Neutral sphingomyelinase (N-SMase) (IC50 = 1 μM)

TNF-induced sphingomyelin (SM) hydrolysis was partially inhibited by GW4869 (10 μM), and SM loss was entirely prevented by the compound at 20 μM. The addition of 10–20 μM GW4869 completely inhibited the initial accumulation of ceramide; however, this effect was partially lost at later time points (24 hours). The reduction in glutathione is preceded by the effects of GW4869. Cell death is markedly inhibited by GW4869 in a dose-dependent manner [1]. GW4869 (10 or 20 μM) prevents macrophages from producing proinflammatory cytokines and releasing exosomes. The release of mature exosomes from multivesicular bodies (MVBs) and ceramide-mediated inward budding are both inhibited by GW4869 [2]. In hepatic stellate cells, GW4869 can also undo the suppression of CCN2 3'-UTR activity caused by miR-214-rich exosomes [3]. Precautions for dissolution: GW4869 is typically prepared as a stock solution in DMSO and stored at -80°C in aliquots.

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A high throughput screen for neutral, magnesium-dependent sphingomyelinase (SMase) was performed. One inhibitor discovered in the screen, GW4869, functioned as a noncompetitive inhibitor of the enzyme in vitro with an IC(50) of 1 microm. It did not inhibit acid SMase at up to at least 150 microm. The compound was then evaluated for its ability to inhibit tumor necrosis factor (TNF)-induced activation of neutral SMase (N-SMase) in MCF7 cells. GW4869 (10 microm) partially inhibited TNF-induced sphingomyelin (SM) hydrolysis, and 20 microm of the compound was protected completely from the loss of SM. The addition of 10-20 microm GW4869 completely inhibited the initial accumulation of ceramide, whereas this effect was partially lost at later time points (24 h). These data therefore support the inhibitory action of GW4869 on N-SMase not only in vitro but also in a cellular model. The addition of GW4869 at both 10 and 20 microm did not modify cellular glutathione levels in response to TNF, suggesting that the action of GW4869 occurred downstream of the drop in glutathione, which was shown previously to occur upstream of the activation of N-SMase. Further, whereas TNF treatment also caused a 75% increase of de novo synthesized ceramide after 20 h of incubation, GW4869, at either 10 or 20 microm, had no effect on this pathway of ceramide generation. In addition, GW4869 did not significantly impair TNF-induced NF-kappaB translocation to nuclei. Therefore, GW4869 does not interfere with other key TNF-mediated signaling effects. GW4869 was able, in a dose-dependent manner, to significantly protect from cell death as measured by nuclear condensation, caspase activation, PARP degradation, and trypan blue uptake. These protective effects were accompanied by significant inhibition of cytochrome c release from mitochondria and caspase 9 activation, therefore localizing N-SMase activation upstream of mitochondrial dysfunction. In conclusion, our results indicate that N-SMase activation is a necessary step for the full development of the cytotoxic program induced by TNF.[1]

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GW4869 inhibits both exosome release and pro-inflammatory cytokine production in macrophages [2]
Next, we sought to determine if blockade of exosome generation would diminish the LPS-mediated pro-inflammatory responses in RAW264.7 macrophages. GW4869 has been successfully used to block the secretion of exosomes from HEK293 cells, cardiomyocytes, liver cells and lung epithelial cells. In this study, we treated RAW264.7 macrophages with two different doses of GW4869 (10 μM and 20 μM). We observed that LPS-triggered exosome generation was remarkably attenuated in macrophages upon pre-treatment of macrophages with 10 μM GW4869, as evidenced by a 22% reduction in the activity of AChE (Fig. 3A). Such attenuation was further enhanced by treatment with 20 μM GW4869 (Fig. 3A). To test the possible toxicity of GW4869, we measured the levels of lactase dehydrogenase (LDH), a marker of cell injury, in the supernatants of macrophages upon exposure to 10 μM and 20 μM GW4869, as well as 0.005% DMSO (solution used to dissolve GW4869) for 24 h. Our results showed that LDH levels were similar in 0.005% DMSO, 10μM GW4869, 20μM GW4869 and non-treated groups (Fig. 3B). This suggests that 0.005% DMSO and GW4869 do not have toxic effects on macrophages. Assessment of pro-inflammatory cytokines revealed a significant decrease in the levels of TNF-α, IL-1β and IL-6 by 16.4%, 62%, and 15.6% respectively, in the supernatants of GW4869-treated macrophages upon LPS challenge, compared to control macrophages treated with LPS only (Figs. 3C–E). Collectively, these data indicate that GW4869 suppresses the endotoxin-triggered generation of exosomes in RAW264.7 macrophages and subsequently, decreases the production of pro-inflammatory cytokines.

