Modified Lichong decoction intervenes in colorectal cancer by modulating the intestinal flora and the Wnt/ß-catenin signaling pathway.

BACKGROUND: The pathogenesis and treatment of colorectal cancer (CRC) continue to be areas of ongoing research, especially the benefits of traditional Chinese medicine (TCM) in slowing the progression of CRC. This study was conducted to investigate the effectiveness and mechanism of action of modified Lichong decoction (MLCD) in inhibiting CRC progression. METHODS: We established CRC animal models using azoxymethane/dextran sodium sulfate (AOM/DSS) and administered high, medium, or low doses of MLCD or mesalazine (MS) for 9 weeks to observe MLCD alleviation of CRC. The optimal MLCD dose group was then subjected to metagenomic and RNA sequencing (RNA-seq) to explore the differentially abundant flora and genes in the control, model and MLCD groups. Finally, the mechanism of action was verified using WB, qRT‒PCR, immunohistochemistry and TUNEL staining. RESULTS: MLCD inhibited the progression of CRC, and the optimal effect was observed at high doses. MLCD regulated the structure and function of the intestinal flora by decreasing the abundance of harmful bacteria and increasing that of beneficial bacteria. The differentially expressed genes were mainly associated with the Wnt/ß-catenin pathway and the cell cycle. Molecular biology analysis indicated that MLCD suppressed the Wnt/ß-catenin pathway and the epithelial-mesenchymal transition (EMT), inhibited abnormal cell proliferation and promoted intestinal epithelial cell apoptosis. CONCLUSION: MLCD mitigated the abnormal growth of intestinal epithelial cells and promoted apoptosis, thereby inhibiting the progression of CRC. This inhibition was accomplished by modifying the intestinal microbiota and disrupting the Wnt/ß-catenin pathway and the EMT. Therefore, MLCD could serve as a potential component of TCM prescriptions for CRC treatment.

Keratinocyte-to-macrophage communication exacerbate psoriasiform dermatitis via LRG1-enriched extracellular vesicles.

Rationale: Macrophage-associated inflammation and keratinocytes excessive proliferation and inflammatory cytokines secretion induced by stimulation play an important role in the progression of psoriasiform dermatitis. However, how these two types of cells communicate remains obscure. Methods: We induced a mouse model with experimental psoriasiform dermatitis by Imiquimod (IMQ). To investigate whether damaged keratinocytes promote macrophage polarization and accelerate skin lesions by releasing extracellular vesicle (EV), purified EV were isolated from the primary epidermis of 5-day IMQ-induced psoriasiform dermatitis model mice, and then fluorescence-labeled the EV with PKH67. The EV was injected into the skin of mice treated with IMQ or vehicle 2 days in situ. In addition, we established a co-culture system of the human monocytic cell line (THP-1) and HaCaT, and THP-1/HaCaT conditioned media culture model in vitro respectively. Subsequently, we evaluated the effect of Leucine-rich α-2-glycoprotein 1 (LRG1)-enriched EV on macrophage activation. Results: We demonstrated macrophages can significantly promote keratinocyte inflammation and macrophage polarization may be mediated by intercellular communication with keratinocytes. Interestingly, IMQ-induced 5-day, keratinocyte-derived EV recruited macrophage and enhanced the progression of skin lesions. Similar to results in vivo, EV released from M5-treated HaCaT significantly promotes Interleukin 1ß (IL-1ß) and Tumor necrosis factor α (TNF-α) expression of THP-1 cells. Importantly, we found that LRG1-enriched EV regulates macrophages via TGF beta Receptor 1 (TGFßR1) dependent process. Conclusion: Our findings indicated a novel mechanism for promoting psoriasiform dermatitis, which could be a potential therapeutic target.

Regulatory factor X1 induces macrophage M1 polarization by promoting DNA demethylation in autoimmune inflammation.

Abnormal macrophage polarization is generally present in autoimmune diseases. Overwhelming M1 macrophage activation promotes the continuous progression of inflammation, which is one of the reasons for the development of autoimmune diseases. However, the underlying mechanism is still unclear. Here we explore the function of Regulatory factor X1 (RFX1) in macrophage polarization by constructing colitis and lupus-like mouse models. Both in vivo and in vitro experiments confirmed that RFX1 can promote M1 and inhibit M2 macrophage polarization. Furthermore, we found that RFX1 promoted DNA demethylation of macrophage polarization-related genes by increasing APOBEC3A/Apobec3 expression. We identified a potential RFX1 inhibitor, adenosine diphosphate (ADP), providing a potential strategy for treating autoimmune diseases.

