Macrophage KLF15 prevents foam cell formation and atherosclerosis via transcriptional suppression of OLR-1.

BACKGROUND: Macrophage-derived foam cells are a hallmark of atherosclerosis. Scavenger receptors, including lectin-like oxidized low-density lipoprotein (LDL) receptor-1 (OLR-1), are the principal receptors responsible for the uptake and modification of LDL, facilitating macrophage lipid load and the uptake of oxidized LDL by arterial wall cells. Krüppel-like factor 15 (KLF15) is a transcription factor that regulates the expression of genes by binding to the promoter during transcription. Therefore, this study aimed to investigate the precise role of macrophage KLF15 in atherogenesis. METHODS: We used two murine models of atherosclerosis: mice injected with an adeno-associated virus (AAV) encoding the Asp374-to-Tyr mutant version of human PCSK9, followed by 12 weeks on a high-fat diet (HFD), and ApoE-/-- mice on a HFD. We subsequently injected mice with AAV-KLF15 and AAV-LacZ to assess the role of KLF15 in the development of atherosclerosis in vivo. Oil Red O, H&E, and Masson's trichome staining were used to evaluate atherosclerotic lesions. Western blots and RT-qPCR were used to assess protein and mRNA levels, respectively. RESULTS: We determined that KLF15 expression was downregulated during atherosclerosis formation, and KLF15 overexpression prevented atherosclerosis progression. KLF15 expression levels did not affect body weight or serum lipid levels in mice. However, KLF15 overexpression in macrophages prevented foam cell formation by reducing OLR-1-meditated lipid uptake. KLF15 directly targeted and transcriptionally downregulated OLR-1 levels. Restoration of OLR-1 reversed the beneficial effects of KLF15 in atherosclerosis. CONCLUSION: Macrophage KLF15 transcriptionally downregulated OLR-1 expression to reduce lipid uptake, thereby preventing foam cell formation and atherosclerosis. Thus, our results suggest that KLF15 is a potential therapeutic target for atherosclerosis.

Diterbutyl phthalate attenuates osteoarthritis in ACLT mice via suppressing ERK/c-fos/NFATc1 pathway, and subsequently inhibiting subchondral osteoclast fusion.

Osteoarthritis (OA) is the most common arthritis with a rapidly increasing prevalence. Disease progression is irreversible, and there is no curative therapy available. During OA onset, abnormal mechanical loading leads to excessive osteoclastogenesis and bone resorption in subchondral bone, causing a rapid subchondral bone turnover, cyst formation, sclerosis, and finally, articular cartilage degeneration. Moreover, osteoclast-mediated angiogenesis and sensory innervation in subchondral bone result in abnormal vascularization and OA pain. The traditional Chinese medicine Panax notoginseng (PN; Sanqi) has long been used in treatment of bone diseases including osteoporosis, bone fracture, and OA. In this study we established two-dimensional/bone marrow mononuclear cell/cell membrane chromatography/time of flight mass spectrometry (2D/BMMC/CMC/TOFMS) technique and discovered that diterbutyl phthalate (DP) was the active constituent in PN inhibiting osteoclastogenesis. Then we explored the therapeutic effect of DP in an OA mouse model with anterior cruciate ligament transaction (ACLT). After ACLT was conducted, the mice received DP (5 mg·kg-1·d-1, ip) for 8 weeks. Whole knee joint tissues of the right limb were harvested at weeks 2, 4, and 8 for analysis. We showed that DP administration impeded overactivated osteoclastogenesis in subchondral bone and ameliorated articular cartilage deterioration. DP administration blunted aberrant H-type vessel formation in subchondral bone marrow and alleviated OA pain assessed in Von Frey test and thermal plantar test. In RANKL-induced RAW264.7 cells in vitro, DP (20 µM) retarded osteoclastogenesis by suppressing osteoclast fusion through inhibition of the ERK/c-fos/NFATc1 pathway. DP treatment also downregulated the expression of dendritic cell-specific transmembrane protein (DC-STAMP) and d2 isoform of the vacuolar (H+) ATPase V0 domain (Atp6v0d2) in the cells. In conclusion, we demonstrate that DP prevents OA progression by inhibiting abnormal osteoclastogenesis and associated angiogenesis and neurogenesis in subchondral bone.

Macrophage NFATc3 prevents foam cell formation and atherosclerosis: evidence and mechanisms.