In mice, GW4869 (2.5 μg/g, i.p.) suppresses exosome release, which prevents the production of pro-inflammatory cytokines and cardiac inflammation when stimulated by LPS. Mice's survival rate is increased and LPS-induced myocardial dysfunction is lessened by GW4869 [2]. In CLP mice, GW4869 (2.5 μg/g, ip) inhibits the production of pro-inflammatory cytokines and cardiac inflammation [2].

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Inhibition of exosome release blocks LPS-stimulated pro-inflammatory cytokine production and cardiac inflammation in mice [2]
To determine the in vivo effects of GW4869, WT mice were injected i.p. with either PBS or GW4869 (2.5μg/g) for 1 h, followed by LPS injection at one dose of 25μg/g. 12 h later, levels of serum exosomes and pro-inflammatory cytokines, as well as myocardial inflammation were assessed. We noticed that, under basal conditions, GW4869 treatment significantly decreased exosome levels by 37% in sera, compared to levels collected from control mice (Fig. 4A). At 12 h after LPS injection, the levels of circulating exosomes were increased significantly compared to PBS-controls, as evidenced by a 1.7-fold elevation in the AChE activity (Fig. 4A). However, such LPS-induced elevation of serum exosomes was significantly inhibited in mice subjected to pre-treatment with GW4869, compared to the PBS+LPS group (Fig. 4A). Accordingly, pre-treatment with GW4869 resulted in a significant decrease in endotoxin-triggered production of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) in the serum, as evidenced by a 17% reduction of TNF-α (Fig. 4B), 11% decrease of IL-1β (Fig. 4C), and 28% reduction of IL-6 (Fig. 4D), compared to the PBS+LPS group. Also, we observed that MPO activity was significantly reduced by 12% in the hearts of mice pre-treated with GW4869 upon LPS challenge, compared to mice pre-treated with PBS. This indicates that LPS-caused infiltration of neutrophils into the myocardium is suppressed by GW4869 (Fig. 4E). Taken together, our results suggest that pre-treatment of WT mice with GW4869 could attenuate LPS-triggered production of exosomes and pro-inflammatory cytokines in the blood which subsequently reduces myocardial inflammation.

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GW4869 mitigates LPS-caused myocardial dysfunction and improves survival in mice [2]
It is well recognized that the production of circulating pro-inflammatory cytokines mediates myocardial dysfunction during septic shock. Given the inhibitory effects of GW4869 on production of pro-inflammatory cytokines and cardiac inflammation, we next evaluated the effects of GW4869 on endotoxin-induced cardiac dysfunction. WT mice were treated with either PBS or GW4869 (2.5μg/g) for 1 h, followed by LPS injection at one does of 25μg/g. Cardiac function was assessed by echocardiography. Consistent with previous observations, cardiac function was depressed in our endotoxemic mice compared to controls (Figs. 5A–C). However, pre-treatment of mice with GW4869 evoked an improvement of cardiac function, evidenced by a significant increase in left ventricular ejection fraction (EF%) and fractional shortening (FS%), compared to LPS-injected mice pre-treated with PBS controls (Figs. 5A–C, and Supplemental Table 1). At 36 h post-LPS injection, only 16.7% of mice pre-treated with PBS (n=12) survived whereas 66.7% of mice pre-treated with GW4869 (n=9) survived (Fig. 5D). Collectively, our data indicate that blockade of exosome generation with GW4869 diminishes the endotoxin-caused myocardial dysfunction and decreases mortality in mice.

