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Suppressive effects of polyozellin on TGFBIp-mediated septic responses in human endothelial cells and mice
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Available online at www.sciencedirect.com
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Original Research
Suppressive effects of polyozellin on TGFBIpmediated septic responses in human endothelial cells and mice Byeongjin Jung, Eun-Ju Yang, Jong-Sup Bae⁎ College of Pharmacy, CMRI, Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu 702-701, Republic of Korea
ARTI CLE I NFO
A BS TRACT
Article history:
Polyozellus multiplex (Thelephoraceae) is a wild mushroom in Korea and Japan and is usually
Received 4 October 2015
harvested in early autumn for food. Polyozellin, a major constituent of the edible
Revised 8 December 2015
mushroom P multiplex, has been known to exhibit biological activities such as
Accepted 16 December 2015
antioxidative and anti-inflammatory effects. Transforming growth factor β–induced protein (TGFBIp) is an extracellular matrix protein whose expression in several cell types is greatly increased by TGF-β. TGFBIp is released by human umbilical vein endothelial cells
Keywords:
and functions as a mediator of experimental sepsis. We hypothesized that polyozellin could
Polyozellin
reduce TGFBIp-mediated severe inflammatory responses in human endothelial cells and
TGFBIp
mice. Here, we investigated the antiseptic effects and underlying mechanisms of
Sepsis
polyozellin against TGFBIp-mediated septic responses. Polyozellin effectively inhibited
Severe inflammation
lipopolysaccharide-induced release of TGFBIp and suppressed TGFBIp-mediated septic
Mice
responses. In addition, polyozellin suppressed cecal ligation and puncture–induced sepsis lethality and pulmonary injury. In conclusion, polyozellin suppressed TGFBIp-mediated and cecal ligation and puncture–induced septic responses. Therefore, polyozellin could be a potential therapeutic agent for treatment of various severe vascular inflammatory diseases via inhibition of the TGFBIp signaling pathway. © 2016 Elsevier Inc. All rights reserved.
1.
Introduction
Transforming growth factor β–induced protein (TGFBIp) is an extracellular matrix protein that can be highly expressed in various cell types [1–3]. TGFBIp contains an N-terminal secretory signal peptide, followed by a cysteine-rich domain, 4 internal homologous repeats (FAS1 domain), and a C-terminal
tripeptide Arg-Gly-Asp motif [1]. Several studies suggest that TGFBIp is involved in cell growth, cell differentiation, wound healing, tumorigenesis, and apoptosis [2–4]. Very recently, we reported that TGFBIp is a promising therapeutic target for the treatment of severe vascular inflammatory diseases, such as sepsis and septic shock [3,5]. In fact, blocking TGFBIp, even at later times after the onset of infection, has been shown to
Abbreviations: ALT, alanine transaminase; AST, aspartate transaminase; BUN, blood urea nitrogen; CAM, cell adhesion molecule; CLP, cecal ligation and puncture; HUVEC, human umbilical vein endothelial cell; LPS, lipopolysaccharide; POZ, polyozellin; TGFBIp, transforming growth factor β-induced protein. ⁎ Corresponding author. College of Pharmacy, Research Institute of Pharmaceutical Sciences, Kyungpook National University, 80 Dahakro, Buk-gu, Daegu 702-701, Republic of Korea. Tel.: +82 53 950 8570; fax: +82 53 950 8557. E-mail address: [email protected] (J.-S. Bae). http://dx.doi.org/10.1016/j.nutres.2015.12.009 0271-5317/© 2016 Elsevier Inc. All rights reserved.
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rescue mice from lethal sepsis [5]. TGFBIp also acts as a lethal mediator in conditions such as sepsis, in which serum TGFBIp levels are substantially increased [3,5]. Once released into the extracellular milieu, TGFBIp can bind to cell surface receptors, such as integrins αvβ3 and αvβ5, in human endothelial cells [6]. Polyozellus multiplex (PM; Thelephoraceae) is a wild mushroom in Korea and Japan and is usually harvested in early autumn for food. The chemical components of PM, polyozellin (POZ) [7], thelephoric acid [8,9], polyozellic acid [8], kynapcin-9 [8], kynapcin-12 [10], kynapcin-13 [11], kynapcin-24 [12], and kynapcin-28 [11], have been isolated and characterized. Moreover, PM shows a variety of biological activities such as anticancer properties [13], inhibitory effects on proryl endopeptidase activity [7], and lipid peroxidation [14]. In HT-29 intestinal epithelial cells derived from human, POZ had an inhibitory effect on interleukin-8 and matrix metalloproteinase-7 production mediated by tumor necrosis factor α [15]. In addition, POZ has demonstrated antioxidative [16] and prolyl endopeptidase inhibitory activities [7] in a cell-free system. However, to the best of our knowledge, the effects of POZ on TGFBIp-mediated septic responses have not been studied yet. Based on previous reports, which demonstrated the potential effects of TGFBIp on vascular inflammatory responses [3,5] and antioxidant activities of POZ [16], we hypothesized that treatment with POZ would suppress TGFBIp- or cecal ligation and puncture (CLP)–induced septic responses in human endothelial cells and mice. To test our hypothesis, we used human endothelial cells and male C57BL/6 mice. The in vitro experimental design with exposure of human umbilical vein endothelial cells (HUVECs) to lipopolysaccharide (LPS) enables us to determine the effects of POZ on the secretion of TGFBIp protein and expression of TGFBIp messenger RNA (mRNA) and its receptors (integrin αvβ3 and αvβ5). To investigate the antiseptic effects of POZ in TGFBIpmediated septic responses, the following experimental techniques were used: hyperpermeability assessment, F-actin staining, assessing expression of cell adhesion molecules (CAMs), and determining adhesion and migration of leukocytes to HUVECs. For the in vivo assessment, CLP-induced septic mice were used because this model more closely resembles human sepsis [17]. In the CLP-induced septic model, we also tested the effects of POZ on lethality, pulmonary injury, and several organ injury markers.