Macrophage: Key player in the pathogenesis of autoimmune diseases.

The macrophage is an essential part of the innate immune system and also serves as the bridge between innate immunity and adaptive immune response. As the initiator and executor of the adaptive immune response, macrophage plays an important role in various physiological processes such as immune tolerance, fibrosis, inflammatory response, angiogenesis and phagocytosis of apoptotic cells. Consequently, macrophage dysfunction is a vital cause of the occurrence and development of autoimmune diseases. In this review, we mainly discuss the functions of macrophages in autoimmune diseases, especially in systemic lupus erythematosus (SLE), rheumatic arthritis (RA), systemic sclerosis (SSc) and type 1 diabetes (T1D), providing references for the treatment and prevention of autoimmune diseases.

Economic evaluation of FLOT and ECF/ECX perioperative chemotherapy in patients with resectable gastric or gastro-oesophageal junction adenocarcinoma.

OBJECTIVE: The perioperative chemotherapy with fluorouracil, leucovorin, oxaliplatin plus docetaxel (FLOT) was recommended by the Chinese Society of Clinical Oncology Guidelines for gastric cancer (2018 edition) for patients with resectable gastric or gastro-oesophageal junction adenocarcinoma (class IIA). However, the economic impact of FLOT chemotherapy in China remains unclear. The analysis aimed to compare the cost-effectiveness of FLOT versus epirubicin, cisplatin plus fluorouracil or capecitabine (ECF/ECX) in patients with locally advanced resectable tumours. DESIGN: We developed a Markov model to compare the healthcare and economic outcomes of FLOT and ECF/ECX in patients with resectable gastric or gastro-oesophageal junction adenocarcinoma. Costs were estimated from the perspective of Chinese healthcare system. Clinical and utility inputs were derived from the FLOT4 phase II/III clinical trial and published literature. Sensitivity analyses were employed to assess the robustness of our result. The annual discount rate for costs and health outcomes was set at 5%. OUTCOME MEASURES: The primary outcome of incremental cost-effectiveness ratios (ICERs) was calculated as the cost per quality-adjusted life years (QALYs). RESULTS: The base-case analysis found that compared with ECF/ECX, the use of FLOT chemotherapy was associated with an additional 1.08 QALYs, resulting in an ICER of US$851/QALY. One-way sensitivity analysis results suggested that the HR of overall survival and progression-free survival had the greatest impact on the ICER. Probabilistic sensitivity analysis demonstrated that FLOT was more likely to be cost-effective compared with ECF/ECX at a willingness-to-pay threshold of US$31 513/QALY. CONCLUSIONS: For patients with locally advanced resectable tumours, the FLOT chemotherapy is a cost-effective treatment option compared with ECF/ECX in China. TRIAL REGISTRATION NUMBER: NCT01216644.

Iron-dependent epigenetic modulation promotes pathogenic T cell differentiation in lupus.

The trace element iron affects immune responses and vaccination, but knowledge of its role in autoimmune diseases is limited. Expansion of pathogenic T cells, especially T follicular helper (Tfh) cells, has great significance to systemic lupus erythematosus (SLE) pathogenesis. Here, we show an important role of iron in regulation of pathogenic T cell differentiation in SLE. We found that iron overload promoted Tfh cell expansion, proinflammatory cytokine secretion, and autoantibody production in lupus-prone mice. Mice treated with a high-iron diet exhibited an increased proportion of Tfh cell and antigen-specific GC response. Iron supplementation contributed to Tfh cell differentiation. In contrast, iron chelation inhibited Tfh cell differentiation. We demonstrated that the miR-21/BDH2 axis drove iron accumulation during Tfh cell differentiation and further promoted Fe2+-dependent TET enzyme activity and BCL6 gene demethylation. Thus, maintaining iron homeostasis might be critical for eliminating pathogenic Th cells and might help improve the management of patients with SLE.

Insufficient Iron Improves Pristane-Induced Lupus by Promoting Treg Cell Expansion.