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GW4869 blocks the production of pro-inflammatory cytokines and cardiac inflammation in CLP mice [2]
We next examined whether the GW4869-mediated protective effects can be replicated in a clinical-relevant septic model, induced by cecal ligation and puncture (CLP). WT mice were injected with PBS or GW4869 (2.5μg/g), followed by CLP or sham surgery. The levels of exosomes and pro-inflammatory cytokines in the serum, as well as myocardial inflammation, were determined at 12 h after the surgery. In sham-surgery controls, pretreatment with GW4869 decreased exosome concentration by 33% compared to mice injected with PBS (Fig. 6A). Similar to endotoxin challenge, CLP surgery caused a significant elevation in the levels of serum exosomes, as measured by AChE activity (Fig. 6A). Importantly, such CLP-stimulated exosome release was significantly inhibited by pre-treatment of CLP mice with GW4869, compared to CLP mice pre-treated with PBS (Fig. 6A). The results of pro-inflammatory cytokine analysis in the serum revealed a significant decrease in the levels of TNF-α by 85% (Fig. 6B) and IL-6 by 46% (Fig. 6C) in CLP-mice pre-treated with GW4869, compared to the PBS+CLP group. CLP-induced cardiac inflammation, assessed by MPO activity, was greatly alleviated in the GW4869+CLP group, compared to the PBS+CLP samples (Fig. 6D). Hence, our data suggest that GW4869 could inhibit the CLP-triggered production of pro-inflammatory cytokines in serum and myocardial inflammation.

Specificity of GW4869 on Enzyme Inhibition [1]
Partially purified rat brain N-SMase and lyso-PAF PLC were incubated in the absence or presence of GW4869 and PS (100 μm), and SM hydrolysis was determined as previously described. In the case ofBacillus cereus N-SMase, PS was not included in the reaction mixture, since it does not affect the bacterial enzymatic activity. B. cereus phosphatidylcholine-PLC was incubated in the presence or absence of GW4869 in a reaction mixture containing 100 mm Tris, pH 7.2, 25% glycerol, 20 mm p-nitrophenyl/phosphorylcholine, and production of p-nitrophenol was quantified spectrophotometrically at 410 nm. Protein phosphatase 2A from bovine kidney was incubated in the presence or absence of GW4869 in buffer containing 50 mm Tris, pH 7.4, 1 mmdithiothreitol, 100 μm MnCl2, and 20% glycerol, and phosphatase activity was measured as described by Jones and Hannun. In all assays, 10 μm GW4869 was used, and 30-min preincubation with the enzymes preceded the addition of the substrate.

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Exosome binding assays [3]
Exosomes from control or SYTO-RNA-labeled HSC or that contained Cy3-miR-199a-5p were labeled for 1 hr with 4 μM of the fluorescent lipophilic membrane dyes PKH26 or PKH67, according the manufacturer’s specifications. Exosomes (0- 4 μg/ml) or free Cy3-labeled miR-199a-5p (1 μM) were added for up to 48 hrs to primary mouse HSC or hepatocytes which were then washed in PBS and imaged using a confocal microscope or lysed in lysis buffer and measured at 590/540 nm using a Spectra Max® M2 microplate reader to assess levels of PKH26 flourescence. Prior to exosome addition in some experiments, HSC were stained with PKH67 and hepatocytes were stained with far red. In some binding experiments, HSC were pre-treated or co-incubated with 0-100 μg/ml RGD or RGE tripeptides, 0-100 μM EDTA 0-10 μM sodium chlorate, 0-10 μM sodium sulfate, 0-10 μg/ml rabbit anti-mouse integrin αvβ3 IgG or 0-20 μg/ml rat anti-mouse integrin α5β1 IgG or 0-10 μg/ml rat anti-mouse integrin αM, (CD11b). For antibody studies, non-immune IgG was used as a negative control.