2.
Methods and materials
2.1.
Reagents
whole blood (15 mL) obtained by venipuncture from 5 healthy volunteers and maintained as previously described [20].
2.3.
2.2.
Cell culture
Primary HUVECs were obtained from Cambrex Bio Science (Charles City, IA, USA) and maintained as described previously [6,18,19]. All experiments were carried out with HUVECs at passages 3 to 5. Human neutrophils were freshly isolated from
Animals and husbandry
Male C57BL/6 mice (6-7 weeks old, weighing 27 g), purchased from Orient Bio Co (Sungnam, Republic of Korea), were used after a 12-day acclimatization period. Animals were housed 5 per polycarbonate cage under controlled temperature (20°C-25°C) and humidity (40%-45%) and a 12:12-hour light/dark cycle. Animals received a normal rodent pellet diet and water ad libitum during acclimatization. Mice were euthanized by CO2 anesthesia followed by cervical dislocation [21]. All animals were treated in accordance with the Guidelines for the Care and Use of Laboratory Animals issued by Kyungpook National University (institutional review board no. KNU 2014-13).
2.4.
Cecal ligation and puncture
For induction of sepsis, male mice were anesthetized with 2% isoflurane (Forane; JW Pharmaceutical, Seoul, South Korea) in oxygen delivered via a small rodent gas anesthesia machine (RC2; Vetequip, Pleasanton, CA, USA), first in a breathing chamber and then via a facemask. They were allowed to breath spontaneously during the procedure. The CLP-induced sepsis model was prepared as previously described [6]. This protocol was approved by the Animal Care Committee at Kyungpook National University prior to conducting the study (institutional review board no. KNU 2012-13).
2.5.
Cell viability assay
3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) was used as an indicator of cell viability as previously described [22]. Cells were grown in 96-well plates at a density of 5 × 103 cells/well. After 24 hours, cells were washed with fresh medium, followed by treatment with POZ. After an incubation period of 48 hours, cells were washed and 100 μL of MTT (1 mg/ mL) was added, followed by incubation for 4 hours. Finally, dimethyl sulfoxide (150 μL) was added in order to solubilize the formazan salt formed, and the amount of formazan salt was determined by measuring the optical density at 540 nm using a microplate reader (Tecan Austria GmbH, Grödig, Austria).
2.6.
Polyozellin was isolated from the methanolic extract of PM by performing column chromatography, as in our previous study [7,16]. Evans blue and crystal violet were obtained from Sigma (St Louis, MO, USA). Vybrant DiD (used at 5 μM) was obtained from Invitrogen (Carlsbad, CA, USA). TGFBIp protein was purified as described previously [6].
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Enzyme-linked immunosorbent assay for TGFBIp
TGFBIp concentrations in cell culture media or mouse serum were determined by competitive enzyme-linked immunosorbent assay (ELISA), as described previously [3,6]. Ninety-six– well plastic flat microtiter plates (Corning, NY, USA) were coated with TGFBIp protein in 20 mM carbonate-bicarbonate buffer (pH 9.6) with 0.02% sodium azide overnight at 4°C. The plates were rinsed 3 times with phosphate-buffered saline (PBS)–0.05% Tween 20 (PBS-T) and kept at 4°C. Lyophilized culture media was preincubated with anti-TGFBIp antibodies (diluted 1:2000 in PBS-T) in 96-well plastic round microtiter plates for 90 minutes at 37°C. Preincubated samples were transferred to a precoated plate and incubated for 30 minutes at room temperature. The plates were rinsed 3 times with PBS-T and incubated for 90 minutes at room temperature with
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peroxidase-conjugated antirabbit IgG antibodies (diluted 1:2000 in PBS-T; Amersham Pharmacia Biotech, Uppsala, Sweden). The plates were rinsed 3 times with PBS-T and incubated for 60 minutes at room temperature in the dark with 200 μL substrate solution (100 μg/mL o-phenylenediamine and 0.003% H2O2). After stopping the reaction with 50 μL of 8 N H2SO4, the absorbance was read at 490 nm.
