Compared with the TGF-1 team of LX-two cells, LX-2 cells cultured with simvastatin by yourself or with equally simvastatin and TGF-1 experienced less iNOS, -SMA, and Collagen expressions (B, C, F~I) and far more eNOS expression (D, E). ( P<0.05, compared with the control group P<0.05, compared with LX-2 cells treated with TGF-1 ^ P<0.05, compared with LX-2 cells treated with simvastatin.).Figure 6. 9004-82-4 chemical informationRepresentative graphs and bar charts of the mRNA and protein expressions of iNOS, eNOS, -SMA, and Collagen in LX-2 cells cultured with L-NAME and simvastatin. L-NAME served as a NOS inhibitor, it inhibited both iNOS and eNOS expression (A~E) and induced more -SMA, and Collagen expressions (F~I). Simvastatin had antagonistic effect on the activation of LX-2 cells induced by L-NAME via increasing the eNOS expression (D, E). ( P<0.05, compared with the control group P<0.05, compared with LX-2 cells treated with L-NAME ^ P<0.05, compared with LX-2 cells treated with simvastatin.).enhanced in rheumatoid arthritis synovial fibroblasts and simvastatin could increase SIRT1 expression, indicating that simvastatin might be beneficial to inflammatory arthritis through its up-regulation of SIRT1/FoxO3a signaling in synovial fibroblasts [34]. In addition, with the progress of NAFLD, microRNA-34a, apoptosis and acetylated p53 were found to be increased gradually in the hepatic tissues, while SIRT1 decreased [35]. Therefore, statins, particularly simvastatin, might play a crucial role in ameliorating the progression of NAFLD, such as hepatic steatosis, inflammation and fibrosis, via regulating microRNA-34a/Sirtuin-1 pathway. Recent studies have reported that simvastatin decreased hepatic venous pressure gradient and improved liver perfusion in patients with cirrhosis [36]. And simvastatin attenuated pressure response to volume expansion and improved the vascular disturbances contribute to portal hypertension by selectively increased eNOS expression and NO availability in cirrhotic liver [37]. In vitro, simvastatin inhibited the proliferation of HSC and decrease the expressions of collagen I, III, and IV, but the specific mechanism was unclear [20]. So whether this effect is associated with the regulation of NOS needs to elucidate. iNOS can be increased by many inflammatory agents. In our study, TGF-1 could activate the quiescent LX-2 cells by increasing the expression of iNOS and decreasing the expression of eNOS. L-NAME, with a dose of 100 , was used to inhibit the expression of both eNOS and iNOS, especially eNOS. And we observed that L-NAME activated HSC by decreasing the expression of eNOS and iNOS. Therefore, eNOS was considered to play a more important role than iNOS in the mechanism of HSC's activation. We found that simvastatin increased eNOS expression and decreased iNOS expression both in hepatic tissue and in cultured LX-2 cells. And simvastatin could attenuat L-NAME's effect on eNOS, increased eNOS expression, and maintained the low level expression of iNOS. Therefore, simvastatin inhibited the activation of quiescent LX-2 cells by regulating the NOS expression.Simvastatin ameliorates the progression of NASH-related hepatic fibrosis. It decreases the hepatic lipid deposition, attenuates hepatic inflammation, and inhibits the development of fibrosis by inhibiting the activation of HSC via modulating iNOS and eNOS. The beneficial effects of simvastatin should be further confirmed in long-term clinical trials for NASH-related fibrosis.Trophoblast stem cells differentiate into two cell types in humans, villous trophoblasts and extravillous trophoblasts (EVTs). Villous trophoblasts are composed of cytotrophoblasts and syncytiotrophoblasts. During intact placentation, the placenta is constructed under low oxygen (2% O2) and low glucose concentrations (1 mM) until 11 weeks of gestation [1,2,3,4]. Intra-placental oxygen tension is rapidly increased after 12 weeks because endovascular EVTs invade the uterine spiral arteries, replace endothelial cells, and participate in the degradation of tunica media smooth muscle cells [5]. This remodeling of the spiral arteries is essential for allowing proper placental perfusion to sustain fetal growth. Failed interstitial and endovascular trophoblast invasion may lead to inadequate transformation of the spiral arteries, resulting in preeclampsia or fetal growth restriction (FGR) [6,7,8]. However, the causes of invasion failure by EVTs in early human placenta are still unknown. Autophagy is an intracellular bulk degradation system through which cytoplasmic components are degraded in lysosomes, resulting in energy production [9]. It occurs ubiquitously at basal levels in all eukaryotic cells and contributes to the routine turnover of cytoplasmic components. The autophagy activation in response to environmental