Cell Viability Assay[1]
Cell Types: MCF7 human breast cancer cells.
Tested Concentrations: 10-20 μM.
Incubation Duration: 30 min (then treated with TNF (3 nM) followed).
Experimental Results: Dramatically inhibited TNF-induced SM hydrolysis, whereas 20 μM of the compound protected completely from the loss of SM.

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Cell Viability Assay[2]
Cell Types: Fresh RAW264.7 macrophages.
Tested Concentrations: 10 or 20 μM.
Incubation Duration: 2 hrs (hours) (then treated with 1 μg/mL LPS incubation ).
Experimental Results: LPS-triggered exosome generation was remarkably attenuated in macrophages upon pre-treatment of macrophages with 10 μM GW4869, as evidenced by a 22% reduction in the activity of AChE. Such attenuation was further enhanced by treatment with the dose of 20 μM.

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Cell Culture and GW4869 Treatment [1]
MCF7 human breast cancer cells were routinely cultured in RPMI 1640 containing 10% FBS at 37 °C in 5% CO2. Unless otherwise indicated, for treatment, cells were seeded at 1.7 × 106 cells/10-cm culture dish in 10 ml of complete medium; after 24 h, the medium was replaced with 7 ml of RPMI 1640 containing 2% FBS and 25 mm Hepes, pH 7.5, and the cells were rested for 2 h prior to treatment. GW4869 was routinely stored at −80 °C as a 1.5 mm stock suspension in Me2SO. Right before use, the suspension was solubilized by the addition of 5% methane sulfonic acid (MSA) (2.5 μl of 5% MSA in sterile double-distilled H2O were added to 50 μl of GW4869 stock suspension; therefore, the concentration of the GW4869 stock solution at the time of the experiments was 1.43 mm). The suspension was mixed and warmed up at 37 °C until clear. Cells were preincubated with the inhibitor for 30 min prior to treatment with TNF. Control cells were treated with Me2SO containing 5% MSA, similarly to the samples receiving the GW4869 solution. When different doses of GW4869 were tested, amounts of vehicle solution were added in order to equal the volume of GW4869 used for the highest dose.

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Sphingomyelin Measurement [1]
Cells were seeded at 0.1 × 106 cells/10-cm dish in 8 ml of complete growth medium. After 48 h, the cells were labeled with [methyl-3H]choline chloride (1 μCi/ml final concentration in 10 ml of growth medium/plate). After ∼60 h, the cells were chased with 10 ml of complete medium for 90 min. Then the cells were washed once with 5 ml of PBS, and 7 ml of medium containing 2% FBS and 25 mm Hepes, pH 7.5, were added. After resting the cells for approximately 1 h, preincubation for 30 min with GW4869 was started, and TNF treatment followed. At the appropriate time points, the medium from each plate was collected, and the cells were washed once with 2 ml of ice-cold PBS. Cells were scraped on ice in 2 ml of PBS, and each plate was washed with an additional 2 ml of PBS. Cells and washes were pooled with the medium and centrifuged for 5 min at 2000 × g (4 °C). The cell pellets were stored at −80 °C. On the day of the measurement, the pellets were resuspended in 600 μl of double-distilled H2O by vortexing and sonication. Aliquots of cell lysates were used for protein determination, and 250 μl in duplicate were used for SM determination as described by Andrieu et al.