2.7.
antimouse IgG antibodies (Sigma) for 1 hour. The cells were washed again 3 times and developed using ophenylenediamene substrate (Sigma). Colorimetric analysis was performed by measuring absorbance at 490 nm. All measurements were performed in triplicate wells. The same experimental procedures were used for monitoring the cell surface expression of αvβ3 and αvβ5 using specific antibodies obtained from EMD Millipore (Billerica, MA, USA).
Permeability assay in vitro 2.10.
Permeability was quantitated by spectrophotometric measurement of the flux of Evans blue–bound albumin across functional HUVEC monolayers using a modified 2-compartment chamber model, as previously described [6]. Briefly, HUVECs were plated (5 × 104 cells/well) in transwells with a pore size of 3 μm and a diameter of 12 mm for 3 days. The confluent monolayers were treated with TGFBIp (5 μg/mL for 6 hours) followed by incubation with POZ for 6 hours.
2.8.
Immunofluorescence staining
HUVECs were grown to confluence on glass coverslips coated with 0.05% poly-L-lysine in complete media containing 10% fetal bovine serum and maintained for 48 hours. Cells were then stimulated with TGFBIp (5 μg/mL) for 16 hours with or without 6 hours POZ (10 or 20 μM). For cytoskeletal staining [23], the cells were fixed in 4% formaldehyde in PBS (vol/vol) for 15 minutes at room temperature, permeabilized in 0.05% Triton X-100 in PBS for 15 minutes, and blocked in blocking buffer (5% bovine serum albumin in PBS) overnight at 4°C. Then, the cells were incubated with F-actin–labeled fluorescein phalloidin (F 432; Molecular Probes, Invitrogen) and visualized by confocal microscopy at a ×630 magnification (TCS-Sp5; Leica Microsystem, Wetzlar, Germany).
2.9.
Migration assay in vitro
Migration assays were performed in transwell plates with a diameter of 6.5 mm, with filters having a pore size of 8 μm. HUVECs (6 × 104) were cultured for 3 days in order to obtain confluent endothelial monolayers. Before the addition of human neutrophils to the upper compartment, cell monolayers were treated with TGFBIp (5 μg/mL for 6 hours) followed by treatment with POZ for 6 hours. Cells in the upper chamber of the filter were aspirated, and nonmigrating cells on top of the filter were removed using a cotton swab. Human neutrophils on the lower side of the filter were fixed with 8% glutaraldehyde and stained with 0.25% crystal violet in 20% methanol (wt/vol). Each experiment was repeated in duplicate wells, and within each well, 9 randomly selected high-power microscopic fields (×200) were counted and expressed as a migration index.
2.11.
Cell-cell adhesion assay
Adherence of human neutrophils to endothelial cells was evaluated by fluorescent labeling of human neutrophils as previously described [25–27]. Briefly, purified human neutrophils (1.5 × 106 cells/mL, 200 μL/well) were labeled with Vybrant DiD dye and then added to washed and stimulated HUVECs. HUVEC monolayers were treated with TGFBIp (5 μg/mL) for 6 hours followed by treatment with POZ for 6 hours.
Expression of CAMs and receptor expression 2.12.
Expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and E-selectin on HUVECs was determined by whole-cell ELISA, as described previously [6,24]. Briefly, confluent monolayers of HUVECs were treated with TGFBIp (5 μg/mL) for 6 hours followed by POZ (10 or 20 μM) for another 6 hours (Table). The medium was removed, and cells were washed with PBS and fixed with 50 μL of 1% paraformaldehyde for 15 minutes at room temperature. After washing, 100 μL of mouse antihuman monoclonal antibodies (VCAM-1, ICAM-1, and E-selectin; Temecula, CA, USA; 1:50 each) were applied. After 1 hour (37°C, 5% CO2), the cells were washed 3 times, followed by application of 100 μL of 1:2000 peroxidase-conjugated
Table – POZ content of the experimental diets fed to mice Dose (μM)
Dose (μg/mouse)
Dose (μg/kg)
10 μM 20 μM
8.8 17.5
325.9 648.1
The average weight of mouse is 27 g, and the average blood volume is 2 mL.