Trace element iron affects T cell biology, but the knowledge about the role of iron in regulating Treg cell expansion is limited. Treg cells play an important role in keeping peripheral T cell tolerance, increasing Treg cell expansion is a promising therapeutic method for SLE. Here we showed that iron deficiency promotes Treg cell expansion by reducing ROS accumulation, improving the disease progression of pristane-induced lupus. Increased oxidative stress inhibits Treg cell differentiation by inducing cell apoptosis. Our data suggest that altering iron metabolism promotes Treg cell expansion by preventing oxidation-induced cell death, which may provide a potential therapeutic strategy for SLE.

Sulforaphane Ameliorates the Severity of Psoriasis and SLE by Modulating Effector Cells and Reducing Oxidative Stress.

Background: Sulforaphane, which is found in cruciferous vegetables, has been reported to have anti-inflammatory, antioxidant, and antitumour activities. However, whether sulforaphane has therapeutic effects on inflammatory or autoimmune skin diseases, including psoriasis and systemic lupus erythematosus (SLE), is unclear. Methods: The therapeutic effects of sulforaphane were analyzed in Imiquimod (IMQ)-induced psoriasis-like mice and lupus-prone MRL/lpr mice. In IMQ-induced psoriasis-like mice treated with sulforaphane (55.3 and 110.6 µmol/kg) or vehicle control, the pathological phenotypes were assessed by the psoriasis area and severity index (PASI) score, haematoxylin-eosin staining (H&E) and quantifying of acanthosis and dermal inflammatory cell infiltration. The proportions of T cell subsets in draining lymph nodes (dLNs) and spleens were examined by flow cytometry. In MRL/lpr mice treated with sulforaphane (82.9 µmol/kg) or vehicle control, mortality and proteinuria were observed, and the glomerular pathology was examined by H&E staining. C3 and IgG depositions in kidney sections were examined by immunofluorescence staining. The proportions of plasma cells, follicular helper T (Tfh) cells, neutrophils and dendritic cells in the dLNs and spleens were examined by flow cytometry. Finally, we examined the Malondialdehyde (MDA) concentration by thiobarbituric acid reactive substance assay and the expression of Prdx1, Nqo1, Hmox1, and Gss by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Results: Sulforaphane ameliorated the skin lesions in IMQ-induced psoriasis-like mice and the renal damage in lupus-prone MRL/lpr mice. In IMQ-induced psoriasis-like mice, sulforaphane reduced the proportions of Th1 and Th17 cells and increased the expression of antioxidant gene Prdx1. In lupus-prone MRL/lpr mice, sulforaphane increased the lifespan and the expression of Prdx1, and decreased the proportions of plasma cells, Tfh cells, neutrophils, and dendritic cells in the dLNs and spleens and the concentration of MDA. Conclusion: Sulforaphane has significant therapeutic effects on IMQ-induced psoriasis-like mice and lupus-like MRL/Lpr mice by reducing inflammatory and autoimmune-related cells and oxidative stress. These findings provide new evidence for developing natural products to treat inflammatory and autoimmune diseases.

Screening for Active Compounds Targeting Human Natural Killer Cell Activation Identifying Daphnetin as an Enhancer for IFN-γ Production and Direct Cytotoxicity.

Natural killer (NK) cells are a potent weapon against tumor and viral infection. Finding active compounds with the capacity of enhancing NK cell effector functions will be effective to develop new anti-cancer drugs. In this study, we initially screened 287 commercially available active compounds by co-culturing with peripheral blood mononuclear cells (PBMCs). We found that five compounds, namely, Daphnetin, MK-8617, LW6, JIB-04, and IOX1, increased the IFN-γ+ NK cell ratio in the presence of IL-12. Further studies using purified human primary NK cells revealed that Daphnetin directly promoted NK cell IFN-γ production in the presence of IL-12 but not IL-15, while the other four compounds acted on NK cells indirectly. Daphnetin also improved the direct cytotoxicity of NK cells against tumor cells in the presence of IL-12. Through RNA-sequencing, we found that PI3K-Akt-mTOR signaling acted as a central pathway in Daphnetin-mediated NK cell activation in the presence of IL-12. This was further confirmed by the finding that both inhibitors of PI3K-Akt and its main downstream signaling mTOR, LY294002, and rapamycin, respectively, can reverse the increase of IFN-γ production and cytotoxicity in NK cells promoted by Daphnetin. Collectively, we identify a natural product, Daphnetin, with the capacity of promoting human NK cell activation via PI3K-Akt-mTOR signaling in the presence of IL-12. Our current study opens up a new potential application for Daphnetin as a complementary immunomodulator for cancer treatments.