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Measurement of Ceramide Generated by de Novo Biosynthesis [1]
Cells were seeded at 1.7 × 106cells/plate in complete growth medium. After 24 h, the medium was replaced with 6 ml of RPMI 1640 containing 2% FBS and 25 mm Hepes, pH 7.5. Right before the addition of GW4869, [3H]palmitate was added to the cells in 1 ml of the same medium to a final activity of 1 μCi/ml. After ∼21 h of treatment with TNF, the medium was collected, and the plates were washed once with PBS that was combined with the medium and centrifuged at 2000 × g for 5 min at 4 °C to collect floating cells. Cells were scraped off the plate with methanol and combined with the floaters. Lipids were extracted by the method of Bligh and Dyer. One ml of the organic phase was used for ceramide determination by separation of lipids through thin layer chromatography (chloroform, methanol, 4.2 n ammonium hydroxide; 4:1:0.1; v/v/v), and 0.35 ml in duplicates were used for determination of inorganic phosphate used to normalize the ceramide values. The ceramide band was identified by comparison with an authentic standard, and radioactivity was quantified in a scintillation counter.

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MTT Assay [1]
5 × 103 cells/well were seeded in a 96-well plate in 75 μl of RPMI containing 2% FBS and 25 mmHepes, pH 7.5. After 24 h, first GW4869 was added in 15 μl of medium/well and incubated for 30 min and then TNF was added in 10 μl/well (total volume of 100 μl/well). At the indicated time points, 25 μl of MTT stock solution (5 mg/ml in PBS) were added to each well and incubated at 37 °C in 5% CO2 for 3 h. Subsequently, cells were solubilized by the addition of 100 μl of lysis buffer (20% SDS (w/v), 50%N,N-dimethylformamide (v/v), 0.8% acetic acid (v/v), pH 4.6–4.8) to each well. The production of the formazan dye was quantitated by measuring the OD at 595 nm with a multiwell plate reader. We have noticed that the efficacy of TNF to induce morphological changes was significantly slower in experiments carried out in the 96-well plates (as for the MTT) compared with those performed in 10-cm Petri dishes (all other experiments).

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Treatment of Macrophages with Exosomes or GW4869 [2]
Fresh RAW264.7 macrophages were plated in 100 mm petri dishes at 1.2×106 cells/dish, and treated with either culture media containing 20 μg of exosomes isolated from non-LPS treated macrophages (non-LPS exosomes) or 20 μg of exosomes isolated from LPS treated macrophages (LPS exosomes), or exosome-free media. The culture supernatants were harvested for cytokine ELISA assays at 10 min and 24 h. For inhibition of exosome generation, macrophages were pre-treated with either culture media containing 10 μM or 20 μM GW4869 for 2 h prior to treatment with 1 μg/ml LPS incubation, Culture supernatants were collected after 24 h for AChE activity assay and cytokine measurement. GW4869 was initially dissolved in DMSO into a stock solution of 5 mM GW4869 before dilution in culture supernatant to achieve 10 μM or 20 μM GW4869 concentration in cell culture condition (Note: final DMSO concentration is 0.005%). To determine the possible toxicity of DMSO or GW4869, fresh RAW 264.7 macrophages (1.2×106 cells/dish) were incubated in culture media containing 0.005% DMSO, 10 μM GW4869 and 20 μM GW4869 for 24 h. Cell injury was determined by measuring the release of lactase dehydrogenase (LDH) into the culture media, using a LDH detection kit according to the manufacturer’s protocol.

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HSC co-culture system [3]
One well of a 2-well micro-culture system received exosome donor P6 HSC that had been transfected with 100nM pre-mir-214. Some cells were cultured with 10 μM GW4869, an inhibitor of neutral sphingomyelinase 2 which is required for exosome biogenesis. After 12 hrs, the other well was seeded with P6 HSC transfected with parental miR-Selection Fire-Ctx lentivector or the same vector containing either wild type or mutant CCN2 3’-UTR lacking the miR-214 binding site. After 12 hrs, direct communication between the cells was initiated and proceeded for 24hrs. In some experiments, 100 μg/ml heparin sulfate or 100 μg/ml chondroitin sulfate were included in the culture medium. Luciferase activity was measured in triplicate using the Dual Luciferase Reporter Assay System. Firefly luciferase activity in pre-mir-21- transfected cells was compared to that in non-transfected cells, with Renilla luciferase activity used for normalization.