In vivo permeability and leukocyte migration assay
For the in vivo study, male mice were anesthetized with 2% isoflurane (Forane; JW Pharmaceutical) in oxygen delivered via a small rodent gas anesthesia machine (RC2; Vetequip), first in a breathing chamber and then via a facemask. They were allowed to breath spontaneously during the procedure. Mice were treated with TGFBIp (0.1 mg/kg, intravenously) for 6 hours followed by treatment with POZ (8.8 or 17.5 μg/mouse) for 6 hours. For the in vivo permeability assay [23], 1% Evans blue dye solution in normal saline was injected intravenously into each mouse. Thirty minutes later, mice were euthanized and peritoneal exudates were collected by washing cavities with 5 mL of normal saline and centrifuging at 200g for 10 minutes. Absorbance of supernatants was read at 650 nm. Vascular permeabilities are expressed as μg of dye/mouse that leaked into the peritoneal cavity and were determined using a standard curve, as previously described [28,29]. For assessment of leukocyte migration [23], mice were euthanized after 6 hours and peritoneal cavities were washed with 5 mL of normal saline. Samples (20 μL) of peritoneal fluids obtained were mixed with 0.38 mL of Turk solution (0.01% crystal violet in 3% acetic acid), and the numbers of leukocytes were counted under a light microscope.
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2.13. Hematoxylin and eosin staining and histopathologic examination Male C57BL/6 mice underwent CLP and were administered POZ (8.8 or 17.5 μg/mouse) intravenously at 12 hours and 50 hours after CLP (n = 5). Mice were euthanized 96 hours after CLP. To analyze the phenotypic changes in the mice lungs [23], lung samples were removed from each mouse, washed 3 times in PBS (pH 7.4) to remove remaining blood, and fixed in 4% formaldehyde solution (Junsei, Tokyo, Japan) in PBS (pH 7.4) for 20 hours at 4°C. After fixation, the samples were dehydrated through a series of ethanol, embedded in paraffin, sectioned at 4-μm thickness, and placed on a slide. The slides were deparaffinized in a 60°C oven, rehydrated, and stained with hematoxylin (Sigma). To remove overstaining, the slides were quickly dipped 3 times in 0.3% acid alcohol and counterstained with eosin (Sigma). The excess stain was removed using a series of ethanol and xylene, and coverslips were placed on the slides. Light microscopic analyses of lung specimens were performed by blinded observation to evaluate pulmonary architecture, tissue edema, and infiltration of the inflammatory cells as previously defined [30]. The results were classified into 4 grades, where grade 1 represented normal histopathology; grade 2 indicated minimal neutrophil leukocyte infiltration; grade 3 represented moderate neutrophil leukocyte infiltration, perivascular edema formation, and partial destruction of pulmonary architecture; and, finally, grade 4 included dense neutrophil leukocyte infiltration, abscess formation, and complete destruction of pulmonary architecture.
2.14.
Measurements of organ injury markers
Plasma levels of aspartate transaminase (AST), alanine transaminase (ALT), blood urea nitrogen (BUN), and creatinine were measured using commercial assay kits (Pointe Scientific, Lincoln Park, MI, USA).
2.15.
Statistical analyses
All experiments were performed independently at least 3 times. Values are expressed as means ± SEM. The statistical significance of differences between test groups was evaluated by 1-way analysis of variance and Tukey post hoc test. Kaplan-Meier survival analysis was performed for evaluation of overall survival rates. A power analysis was performed using “R” software (Bell Laboratories, Murray Hill, NJ). SPSS for Windows, version 16.0 (SPSS, Chicago, IL, USA), was used to perform statistical analysis, and statistical significance was accepted for P values less than .05.
3.
Results and discussion
3.1.