UHRF1 downregulation promotes T follicular helper cell differentiation by increasing BCL6 expression in SLE.

BACKGROUND: Transcription factor B cell lymphoma 6 (BCL6) is a master regulator of T follicular helper (Tfh) cells, which play a crucial role in the pathogenesis of systemic lupus erythematosus (SLE). However, the mechanisms by which BCL6 expression is regulated are poorly understood. Ubiquitin-like with PHD and RING finger domains 1 (UHRF1) is an important epigenetic factor that regulates DNA methylation and histone modifications. In the present study, we assessed whether UHRF1 can regulate BCL6 expression and influence the differentiation and proliferation of Tfh cells. RESULTS: Compared to healthy controls, the mean fluorescence intensity of UHRF1 (UHRF1-MFI) in Tfh cells from SLE patients was significantly downregulated, whereas that of BCL6 (BCL6-MFI) was significantly upregulated. In vitro, UHRF1 knockdown led to BCL6 overexpression and promoted Tfh cell differentiation. In contrast, UHRF1 overexpression led to BCL6 downregulation and decreased Tfh cell differentiation. In vivo, conditional UHRF1 gene knockout (UHRF1-cKO) in mouse T cells revealed that UHRF1 depletion can enhance the proportion of Tfh cells and induce an augmented GC reaction in mice treated with NP-keyhole limpet hemocyanin (NP-KLH). Mechanistically, UHRF1 downregulation can decrease DNA methylation and H3K27 trimethylation (H3K27me3) levels in the BCL6 promoter region of Tfh cells. CONCLUSIONS: Our results demonstrated that UHRF1 downregulation leads to increased BCL6 expression by decreasing DNA methylation and H3K27me3 levels, promoting Tfh cell differentiation in vitro and in vivo. This finding reveals the role of UHRF1 in regulating Tfh cell differentiation and provides a potential target for SLE therapy.

Bazedoxifene inhibits PDGF-BB induced VSMC phenotypic switch via regulating the autophagy level.

There is increasing evidence that Bazedoxifene, as an FDA-approved selective estrogen inhibitor, approved by FDA, not only inhibits estrogen receptors, but also has other pharmacological effects. The purpose of this study was to investigate the effects of Bazedoxifene on the functional changes of vascular smooth muscle cells (VSMCs) after PDGF-BB stimulation. VSMCs were divided into control group, PDGF-BB treatment group, and PDGF-BB treatment group with different concentrations of Bazedoxifene. CCK-8 and EdU staining were used to determine the VSMCs viability and proliferation. Western blot was used to detect the expressions of vimentin, SMA, ERK, p-ERK, STAT3, p-STAT3, AKT, p-AKT, and LC3 I/II. Wound healing method was used to detect the migration of VSMCs. PDGF-BB treatment significantly enhanced the viability and proliferation of VSMCs as indicated by CCK-8 and EdU assays (P < 0.01), while Bazedoxifene pretreatment could reduce the increased viability and proliferation of VSMCs caused by PDGF-BB (P < 0.05). Wound healing test also showed Bazedoxifene significantly attenuated the migration in the PDGF-BB stimulated VSMCs (P < 0.01). PDGF-BB also induced the phenotypic switch and decreased the autophagy level in VSMCs, manifested as a reduction in vimentin, SMA, and LC3 II (P < 0.01). These effects of PDGF-BB were partially reversed by Bazedoxifene (P < 0.05). Bazedoxifene may inhibit the proliferation and migration of VSMCs through up-regulate the autophagy level after PDGF-BB stimulation.

Tanshinone â ¡A inhibits homocysteine-induced proliferation of vascular smooth muscle cells via miR-145/CD40 signaling.