stress is required for cell survival in yeast as well as in mammals [10]. Hypoxia inducible factor (HIF) is well known to be a central transcription factor that enables adaptive responses to hypoxic stress under normal and pathological conditions by activating a large number of genes responsible for oxygen delivery, angiogenesis, cell proliferation, cell differentiation, and metabolism [11,12]. Phenotypic changes in EVTs, from a proliferative type to an invasive type, in the early placenta are well known to be regulated through HIF1 transcription factors, which suggests that HIF1a is a master regulator for EVT differentiation [13]. Recent reports have indicated that hypoxia can activate a lysosomal degradation pathway known as autophagy, which mediates both selective and bulk degradation of proteins, cytoplasmic content, and organelles. Regarding the correlation between autophagy and EVT functions, we previously reported that autophagy was essential for the invasion of EVTs and EVT-vascular remodeling under physiological hypoxia, which is necessary for the first stage of placentation in vitro and in vivo [14]. Autophagy also plays an increasingly recognized role in quality control during hypoxia by removing mitochondria that may otherwise become cytotoxic [15]. The HIF1a expression levels in the human placenta are high at 7 weeks of gestation when oxygen tension is low, and decrease at around 12 weeks of gestation when placental oxygen tension increases [16]. However, continuous exposure to hypoxia in the early stage of pregnancy has been shown to induce preeclampsialike symptoms in IL-10 knockout mice [17], suggesting that severe hypoxia itself could cause preeclampsia. During early-onset human preeclampsia, the placenta is exposed to severe hypoxia independently of intervillous maternal blood-oxygen tension, due to a loss of the placenta's ability to adapt to variations in oxygen tension [18]. Although we have reported that impairment of autophagy by soluble endoglin contributes to invasion failure under physiological hypoxia, it remains unclear how severe hypoxia, which is lower than physiological hypoxia, affects the functions in EVTs with or without autophagy. In this study we show that overexpression of HIF1a decreases the invasiveness of autophagy-deficient HTR8/SVneo cells by suppressing cellular adenosine triphosphate (ATP) levels. Autophagy-deficient HTR8/SVneo cells with overexpression of HIF1a also expressed purinergic receptor P2X ligand-gated ion channel 7 (P2RX7). Furthermore, ATP treatment recovered the invasive nature of autophagy-deficient HTR8/SVneo cells. These results suggest that autophagy supplies cellular energy for EVTs to protect them from HIF1a-induced energy depletion.Cells were subsequently stained with the LC3 antibody. The incidence of autophagy was estimated by quantifying the number of LC3 puncta within LC3-stained cells by manually counting five independent visual fields using a confocal microscope (LSM700, Carl Zeiss, Oberkochen, Germany). At least 5 cells per 40 high power fields were counted in ten randomly chosen fields, and these experiments were independently performed at least three times.An invasion assay was performed using a BD BioCoat Growth Factor Reduced Matrigel Invasion Chamber (354483, BD Biosciences, San Jose, CA, USA) according to the manufacturer's instructions. Cells were plated in the upper insert at 56104/well and incubated in DMEM with or without CoCl2 (250 mM). When HTR8/SVneo cells were treated with CoCl2, the expression of HIF-1a was increased in a dose-dependent manner (Figure S1a). To analyze the effect of severe hypoxia in HTR8/SVneo cells, 250 mM CoCl2, which has been shown to induce higher HIF-1a expression than under a 2% oxygen tension condition (Fig. 1a) without affecting proliferation (Fig. 2e), was used in our assay. These cells were incubated for 24 or 48 h, and then the upper surface of the membrane in each insert was gently scrubbed with a cotton swab to remove all of the non-invading cells. Cells on the under surfaces of the membranes were fixed in 100% methanol (131-01826, Wako Pure Chemical Industries Ltd., Osaka, Japan), and stained with 0.05% Toluidine Blue Solution (206-14555, Wako Pure Chemical Industries Ltd.). For each experiment, the number of cells in seven randomly chosen fields of each filter was estimated by manual counting, and these experiments were independently performed at least three times.CoCl2 (Fluka Biochemika Ltd., Buchs, Switzerland) was purchased from Fluka Biochemika Ltd.. Rpamycin (R8781, 100 or 500 nM), an activator of autophagy, and three-methyladenine (3-MA, 5 mM, M9281), an inhibitor of autophagy, were purchased from Sigma-Aldrich (St. Louis, MO, USA). The following antibodies (Ab) were used: rabbit polyclonal Ab for MAP1LC3B (PM036, MBL, Nagoya, Japan), rabbit monoclonal Ab for P2RX7 (ab109246, Abcam Inc., Cambridge, MA, USA), mouse monoclonal Ab for HIF1-a (H72320, BD Pharmingen, Franklin Lakes, NJ, USA) and mouse monoclonal Ab for a-tubulin (T8203, Sigma-Aldrich). The protease inhibitors E64d (4321-v Peptide Institute, Osaka, Japan) and pepstatin A (4397, Peptide Institute) were purchased from the Peptide Institute Inc.Cell migration assay was carried out using the cell migration assay system (CBA-100, Cell Biolabs, Inc., San Diego, CA, USA) equipped with 8-mm pore size of migration chambers. Methods used in this assay were similar to Matrigel invasion assay except that the transwell insert was not coated with Matrigel. After 48 h of incubation, migrated cell numbers were calculated. For each experiment, the number of cells in seven randomly chosen fields of each filter was estimated by manual counting, and these experiments were independently performed at least three times.The EVT cell lines HTR8/SVneo (a gift from Dr. Charles H. Graham, Department of Anatomy and Cell Biology, Queen's University, Ontario, Canada) and HchEpC1b were used in this study [19,20]. The constructed autophagy-deficient cell line, HTR8-ATG4BC74A mutant cells, and the control vector-infected cell line, HTR8-mStrawberry cells, were also used. The procedures for constructing the vectors were reported previously [21]. The expression of mStrawberry was confirmed by fluorescence microscopy. HTR8/SVneo cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 mg/ml streptomycin (15140, Life Technologies, Carlsbad, CA, USA) at 37uC in a 5% CO2 atmosphere. HchEpC1b cells were cultured in RPMI1640 supplemented with 10% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin. To mimic severe hypoxic conditions, cells were plated on a 35-mm dish at 26105 cells/dish, and, after 24 h, were cultured in medium containing CoCl2 (250 mM, Fluka Biochemika Ltd.) under a 5% CO2 atmosphere at 37uC.Cells were washed with cold PBS, harvested and lysed in a lysis buffer (25 mM HEPES-NaOH, 1 mM EDTA, 1 mM DTT, 400 mM NaCl, 1.5 mM MgCl2, 0.1% Triton X-100, 0.1% protease inhibitor cocktail (Sigma-Aldrich), and 0.1% PMSF (Sigma-Aldrich)). The lysates were centrifuged at 14000 rpm for 15 min at 4uC, and the protein concentration of the supernatant was determined with a BCA protein assay kit (Bio-Rad Laboratories Inc., Hercules, CA, USA) according to the manufacturer's instructions. Protein samples were mixed with a 26sample buffer (Wako Pure Chemical Industries Ltd.), and samples were heated at 95uC for 3 min. Equal amounts of protein were then subjected to 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes. The membranes were then incubated overnight at 4uC in a blocking buffer (tris buffered saline with 0.1% Tween 20 and 5% bovine serum albumin 5% BSA/TBST), before being washed with PBST and then incubated for 2 h at room temperature with the following antibodies: anti-LC3 (1:1000 MBL), anti-HIF1a (1:500 BD Pharmingen), and anti-a-tubulin (1:1000 SigmaAldrich, diluted with 5% BSA/TBST). Finally, they were incubated for 1 h with secondary antibodies labeled with For the quantitative analysis of MAP1LC3B (LC3), the cells were pretreated with the lysosomal protease inhibitors E64d (10 ng/ml) and pepstatin A (10 ng/ml) for 2 h to distinguish cytoplasmic LC3 puncta, and were then fixed with 4% paraformaldehyde-PBS [22]horseradish 2626444peroxidase-conjugated anti-mouse or anti-rabbit antibody (1:1000 Cell Signaling, Beverly, MA, USA) and visualized with an enhanced chemiluminescence detection system (ECL detection kit PIERCE, Rockford, IL, USA).Cells were harvested, washed once, and fixed in 4% paraformaldehyde-PBS for 15 min at room temperature. Cells were then resuspended in PBS solution containing the anti- P2RX7 antibody (1:100 Abcam Inc.) for 30 min at room temperature. Cells were subsequently stained with FITC goat anti-rabbit IgG (1:50 BD Pharmingen) and incubated for 30 min at room temperature. Samples were then analyzed with a FACS Calibur flow cytometer (BD Biosciences) using Cell Quest software as described previously (BD Biosciences) [23]. Negative controls were produced by replacing the primary antibody with normal rabbit serum. To estimate the proportion of dead cells, the harvested cells were suspended in PBS solution containing 5 mg/ml propidium iodide (P3566, Molecular Probes, Eugene, OR, USA), and were incubated for 30 min at room temperature. Samples were also analyzed with a FACS Calibur flow cytometer.A cell proliferation assay was performed with the cell proliferation reagent WST-1 (Roche, Basel, Switzerland) according to the manufacturer’s instructions. 26104 cells/well were incubated in microplates for 24 or 48 h (tissue culture grade, 96 wells, flat bottom Falcon) at a final volume of 100 ml/well for each culture medium.