Effects of POZ on LPS and CLP-mediated release of TGFBIp
Our previous study demonstrated that stimulation of TGFBIp release by LPS from human endothelial cells and 100 ng/mL LPS is sufficient to induce release of TGFBIp [5,6]. Similarly, in the current study, 100 ng/mL LPS stimulated the release of TGFBIp by HUVECs (Fig. 1A). To investigate the effects of POZ on LPS-mediated release of TGFBIp, HUVECs were stimulated with 100 ng/mL LPS for 1 hour, followed by treatment with
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increasing concentrations of POZ for 6 hours. As shown in Fig. 1A, POZ inhibited the release of TGFBIp in HUVECs, with an optimal effective concentration greater than 5 μM. However, in the absence of LPS pretreatment, POZ did not affect TGFBIp release (Fig. 1A). In order to confirm these effects in vivo, CLP-induced septic mice were used because this model more closely resembles human sepsis than LPSinduced endotoxemia [17]. As shown in Fig. 1B, treatment with POZ resulted in marked inhibition of CLP-induced release of TGFBIp. The average circulating blood volume for mice is 72 mL/kg [31]. Because the average weight of the mice used in the study was 27 g and the average blood volume was 2 mL, the amount of POZ (8.8 or 17.5 μg/mouse) injected yielded a maximum concentration of 10 or 20 μM in the peripheral blood. To determine the molecular mechanism by which POZ inhibited the release of LPS-mediated TGFBIp, we tested the effects of POZ on the transcriptional regulation of TGFBIp by LPS in HUVECs. Thus, we measured the effect of POZ on LPS-induced TGFBIp mRNA levels using real-time quantitative reverse transcriptase polymerase chain reaction. As shown in Fig. 1C, LPS induced an increase in the expression levels of TGFBIp mRNA and treatment with POZ resulted in decreased expression levels of LPS-induced TGFBIp mRNA. Next, we investigated the effects of POZ on expression of the TGFBIp receptors, integrins αvβ3 and αvβ5, in HUVECs [6]. As shown in Fig. 1C, treatment with LPS resulted in a more than 4fold increase in expression of αvβ5 in HUVECs and treatment with POZ resulted in significantly inhibited expression of αvβ5. However, consistent with previous findings [6], the expression of integrin αvβ3 was not changed by LPS [6] or POZ (Fig. 1D). Therefore, the inhibitory effects of POZ on the release of TGFBIp were mediated by suppression of TGFBIp receptor (integrin αvβ5). To assess the cytotoxicity of POZ, cell viability assays were performed in HUVECs treated with POZ for 24 hours. At concentrations up to 50 μM, POZ did not affect cell viability (Fig. 1E). High plasma concentrations of TGFBIp in patients with sepsis are known to be related to the severity of sepsis [6], and pharmacologic inhibition of TGFBIp is known to improve survival in animal models of sepsis [5]. Therefore, prevention of CLP-induced release of TGFBIp by POZ suggests the potential for use of POZ in the treatment of vascular inflammatory diseases.
3.2. Effect of POZ on TGFBIp-mediated vascular barrier disruption A permeability assay was performed to determine the effects of POZ on the barrier integrity of HUVECs. Treatment with 20 μM POZ alone did not alter barrier integrity (Fig. 2A). In contrast, TGFBIp is known to cause cleavage and disruption of endothelial barrier integrity [5,6]. Thus, HUVECs were treated with various concentrations of POZ for 6 hours after the addition of TGFBIp (5 μg/mL). As shown in Fig. 2A, treatment with POZ resulted in a dose-dependent decrease in TGFBIp-mediated disruption of barrier integrity. TGFBIp-mediated vascular permeability in mice was assessed in order to confirm this vascular barrier protective effect in vivo. As shown in Fig. 2B, treatment with POZ resulted in markedly inhibited peritoneal leakage of dye induced by TGFBIp. Cytoskeletal proteins are important for the maintenance of cell integrity and shape [32]. In addition, redistribution of
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Fig. 1 – Effects of POZ on release of TGFBIp and expression of receptors. A, HUVECs were treated with the indicated concentrations of POZ for 6 hours after stimulation with LPS (100 ng/mL, 1 hour), and TGFBIp release was measured by ELISA. B, Male C57BL/6 mice that underwent CLP were administered POZ at 10 or 20 μM/mouse each intravenously 12 hours after CLP (n = 5). Mice were euthanized 24 hours after CLP. Serum TGFBIp levels were measured by ELISA. C, The same as panel A, except that real-time qRT-PCR analysis was performed using specific primers for TGFBIp and actin, as described in the Materials and Methods section. D, Confluent HUVECs were activated with LPS (100 ng/mL, 3 hours), followed by incubation with POZ for 6 hours. Expression of αvβ3 (white bar) and αvβ5 (black bar) was determined by cell-based ELISA. E, Effect of POZ on cellular viability was measured by MTT assay. D = 0.2% DMSO is the vehicle control. Values are expressed as means ± SEM (triplicate, n = 5/group). *P < .05 vs LPS alone (A, C, D) or CLP alone (B). DMSO indicates dimethyl sulfoxide; qRT-PCR, quantitative reverse transcriptase polymerase chain reaction.