Tanshinone IIA (Tan IIA), isolated from the traditional Chinese herb Danshen, exhibits broad cardiovascular protective effects. However, the effect of Tan IIA on Homocysteine (Hcy)-induced proliferation of vascular smooth muscle cells (VSMCs) remains unknown. We herein determined whether Tan IIA exerted anti-proliferative effect in Hcy-treating VSMCs, and further investigated the underlying mechanism (miR-145/CD40 signaling). The results showed that Tan IIA significantly inhibited VSMCs proliferation induced by Hcy in a dose-dependent manner, and reversed the VSMCs injury as indicated by decreased KLF4 and increased Calponin expression. In view of the key role of miR-145 in VSMCs, we further explored the role of miR-145 on the protective effect of Tan IIA against Hcy-induced VSMCs proliferation. The miR-145 expression was down-regulated and its targeted gene CD40 was up-regulated in Hcy-treating VSMCs, while the Tan IIA reversed the effect of Hcy, suggesting the miR-145/CD40 may be involve in the protective effect of Tan IIA. To determine the speculation, miR-145 inhibitor was used to inhibit miR-145 expression. The results indicated that miR-145 inhibitor can suppress the protective effects of Tan IIA against Hcy-induced VSMCs proliferation. Collectively, present study demonstrates that Tan IIA inhibits Hcy-induced proliferation of VSMCs via miR-145/CD40 signaling.

The miR-145 rs353291 C allele increases susceptibility to atherosclerosis.

Atherosclerosis (AS) is one of the leading causes of death, with variable frequencies in different populations. To this end, we examined whether AS is associated with the miR-145 rs353291 polymorphism and specific rs353291 alleles. The distribution of the rs353291 C allele and CC genotypes was significantly higher in AS patients than in controls. Compared to individuals with the rs353291 TT genotype, carriers of the rs353291 C allele showed a significant increase in the plasma levels of sCD40L, IL-6 and MCP-1 and a decrease in miR-145 expression. After ox-LDL administration, the peripheral blood mononuclear cells (PBMCs) of rs353291 C allele carriers showed significantly increased CD40 and MCP-1 expression compared with those of individuals with the rs353291 TT genotype. These data suggest that the rs353291 C allele may increase susceptibility to atherosclerosis.

Reasonable drug analysis of Listeria monocytogenes meningitis related to mantle cell lymphoma.

We report a case of Listeria meningitis related to mantle cell lymphoma. A clinical pharmacist adjusted repeatedly the patient's anti-infective therapeutic regimen by analyzing the pharmacologic and pharmacokinetic characteristics of antibacterial drugs (such as cefotaxime, meropenem, etc.) due to the patient's repeated fever during hospitalization. To the best of our knowledge, this is the first case of Listeria meningitis related to mantle cell lymphoma treated successfully with meropenem reported in China. This case aims to optimize the anti-infection treatment regimen of Listeria meningitis and to provide a reference for clinicians and clinical pharmacists to use drugs rationally.

Downregulation of BDH2 modulates iron homeostasis and promotes DNA demethylation in CD4+ T cells of systemic lupus erythematosus.

DNA hypomethylation plays an important role in the pathogenesis of systemic lupus erythematosus (SLE). Here we investigated whether 3-hydroxy butyrate dehydrogenase 2 (BDH2), a modulator of intracellular iron homeostasis, was involved in regulating DNA hypomethylation and hyper-hydroxymethylation in lupus CD4+ T cells. Our results showed that BDH2 expression was decreased, intracellular iron was increased, global DNA hydroxymethylation level was elevated, while methylation level was reduced in lupus CD4+ T cells compared with healthy controls. The decreased BDH2 contributed to DNA hyper-hydroxymethylation and hypomethylation via increasing intracellular iron in CD4+ T cells, which led to overexpression of immune related genes. Moreover, we showed that BDH2 was the target gene of miR-21. miR-21 promoted DNA demethylation in CD4+ T cells through inhibiting BDH2 expression. Our data demonstrated that the dysregulation of iron homeostasis in CD4+ T cells induced by BDH2 deficiency contributes to DNA demethylation and self-reactive T cells in SLE.

Down-regulation of MBD4 contributes to hypomethylation and overexpression of CD70 in CD4+ T cells in systemic lupus erythematosus.

