal mucosal fibroblasts (BMFs) were assessed by enzyme-linked immunosorbent assay or Western blot analysis. RESULTS: Thrombin and protease-activated receptor-1 (PAR-1) agonist induced TGF-ß1 activation and Smad3 phosphorylation. Pretreatment with TGF-ß-neutralizing antibody completely inhibited thrombin-induced CCN2 synthesis. Neutralizing antibodies to integrin αv, ß1, αvß3, αvß5, and Rho-associated coiled-coil forming protein kinase (ROCK) inhibitor Y27632 completely blocked thrombin-induced TGF-ß1 activation, Smad3 phosphorylation, and CCN2 synthesis. Epigallocatechin-3-gallate (EGCG) dose-dependently inhibited thrombin-induced TGF-ß1 activation. CONCLUSION: Thrombin induces αvß1, αvß3, and αvß5 integrins-mediated TGF-ß1 activations via ROCK signaling. EGCG inhibits thrombin-induced CCN2 synthesis in BMFs by suppressing latent TGF-ß1 activation.

Epigallocatechin-3-gallate inhibits transforming-growth-factor-ß1-induced collagen synthesis by suppressing early growth response-1 in human buccal mucosal fibroblasts.

BACKGROUND/PURPOSE: Transforming growth factor (TGF)-ß is a key regulator in the pathogenesis of oral submucous fibrosis (OSF). Early growth response (Egr)-1 is essential for fibrotic responses to TGF-ß. Because TGF-ß signaling is cell-type- and context-dependent, we investigated the signaling involved in TGF-ß-induced Egr-1 in primary human buccal mucosal fibroblasts (BMFs). METHODS: TGF-ß-induced Egr-1 and its signaling were assessed by western blotting in BMFs. Egr-1 small interfering RNA was used to define the role of Egr-1 on TGF-ß-induced mRNAs of the α1- and α2-chains of type I collagen (COL1A1 and COL1A2) and acid-soluble collagen production (via Sircol collagen assay). The effects of epigallocatechin-3-gallate (EGCG) on TGF-ß-induced Egr-1 protein and acid-soluble collagen were also evaluated. RESULTS: TGF-ß1 stimulated Egr-1 production in BMFs. Pretreatment with PD98059, SP600125, SB431542, and SIS3, but not SB203580, significantly reduced TGF-ß1-induced Egr-1 protein expression. Genetic targeting of Egr-1 completely inhibited TGF-ß1-induced type I collagen mRNAs and collagen protein expression. EGCG fully inhibited TGF-ß1-induced Egr-1 and TGF-ß1-stimulated production of acid-soluble collagens. CONCLUSION: We conclude that activin receptor-like kinase (ALK)5, Smad3, extracellular signal-regulated kinase, and c-Jun N-terminal kinase are involved in the TGF-ß1-induced Egr-1 protein production in BMFs. Egr-1 mediates TGF-ß1-induced COL1A1 and COL1A2 mRNA expression and acid-soluble collagen production in BMFs. EGCG can block TGF-ß1-induced collagen production by attenuating Egr-1 expression in BMFs. Egr-1 is a key mediator in TGF-ß1-induced pathogenesis of OSF. EGCG may be useful in the prevention or treatment of OSF.

Phenethyl isothiocyanate enhances TRAIL-induced apoptosis in oral cancer cells and xenografts.