the actin cytoskeleton, detachment of cells, and loss of cellcell contact due to cytokine stimulation are all associated with an increased endothelial monolayer permeability [33,34]. Therefore, we next examined the effects of POZ on actin cytoskeletal arrangement in HUVECs by immunofluorescence staining of HUVEC monolayers with F-actin–labeled fluorescein phalloidin. Control HUVECs exhibited a random distribution of F-actin throughout the cells, with some localization of actin filament bundles at the cell boundaries (Fig. 2C). Barrier disruption in HUVECs induced by TGFBIp treatment (5 μg/mL) was accompanied by the formation of paracellular gaps (shown by arrows). In addition, treatment
with POZ (10 or 20 μM) inhibited the formation of TGFBIpinduced paracellular gaps with the formation of dense F-actin rings (Fig. 2C). These results suggest that POZ treatment inhibited the TGFBIp-mediated morphologic changes and gap formation in endothelial cells, which are associated with F-actin redistribution, thereby increasing vascular barrier integrity. Sepsis inducers, such as high-mobility group box 1 and LPS, are known to induce proinflammatory responses by promoting phosphorylation of p38 MAPK [35–39]. Therefore, we determined whether TGFBIp could also enhance the phosphorylation of p38 MAPK and, if so, whether POZ
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Fig. 2 – Effects of POZ on TGFBIp-mediated permeability in vitro and in vivo. A, The effects of posttreatment with different concentrations of POZ for 6 hours on the barrier disruptions caused by TGFBIp (5 μg/mL, 6 hours) were monitored by measuring the flux of Evans blue bound albumin across HUVECs. B, The effects of POZ at 10 or 20 μM/mouse on TGFBIpinduced (0.1 mg/kg, intravenously) vascular permeability in mice were examined by measuring the amount of Evans blue in peritoneal washings (expressed μg/mouse, n = 5). C, Staining for F-actin. HUVEC monolayers grown on glass coverslips were stimulated with TGFBIp for 1 hour, treated with POZ for 6 hours, and stained for F-actin. Arrows indicate intercellular gaps. D, HUVECs were activated with TGFBIp (5 μg/mL, 6 hours), followed by treatment with different concentrations of POZ for 6 hours. The effects of POZ on TGFBIp-mediated expression of phospho p38 were determined by ELISA. Results are expressed as the means ± SEM of at least 3 independent experiments (triplicate, n = 5/group). *P < .05 vs TGFBIp alone.
inhibited TGFBIp-induced activation of p38 MAPK in HUVECs. As shown in Fig. 2D, TGFBIp induced the activation of p38 MAPK, which was significantly inhibited by treatment with POZ. These findings demonstrate inhibition of TGFBIp-mediated endothelial disruption and maintenance of human endothelial cell barrier integrity by POZ in mice treated with TGFBIp.
3.3. Effects of POZ on TGFBIp-mediated CAM expression, neutrophil adhesion, and migration Several studies have reported that TGFBIp enhanced the expression of CAMs, such as ICAM-1, VCAM-1, and E-selectin, on the surfaces of human cells, thereby promoting adhesion and migration of leukocytes across the endothelium to sites of inflammation [3,6,40,41]. According to our findings, TGFBIp induced up-regulation of the surface expression of VCAM-1, ICAM-1, and E-selectin (Fig. 3A), and POZ inhibited this effect, suggesting that the inhibitory effects of POZ on the expression of CAMs are mediated via attenuation of the TGFBIp signaling pathway by POZ. In addition, elevated expression of CAMs corresponded well with enhanced binding of human neutrophils to TGFBIp-activated endothelial cells, followed by their migration. In addition, treatment with POZ resulted in down-
regulation of human neutrophils adherence and their subsequent migration across activated endothelial cells in a concentration-dependent manner (Fig. 3B, C). These results suggest that POZ not only inhibits endotoxin-mediated release of TGFBIp in endothelial cells but also down-regulates the proinflammatory signaling effect caused by the release of TGFBIp, thereby inhibiting amplification of inflammatory pathways by nuclear cytokines. To confirm this effect in vivo, we examined TGFBIp-induced migration of leukocytes in mice. TGFBIp was found to stimulate migration of leukocytes into the peritoneal cavities of mice, and treatment with POZ resulted in a significant reduction of peritoneal leukocyte counts (Fig. 3D). Experiments on CAMs are widely performed in vitro for study of regulation of the interactions between leukocytes and endothelial cells [42,43]. In the current study, treatment with POZ resulted in down-regulation of TGFBIpinduced levels of VCAM-1, ICAM-1, and E-selectin, suggesting that POZ inhibits the adhesion and migration of leukocytes to inflamed endothelium.
3.4.
Protective effect of POZ in CLP-induced septic mortality
Sepsis is a systemic response to serious infection and has a poor prognosis when it is associated with organ dysfunction,
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Fig. 3 – Effects of POZ on TGFBIp-mediated proinflammatory responses. A-C, HUVECs were stimulated with TGFBIp (5 μg/mL) for 6 hours, followed by treatment with POZ for 6 hours. TGFBIp-mediated (A) expression of VCAM-1 (white bar), ICAM-1 (gray bar), and E-selectin (black bar) in HUVECs; (B) adherence of human neutrophils to HUVEC monolayers; and (C) migration of human neutrophils through HUVEC monolayers were analyzed. D, The effects of posttreatment with POZ at 10 or 20 μM/mouse on leukocyte migration into the peritoneal cavities of mice caused by TGFBIp (0.1 mg/kg, intravenously) were analyzed. Values are expressed as means ± SEM (triplicate, n = 5/group). *P < .05 vs TGFBIp.