Animal/Disease Models: 10-12 weeks old Male wild-type C57BL/6 mice (Endotoxin-Challenged Mice)[2].
Doses: 2.5 μg/g.
Route of Administration: IP once (1 h later, followed by an ip injection of LPS ( 2.5 μg/g, 100 μL)).
Experimental Results: Dramatically diminished exosome levels by 37% in sera, compared to levels collected from control mice. At 12 h after LPS injection, the levels of circulating exosomes were increased Dramatically compared to PBS-controls , as evidenced by a 1.7-fold elevation in the AChE activity.

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Animal/Disease Models: 10-12 weeks old Male wild-type C57BL/6 mice (CLP Polymicrobial Sepsis Model)[2].
Doses: 2.5 μg/g.
Route of Administration: IP once (before sham or CLP surgery).
Experimental Results: diminished exosome concentration by 33% compared to mice injected with PBS in sham-surgery controls. CLP-stimulated exosome release was Dramatically inhibited by pre-treatment of CLP mice compared to CLP mice pre- treated with PBS.

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GW4869 Treatment in Endotoxin-Challenged Mice [2]
Wild-type male C57BL/6 mice were studied at 10 to 12 weeks old. The mice were randomly assigned to four groups: PBS, GW4869, PBS+LPS and GW4869+LPS (n=5 per group). GW4869, dissolved in DMSO (0.005%), was intraperitoneally (i.p.) injected at one dose of 2.5μg/g. Mice in the PBS+LPS group were pre-injected i.p. with PBS 1 h prior to an i.p. injection of LPS (25 μg/g). Mice in the group of GW4869+LPS were pre-injected i.p. with GW4869 (2.5μg/g) for 1 h, followed by an i.p. injection of LPS (25 μg/g, 100μl). Mice received injections of PBS to a comparable volume (100μl) as controls. The survival rate of the PBS+LPS and GW4869+LPS groups were monitored every 6 h for a 36 h period.

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Pre-Treatment with GW4869 in CLP Polymicrobial Sepsis Model [2]
Polymicrobial sepsis was surgically induced by cecal ligation and puncture (CLP) as previously described. Wild-type C57BL/6 mice (10–12 week, male) were randomly assigned to four groups: PBS+Sham, GW4869+Sham, PBS+CLP and GW4869+CLP. Before sham or CLP surgery, mice were administered by intraperitoneal (i.p.) injection at one dose of GW4869 (2.5μg/g). Same volume of PBS was injected as controls. For CLP surgery, mice were anesthetized by isoflurane inhalation and ventilated with room air using a rodent ventilator. A 1- to 2-cm midline incision was made below the diaphragm to expose the cecum. The cecum was ligated at 1.0 cm from the tip with a 5-0 sterile silk suture. A single through and through puncture was made at the middle between the ligation and the tip of the cecum with an 18-gauge to induce severe septic injury. After puncturing, the cecum was gently squeezed to extrude a small amount of feces and returned to the abdominal cavity. The abdominal wall incision was closed in layers. After surgery, pre-warmed normal saline (0.05–0.1 ml/g body weight) was administered subcutaneously. Post-operative pain control was managed with subcutaneous injection of bupivacain and buprenorphine. Sham controls were exposed to the same surgery; however, their cecum was neither ligated nor punctured. The survival rate of the PBS+CLP and the GW4869+CLP groups was monitored every 6h for 2 days.

[1]. Inhibition of tumor necrosis factor-induced cell death in MCF7 by a novel inhibitor of neutralsphingomyelinase. J Biol Chem. 2002 Oct 25;277(43):41128-39.

[2]. Blockade of exosome generation with GW4869 dampens the sepsis-induced inflammation and cardiac dysfunction. Biochim Biophys Acta. 2015 Nov;1852(11):2362-71.

[3]. Integrins and heparan sulfate proteoglycans on hepatic stellate cells (HSC) are novel receptors for HSC-derived exosomes. FEBS Lett. 2016 Dec;590(23):4263-4274.