BACKGROUND: Systemic lupus erythematosus (SLE) is an autoimmune disease that is characterized by lymphocytic infiltration and overproduction of autoantibodies, leading to significant morbidity and mortality. However, the pathogenesis of this disorder has not yet been completely elucidated. It has been reported that CD70, a B cell costimulatory molecule encoded by the gene TNFSF7 (tumor necrosis factor ligand superfamily member 7), is overexpressed in CD4+ T cells from patients with SLE due to the demethylation of its promoter. We aimed to investigate the expression patterns of MBD4 (methyl-CpG binding domain protein 4) in CD4+ T cells and its contribution to the pathogenesis of SLE by increasing CD70 expression through epigenetic regulation. RESULTS: Our results showed that the expression of MBD4 was significantly decreased in CD4+ T cells from SLE patients. We verified that transfection of MBD4 siRNA into healthy CD4+ T cells upregulated expression of CD70 and decreased the methylation level of the CD70 promoter. Overexpression of MBD4 inhibited CD70 expression and enhanced the DNA methylation level of CD70 in CD4+ T cells of SLE patients. CONCLUSION: Our results indicated that downregulation of MBD4 contributed to overexpression and hypomethylation of the CD70 gene in SLE CD4+ T cells. This modulation of MBD4 may provide a novel therapeutic approach for SLE.

Epigenetic regulation in monocyte/macrophage: A key player during atherosclerosis.

Atherosclerosis is a chronic inflammatory disease. Recently, a growing body of evidence emphasizes that the monocyte and macrophage differentiation and activation are key processes in the development of atherosclerosis. However, the regulatory mechanism that manipulates the function of monocyte and macrophage is still unclear. Recent years, epigenetic mechanisms have received a wide attention and bring us a new field of vision. More and more evidence shows that epigenetics weighs heavily in atherosclerosis by regulating the function and differentiation states of monocyte and macrophage. In this review, we illuminate the epigenetic regulation mechanisms in monocyte and macrophage and their contributions to inflammatory processes of atherosclerosis to provide new thoughts and find novel targets or biomarkers for atherosclerosis.

Epigallocatechin-3-gallate inhibits homocysteine-induced apoptosis of endothelial cells by demethylation of the DDAH2 gene.

Our previous study showed that hypermethylation of dimethylarginine dimethylaminohydrolase 2 contributes to homocysteine-induced apoptosis of human umbilical vein endothelial cells. Epigallocatechin-3-gallate is a green tea-derived phenol which has been proved beneficial on atherosclerosis. It was demonstrated that epigallocatechin-3-gallate inhibits DNA methyltransferase activity and reactivates methylation-silenced genes in cancer cells. The aim of this study was to address whether epigallocatechin-3-gallate could induce DNA demethylation of the dimethylarginine dimethylaminohydrolase 2 gene, contributing to prevent endothelial cells from apoptosis induced by homocysteine. Human umbilical vein endothelial cells (ATCC, CRL-2480) were treated with homocysteine (1 mM) for 48 hours with or without epigallocatechin-3-gallate (20 µM) or 5-Aza (DNA methyltransferase inhibitor, 5 µM). Apoptosis rate of human umbilical vein endothelial cells was assayed by flow cytometry with an annexin V-FITC apoptosis detection kit. The mRNA and protein expression level of dimethylarginine dimethylaminohydrolase 2 and DNA methyltransferase 1 were detected by real-time PCR and Western blot, respectively. DNA methylation level of dimethylarginine dimethylaminohydrolase 2 was assayed by methylation specific PCR. The binding level of DNA methyltransferase 1 in the promoter of dimethylarginine dimethylaminohydrolase 2 was determined by chromatin immunoprecipitation-quantitative real-time PCR. It was shown that the apoptosis rate was decreased significantly in human umbilical vein endothelial cells treated with homocysteine compared with the control. Furthermore, the mRNA and protein level of dimethylarginine dimethylaminohydrolase 2 were downregulated, the dimethylarginine dimethylaminohydrolase 2 gene promoter was hypermethylated, and the DNA methyltransferase 1 mRNA and protein level were increased in human umbilical vein endothelial cells treated with homocysteine. Chromatin immunoprecipitation-quantitative real-time PCR revealed that homocysteine-induced binding of DNA methyltransferase 1 to the dimethylarginine dimethylaminohydrolase 2 promoter was increased. Pretreatment with epigallocatechin-3-gallate or 5-Aza inhibited such effects of homocysteine. In conclusion, epigallocatechin-3-gallate exerted protective effects on homocysteine-induced apoptosis in human umbilical vein endothelial cells by inhibiting promoter hypermethylation of the dimethylarginine dimethylaminohydrolase 2 gene and inducing dimethylarginine dimethylaminohydrolase 2 expression. These effects may be due to the decreased DNA methyltransferase 1 expression and binding of DNA methyltransferase 1 to the dimethylarginine dimethylaminohydrolase 2 promoter induced by epigallocatechin-3-gallate. This research suggests that modulating the epigenetic processes might be a novel plausible way for treatment of atherosclerosis.