AIMS: Our previous study demonstrated that Ca2+ influx through the Orai1 store-operated Ca2+ channel in macrophages contributes to foam cell formation and atherosclerosis via the calcineurin-ASK1 pathway, not the classical calcineurin-nuclear factor of activated T-cell (NFAT) pathway. Moreover, up-regulation of NFATc3 in macrophages inhibits foam cell formation, suggesting that macrophage NFATc3 is a negative regulator of atherogenesis. Hence, this study investigated the precise role of macrophage NFATc3 in atherogenesis. METHODS AND RESULTS: Macrophage-specific NFATc3 knockout mice were generated to determine the effect of NFATc3 on atherosclerosis in a mouse model of adeno-associated virus-mutant PCSK9-induced atherosclerosis. NFATc3 expression was decreased in macrophages within human and mouse atherosclerotic lesions. Moreover, NFATc3 levels in peripheral blood mononuclear cells from atherosclerotic patients were negatively associated with plaque instability. Furthermore, macrophage-specific ablation of NFATc3 in mice led to the atherosclerotic plaque formation, whereas macrophage-specific NFATc3 transgenic mice exhibited the opposite phenotype. NFATc3 deficiency in macrophages promoted foam cell formation by potentiating SR-A- and CD36-meditated lipid uptake. NFATc3 directly targeted and transcriptionally up-regulated miR-204 levels. Mature miR-204-5p suppressed SR-A expression via canonical regulation. Unexpectedly, miR-204-3p localized in the nucleus and inhibited CD36 transcription. Restoration of miR-204 abolished the proatherogenic phenotype observed in the macrophage-specific NFATc3 knockout mice, and blockade of miR-204 function reversed the beneficial effects of NFATc3 in macrophages. CONCLUSION: Macrophage NFATc3 up-regulates miR-204 to reduce SR-A and CD36 levels, thereby preventing foam cell formation and atherosclerosis, indicating that the NFATc3/miR-204 axis may be a potential therapeutic target against atherosclerosis.

Reduced intracellular chloride concentration impairs angiogenesis by inhibiting oxidative stress-mediated VEGFR2 activation.

Chloride (Cl-) homeostasis is of great significance in cardiovascular system. Serum Cl- level is inversely associated with the mortality of patients with heart failure. Considering the importance of angiogenesis in the progress of heart failure, this study aims to investigate whether and how reduced intracellular Cl- concentration ([Cl-]i) affects angiogenesis. Human umbilical endothelial cells (HUVECs) were treated with normal Cl- medium or low Cl- medium. We showed that reduction of [Cl-]i (from 33.2 to 16.18 mM) inhibited HUVEC proliferation, migration, cytoskeleton reorganization, tube formation, and subsequently suppressed angiogenesis under basal condition, and VEGF stimulation or hypoxia treatment. Moreover, VEGF-induced NADPH-mediated reactive oxygen species (ROS) generation and VEGFR2 axis activation were markedly attenuated in low Cl- medium. We revealed that lowering [Cl-]i inhibited the expression of the membrane-bound catalytic subunits of NADPH, i.e., p22phox and Nox2, and blunted the translocation of cytosolic regulatory subunits p47phox and p67phox, thereby restricting NADPH oxidase complex formation and activation. Furthermore, reduced [Cl-]i enhanced ROS-associated protein tyrosine phosphatase 1B (PTP1B) activity and increased the interaction of VEGFR2 and PTP1B. Pharmacological inhibition of PTP1B reversed the effect of lowering [Cl-]i on VEGFR2 phosphorylation and angiogenesis. In mouse hind limb ischemia model, blockade of Cl- efflux using Cl- channel inhibitors DIDS or DCPIB (10 mg/kg, i.m., every other day for 2 weeks) significantly enhanced blood flow recovery and new capillaries formation. In conclusion, decrease of [Cl-]i suppresses angiogenesis via inhibiting oxidase stress-mediated VEGFR2 signaling activation by preventing NADPH oxidase complex formation and promoting VEGFR2/PTP1B association, suggesting that modulation of [Cl-]i may be a novel therapeutic avenue for the treatment of angiogenic dysfunction-associated diseases.

Synthesis, Anti-inflammatory Activities and Mechanisms of 3,5- dihydroxycinnamic Acid Derivatives.