[4]. Sphingomyelin Regulates the Activity of Secretory Phospholipase A2 in the Plasma Membrane. J Cell Biochem. 2015 Sep;116(9):1898-907.

Sepsis is an infection-induced severe inflammatory disorder that leads to multiple organ failure. Amongst organs affected, myocardial depression is believed to be a major contributor to septic death. While it has been identified that large amounts of circulating pro-inflammatory cytokines are culprit for triggering cardiac dysfunction in sepsis, the underlying mechanisms remain obscure. Additionally, recent studies have shown that exosomes released from bacteria-infected macrophages are pro-inflammatory. Hence, we examined in this study whether blocking the generation of exosomes would be protective against sepsis-induced inflammatory response and cardiac dysfunction. To this end, we pre-treated RAW264.7 macrophages with GW4869, an inhibitor of exosome biogenesis/release, followed by endotoxin (LPS) challenge. In vivo, we injected wild-type (WT) mice with GW4869 for 1h prior to endotoxin treatment or cecal ligation/puncture (CLP) surgery. We observed that pre-treatment with GW4869 significantly impaired release of both exosomes and pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) in RAW264.7 macrophages. At 12h after LPS treatment or CLP surgery, WT mice pre-treated with GW4869 displayed lower amounts of exosomes and pro-inflammatory cytokines in the serum than control PBS-injected mice. Accordingly, GW4869 treatment diminished the sepsis-induced cardiac inflammation, attenuated myocardial depression and prolonged survival. Together, our findings indicate that blockade of exosome generation in sepsis dampens the sepsis-triggered inflammatory response and thereby, improves cardiac function and survival. [2]

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Exosomes mediate intercellular microRNA delivery between hepatic stellate cells (HSC), the principal fibrosis-producing cells in the liver. The purpose of this study was to identify receptors on HSC for HSC-derived exosomes, which bind to HSC rather than to hepatocytes. Our findings indicate that exosome binding to HSC is blocked by treating HSC with RGD, EDTA, integrin αv or β1 siRNAs, integrin αvβ3 or α5β1 neutralizing antibodies, heparin, or sodium chlorate. Furthermore, exosome cargo delivery and exosome-regulated functions in HSC, including expression of fibrosis- or activation-associated genes and/or miR-214 target gene regulation, are dependent on cellular integrin αvβ3, integrin α5β1, or heparan sulfate proteolgycans (HSPG). Thus, integrins and HSPG mediate the binding of HSC-derived exosomes to HSC as well as the delivery and intracellular action of the exosomal payload. [3]

C30H28N6O2.2HCL.XH2O

577.5

576.18

C, 62.39; H, 5.24; Cl, 12.28; N, 14.55; O, 5.54

6823-69-4

GW4869-13C4; 475570-61-7

16078967

Light yellow to yellow solid powder

4.577

6

4

8

40

856

0

O=C(NC1=CC=C(C2=NCCN2)C=C1)/C=C/C3=CC=C(/C=C/C(NC4=CC=C(C5=NCCN5)C=C4)=O)C=C3.[H]Cl.[H]Cl

NSFKAZDTKIKLKT-CLEIDKRQSA-N

InChI=1S/C30H28N6O2.2ClH/c37-27(35-25-11-7-23(8-12-25)29-31-17-18-32-29)15-5-21-1-2-22(4-3-21)6-16-28(38)36-26-13-9-24(10-14-26)30-33-19-20-34-30;;/h1-16H,17-20H2,(H,31,32)(H,33,34)(H,35,37)(H,36,38);2*1H/b15-5+,16-6+;;

(E)-3-[4-[(E)-3-[4-(4,5-dihydro-1H-imidazol-2-yl)anilino]-3-oxoprop-1-enyl]phenyl]-N-[4-(4,5-dihydro-1H-imidazol-2-yl)phenyl]prop-2-enamide;dihydrochloride

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)