Homocysteine-induced hypermethylation of DDAH2 promoter contributes to apoptosis of endothelial cells.

Homocysteine (Hcy) could induce apoptosis of endothelial cells (ECs). Dimethylarginine dimethylaminohydrolase 2 (DDAH2) is recognized as a protective factor to improve the endothelial function. Defect of DDAH2 has been confirmed to be involved in the Hcy-induced dysfunction of endothelial NO system. This study was to determine whether Hcy could inhibit DDAH2 expression and induce apoptosis of ECs via increasing DNA methylation level of DDAH2 promoter and whether DNA methylation inhibitor 5-azacytidine (5-aza) could attenuate the effect of Hcy on ECs. Firstly, human umbilical vein endothelial cells (HUVECs) were treated by Hcy with or without 5-aza for 48 h. MTT assay showed that Hcy reduced the viability of HUVECs in a dose-dependent manner. The level of asymmetric dimethylarginine (ADMA) and the apoptosis rate of HUVECs treated with Hcy at 1.0 mM were increased significantly compared with that of control. Moreover, we found that mRNA level of DDAH2 was down-regulated and DNA methylation level of DDAH2 promoter was increased significantly in HUVECs treated with Hcy, in concomitance with up-regulated protein level of DNA methyltransferase 1 (DNMT1). Furthermore, we also found that 5-aza could neutralize the effect of Hcy on ECs through up-regulating mRNA level of DDAH2 and inducing demethylation of DDAH2 promoter. In conclusion, hypermethylation of DDAH2 contributes to Hcy induced apoptosis of ECs. Modulating DNA methylation status of DDAH2 promoter may be a potential strategy for treatment of endothelial dysfunction.

Formation and function of ceramide-enriched membrane platforms with CD38 during M1-receptor stimulation in bovine coronary arterial myocytes.

CD38 contains an ADP ribosylcyclase domain that mediates intracellular Ca(2+) signaling by the production of cyclic ADP-ribose (cADPR), but the mechanisms by which the agonists activate this enzyme remain unclear. The present study tested a hypothesis that a special lipid-raft (LR) form, ceramide-enriched lipid platform, contributes to CD38 activation to produce cADPR in response to muscarinic type 1 (M(1)) receptor stimulation in bovine coronary arterial myocytes (CAMs). By confocal microscopic analysis, oxotremorine (Oxo), an M(1) receptor agonist, was found to increase LR clustering on the membrane with the formation of a complex of CD38 and LR components such as GM(1), acid sphingomyelinase (ASMase), and ceramide, a typical ceramide-enriched macrodomain. At 80 microM, Oxo increased LR clustering by 78.8%, which was abolished by LR disruptors, methyl-beta-cyclodextrin (MCD), or filipin. With the use of a fluorescence resonance energy transfer (FRET) technique, 15.5+/-1.9% energy transfer rate (vs. 5.3+/-0.9% of control) between CD38 and LR component, ganglioside M(1) was detected, further confirming the proximity of both molecules. In the presence of MCD or filipin, there were no FRET signals detected. In floated detergent-resistant membrane fractions, CD38 significantly increased in LR fractions of CAMs treated by Oxo. Moreover, MCD or filipin attenuated Oxo-induced production of cADPR via CD38. Functionally, Oxo-induced intracellular Ca(2+) release and coronary artery constriction via cADPR were also blocked by LR disruption or ASMase inhibition. These results provide the first evidence that the formation of ceramide-enriched lipid macrodomains is crucial for Oxo-induced activation of CD38 to produce cADPR in CAMs, and these lipid macrodomains mediate transmembrane signaling of M(1) receptor activation to produce second messenger cADPR.