BACKGROUND: 3,4-dihydroxycinnamic acid and its derivatives exhibit numerous biologic activities. Such activities have not previously been reported for 3,5-dihydroxycinnamic acid derivatives. In this study, ten derivatives of 3,5- dihydroxycinnamic acid were synthesized and their anti-inflammatory activities were tested in 12-O-tetradecanoylphorbol 13-acetate-induced mouse ear edema. Molecular biological studies have shed lights on their anti-inflammatory mechanism. METHODS: Anti-inflammatory activities of ten new synthesized derivatives of 3,5-dihydroxycinnamic acid were tested in 12-O-tetradecanoylphorbol 13-acetate-induced mouse ear edema, and their anti-inflammatory mechanism was studied by ELISA, real-time RT-PCR, MPO assay and AA-induced mouse ear edema. RESULTS: Compound 7 showed a pronounced anti-inflammatory effect and the inhibition rate was 65.6% at a dose of 1.6mg/ear. This compound acted by reducing mRNA and protein synthesis of tumor necrosis factor-α, interleukins 1ß and 6, and also by decreasing the levels of activated neutrophil infiltrates. Furthermore, compound 7 significantly suppressed arachidonic acid-induced edema as well. Cell-based assays showed that compound 7 inhibited the production of cyclooxygenase- 2-catalyzed prostaglandin E2 from lipopolysaccharide-treated RAW 264.7 cells, and also inhibited 5-lipoxygenase production from A23187-treated RBL-1 cells, and consequently reduced leukotriene B4 production. CONCLUSION: This investigation revealed that some of the derivatives of 3,5-dihydroxycinnamic acid exhibit a more pronounced anti-inflammatory effect than 3,4-dihydroxycinnamic acid. Therefore, 3,5-dihydroxycinnamic acid derivatives, especially compound 7, represent potential value for antiinflammatory drug development.

[Helicobacter pylori infection in the gastric mucosa of patients with HIV/AIDS in different clinical stages].

OBJECTIVE: To analyze Helicobacter pylori infection in the gastric mucosa of patients with HIV/AIDS in different clinical stages. METHODS: This study involved 170 patients with HIV/AIDS and 34 HIV-negative patients. All the patients underwent upper endoscopy and antral gastric biopsy to determine the status of Helicobacter pylori infection using aniline red staining and rapid urease test. The patients with HIV/AIDS were stratified based on CD4(+)T lymphocyte counts and clinical setting into asymptomatic HIV infection (A1, A2) group, symptomatic HIV infection (B1, B2) group and AIDS (A3, B3, C1-3) group. RESULTS: The prevalence of Helicobacter pylori infection in HIV/AIDS patients was 16.5% (28/170), and in the 3 groups classified, the infection rates were 23.4% (11/47), 14.0% (8/57), and 13.6% (9/66), respectively; the infection rate was 47.1% (16/34) in the control group. Helicobacter pylori infection rate in the gastric mucosa of the patients with HIV/AIDS in different clinical stages was significantly lower than that of the control group (P<0.05); the infection rates in symptomatic HIV-infected (B1, B2) group and AIDS (A3, B3, C1-3) group were significantly lower than that in asymptomatic HIV-infected (A1, A2) group (P<0.05). CONCLUSION: The low Helicobacter pylori infection rate in HIV/AIDS patients may result from severe immunodeficiency in the gastric mucosa.

Tumor necrosis factor--alpha gene promoter polymorphisms in Chinese patients with nonalcoholic fatty liver diseases.

BACKGROUND: Nonalcoholic Fatty Liver Disease (NAFLD) encompasses a histopathological spectrum of clinical conditions such as simple fatty liver (steatosis), nonalcoholic steatohepatitis (NASH), and a variant that has degrees of fibrosis. Tumor Necrosis Factor-alpha (TNF-alphalpha) is considered essential for NAFLD. Therefore, we investigated the correlation between TNF-alphalpha gene promoter polymorphism and NAFLD in this human study. PATIENTS AND METHODS: The TNF-alpha gene polymorphisms at position -238 and -308 were analyzed in 189 Chinese patients with NAFLD and 138 healthy controls by using polymerase chain reaction and restriction fragment length polymorphism assay. The serum levels of TNF-alpha in both patient and control groups were measured by ELISA. The associations of TNF-alpha polymorphism and serum TNF-alpha, and/or insulin resistance, and/or clinical features were analyzed. RESULTS: The carrier frequencies of TNF-alpha gene polymorphism with G/A mutation at -238 were significantly higher in the patients with NAFLD than those in the control subjects (p < 0.05). However, there were no significant differences between the NAFLD patients and control subjects in the polymorphisms at -308 (p > 0.05). In addition, the serum level of TNF-alpha was markedly higher in the patients with NAFLD than in the control subjects (p < 0.05). There were significant associations between TNF-alpha gene polymorphism in the -238 A allele and increased serum TNF-alpha, insulin resistance, as well as increased body mass index in the NAFLD patients. CONCLUSIONS: The results indicate that the TNF-alpha gene polymorphism at position -238 is associated with susceptibility of nonalcoholic Fatty Liver Disease in a Chinese population.