hypoperfusion, or hypotension [39,44]. Based on the abovedescribed findings, we hypothesized that treatment with POZ would result in reduced mortality in our CLP-induced sepsis mouse model. To investigate the question of whether POZ protects mice from CLP-induced sepsis lethality, POZ was administered to mice after CLP. Twenty-four hours after the operation, animals manifested signs of sepsis such as shivering, bristled hair, and weakness. Administration of POZ (8.8 or 17.5 μg/mouse) 12 hours after CLP did not prevent CLP-induced death (data not shown); therefore, it was administered 2 times (once 12 hours after CLP and once 50 hours after CLP), which resulted in an increase in the survival rate from 30% to 50%, according to the Kaplan-Meier survival analysis (P < .0001; Fig. 4A). This marked survival benefit achieved by administration of POZ suggests that suppression of TGFBIp release and of TGFBIp-mediated inflammatory responses provides a therapeutic strategy for management of sepsis and septic shock.
3.5. Protective effect of POZ against CLP-induced pulmonary and tissue injury To confirm the protective effects of POZ on CLP-induced death, we determined the effects of POZ on CLP-induced pulmonary injury. There were no significant differences between lungs of sham and sham + POZ in light microscopic observations (data not shown). In the CLP group, interstitial edema with massive infiltration of the inflammatory cells into the
interstitium and alveolar spaces were observed, and the pulmonary architecture was severely damaged (Fig. 4B, C). These morphologic changes were less pronounced in the CLP + POZ group (Fig. 4B, C). Systemic inflammation during sepsis frequently causes multiple-organ failure, in which the liver and kidneys are major target organs [45]. Cecal ligation and puncture resulted in significant increases in the plasma levels of ALT and AST (markers of hepatic injury; Fig. 4D), and creatinine and BUN (markers of renal injury; Fig. 4E, F), which were reduced by POZ. Another important marker of tissue injury, lactate dehydrogenase, was also reduced by POZ in CLP-operated mice (Fig. 4G). In this study, we tested the hypothesis that treatment with POZ would suppress TGFBIp- or CLP-induced septic responses in human endothelial cells and mice. The results showed that POZ suppressed LPS-induced secretion and mRNA expression of TGFBIp or CLP-induced TGFBIp secretion. Polyozellin inhibited TGFBIp-mediated hyperpermeability, activation of p38, up-regulation of CAM expression, and adhesion and migration of leukocytes to HUVECs. Finally, POZ protected against CLP-induced lethality and organ damage. Therefore, the data are in accordance with our hypothesis that POZ can protect against TGFBIp- or CLP-induced septic responses in human endothelial cells and mice. The molecular mechanism of the anti-inflammatory effects of POZ against the TGFBIp-mediated septic response may be mediated by the suppression of TGFBIp release and transcriptional suppression of TGFBIp mRNA (Fig. 1A-C), the
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Fig. 4 – Effects of POZ on lethality or pulmonary injury after CLP. A, Male C57BL/6 mice (n = 20) were administered POZ (10 μM/mouse, □; 20 μM/mouse, ■) at 12 hours and 50 hours after CLP. Animal survival was monitored every 6 hours after CLP for 132 hours. Control CLP mice (●) and sham-operated mice (○) were administered sterile saline (n = 20). The Kaplan-Meier survival analysis was used for determination of overall survival rates vs CLP-treated mice. (B) The same as panel A, except that mice were euthanized 96 hours after CLP. Histopathologic scores of the lung tissue were recorded as described in the Materials and Methods section. *P < .05 vs CLP. C, Photomicrographs of lung tissues (hematoxylin and eosin staining, ×200). Sham group (grade 1); CLP group (grade 3); right, CLP + POZ (10 or 20 μM/mouse; grade 2). Illustrations indicate representative images from 3 independent experiments. D-G, The same as panels B and C, except that mice were bled and euthanized. Levels of AST (D), ALT (D), creatinine (E), BUN (F), and lactate dehydrogenase (G) in plasma were measured. Values are expressed as means ± SEM (triplicate, n = 20/group). *P < .05 vs CLP. LDH indicates lactate dehydrogenase. expressions of TGFBIp receptor (integrin αvβ5; Fig. 1D), and TGFBIp-mediated hyperpermeability (Fig. 2A-C) via suppression of the activation of p38 (Fig. 2D). Furthermore, the inhibitory mechanism of POZ on the interaction between leukocytes and endothelial cells is mediated by the inhibition of the expression of CAMs such as VCAM, ICAM, and Eselectin (Fig. 3). The possible main target of POZ in TGFBIpmediated septic response could be the interactions between released TGFBIp and its receptor (integrin αvβ5), because the binding of the ligand (TGFBIp) to its receptor (αvβ5) mediated
severe vascular inflammatory responses downstream such as hyperpermeabililty, adhesion, and migration of leukocytes toward endothelial cells [5,6]. Based on the current findings, POZ has potential as a therapeutic agent for severe vascular inflammatory diseases, such as sepsis and septic shock. The main limitation of this study was the inability to determine the pharmacokinetics of POZ. After POZ is injected intravenously, it undergoes absorption, distribution, metabolism, and excretion, which describes the disposition of a
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pharmaceutical compound within an organism. These 4 criteria all influence the POZ levels, kinetics, and the overall performance of the drug. Thus, additional work will be required to elucidate the pharmacokinetic properties of POZ in vivo. Our results demonstrate that POZ inhibits both LPS and CLP-mediated release of TGFBIp, expression of TGFBIp receptor (integrin αvβ5), and TGFBIp-mediated barrier disruption through increases in barrier integrity and inhibition of CAM expression. In addition, POZ reduces human neutrophil adhesion and migration toward HUVECs. These barrier protective effects of POZ were confirmed in a mouse model, in which treatment with POZ resulted in a reduction in TGFBIp-induced mortality. Our findings indicate that POZ merits use as a potential therapeutic agent for severe vascular inflammatory diseases, such as sepsis and septic shock.