OBJECTIVES: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been regarded as a promising candidate for cancer therapy. However, most of oral cancer cell lines are resistant to the TRAIL-induced cytotoxicity. The aim of this study was to investigate the ability of phenethyl isothiocyanate (PEITC) to sensitize TRAIL-induced apoptosis in TRAIL-resistant oral cancer cells and xenografts. MATERIALS AND METHODS: Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, Western blotting, and a mouse xenograft model were used to study the effects of PEITC and TRAIL on two TRAIL-resistant human oral cancer cells, SAS and Ca9-22. RESULTS: PEITC upregulated death receptor 4 (DR4) and DR5 protein expression and increased reactive oxygen species (ROS) production in both SAS and Ca9-22 cells. Antioxidant N-acetyl-L-cysteine (NAC) and c-Jun NH2-terminal kinase (JNK) inhibitor SP600125 inhibited PEITC-induced DR4 and DR5 expression. Inhibitor experiments showed that PEITC induced apoptosis through ROS-mediated JNK activation and upregulation of DR4 and DR5. Furthermore, treatment with PEITC significantly increased TRAIL-induced apoptosis in both cells. Combined treatment with PEITC and TRAIL had greater effect on the inhibition of tumor growth than either agent alone. CONCLUSIONS: We showed for the first time that PEITC overcomes TRAIL resistance in oral cancer cells and enhance the therapeutic potential of TRAIL in vivo. CLINICAL RELEVANCE: PEITC, either alone or in combination with TRAIL, can be used as a new therapeutic approach for the treatment of oral cancers.

Arecoline stimulated early growth response-1 production in human buccal fibroblasts: suppression by epigallocatechin-3-gallate.

BACKGROUND: Early growth response-1 (Egr-1) protein plays an important role in many human fibrotic diseases. Areca nut chewing is the most important risk factor of oral submucous fibrosis (OSF). METHODS: Egr-1 protein expression in OSF was examined using antibody to Egr-1. Arecoline-induced Egr-1 expression and its signaling pathways were assessed by Western blot analyses in human buccal mucosal fibroblasts (BMFs). RESULTS: Elevated Egr-1 staining was observed in epithelial cells, fibroblast, and inflammatory cells in 7 of 10 OSF cases. Arecoline, a main alkaloid found in the areca nut, stimulated Egr-1 synthesis in BMFs. Pretreatment with antioxidant N-acetyl-L-cysteine, c-Jun NH2-terminal kinase inhibitor SP600125, and extracellular signal-regulated kinase inhibitor PD98059 significantly reduced arecoline-induced Egr-1 synthesis. Epigallocatechin-3-gallate (EGCG) inhibited arecoline-induced Egr-1 synthesis and collagen gel contraction in a dose-responsive manner. CONCLUSION: Constitutive Egr-1 expression during areca nut chewing may play a role in the pathogenesis of OSF. EGCG could be a good candidate for prevention or treatment of OSF.

Correlation of F4-neuroprostanes levels in cerebrospinal fluid with outcome of aneurysmal subarachnoid hemorrhage in humans.

Aneurysmal subarachnoid hemorrhage (aSAH) is one type of hemorrhagic stroke in humans. F(2)-isoprostanes (F(2)-IsoPs) and F(4)-neuroprostanes (F(4)-NPs), derived from arachidonic acid and docosahexaenoic acid (DHA), respectively, are specific markers of lipid peroxidation. We previously demonstrated that F(2)-IsoPs levels in cerebrospinal fluid (CSF) of aSAH patients positively correlated with poor clinical conditions. In this work, we refined F(4)-NPs analysis and investigated the role of potential oxidative damage to neurons in aSAH patients by detecting F(4)-NPs in CSF. [(2)H(4)]-15-F(2t)-IsoP, rather than [(18)O(2)]-17-F(4c)-NP or [(2)H(4)]-PGF(2 alpha), was used as the internal standard for F(4)-NPs analysis. One problem of the use of [(18)O(2)]-17-F(4c)-NP was the potential interference resulting from F(2)-dihomo-IsoPs in CSF. CSF specimens of 15 aSAH patients for up to 10 days and those of 12 non-aSAH controls were analyzed. First day, mean, and peak levels of F(4)-NPs were all significantly higher in aSAH patients than in controls and correlated with the Fisher Scale and 3-month Glasgow Outcome Scale, but only mean levels of F(4)-NPs correlated with Hunt and Hess Grade. The results first demonstrate oxidative damage to DHA in brain tissue following aSAH and suggest that F(4)-NPs in CSF could be a better predictor for outcome of aSAH than F(2)-IsoPs at early time points.