Authorship B.J. and J-S.B. designed the study; B.J. and E-J.Y. conducted the research; B.J., E-J.Y., and J-S.B. analyzed data; and J-S.B. wrote the manuscript. All authors read and approved the final manuscript.
Acknowledgment This research was supported by the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Science, ICT & Future Planning (2014R1A2A1A11049526) and by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute), funded by the Ministry of Health & Welfare, Republic of Korea (Grant No. HI15C0001).
Conflict of interest statement The authors declare no conflicts of interest.
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Available online at www.sciencedirect.com
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Original Research
Suppressive effects of polyozellin on TGFBIpmediated septic responses in human endothelial cells and mice Byeongjin Jung, Eun-Ju Yang, Jong-Sup Bae⁎ College of Pharmacy, CMRI, Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu 702-701, Republic of Korea
ARTI CLE I NFO
A BS TRACT
Article history:
Polyozellus multiplex (Thelephoraceae) is a wild mushroom in Korea and Japan and is usually
Received 4 October 2015
harvested in early autumn for food. Polyozellin, a major constituent of the edible
Revised 8 December 2015
mushroom P multiplex, has been known to exhibit biological activities such as
Accepted 16 December 2015
antioxidative and anti-inflammatory effects. Transforming growth factor β–induced protein (TGFBIp) is an extracellular matrix protein whose expression in several cell types is greatly increased by TGF-β. TGFBIp is released by human umbilical vein endothelial cells
Keywords:
and functions as a mediator of experimental sepsis. We hypothesized that polyozellin could
Polyozellin
reduce TGFBIp-mediated severe inflammatory responses in human endothelial cells and
TGFBIp
mice. Here, we investigated the antiseptic effects and underlying mechanisms of
Sepsis
polyozellin against TGFBIp-mediated septic responses. Polyozellin effectively inhibited
Severe inflammation
lipopolysaccharide-induced release of TGFBIp and suppressed TGFBIp-mediated septic
Mice
responses. In addition, polyozellin suppressed cecal ligation and puncture–induced sepsis lethality and pulmonary injury. In conclusion, polyozellin suppressed TGFBIp-mediated and cecal ligation and puncture–induced septic responses. Therefore, polyozellin could be a potential therapeutic agent for treatment of various severe vascular inflammatory diseases via inhibition of the TGFBIp signaling pathway. © 2016 Elsevier Inc. All rights reserved.
1.
Introduction
Transforming growth factor β–induced protein (TGFBIp) is an extracellular matrix protein that can be highly expressed in various cell types [1–3]. TGFBIp contains an N-terminal secretory signal peptide, followed by a cysteine-rich domain, 4 internal homologous repeats (FAS1 domain), and a C-terminal
tripeptide Arg-Gly-Asp motif [1]. Several studies suggest that TGFBIp is involved in cell growth, cell differentiation, wound healing, tumorigenesis, and apoptosis [2–4]. Very recently, we reported that TGFBIp is a promising therapeutic target for the treatment of severe vascular inflammatory diseases, such as sepsis and septic shock [3,5]. In fact, blocking TGFBIp, even at later times after the onset of infection, has been shown to
Abbreviations: ALT, alanine transaminase; AST, aspartate transaminase; BUN, blood urea nitrogen; CAM, cell adhesion molecule; CLP, cecal ligation and puncture; HUVEC, human umbilical vein endothelial cell; LPS, lipopolysaccharide; POZ, polyozellin; TGFBIp, transforming growth factor β-induced protein. ⁎ Corresponding author. College of Pharmacy, Research Institute of Pharmaceutical Sciences, Kyungpook National University, 80 Dahakro, Buk-gu, Daegu 702-701, Republic of Korea. Tel.: +82 53 950 8570; fax: +82 53 950 8557. E-mail address: [email protected] (J.-S. Bae). http://dx.doi.org/10.1016/j.nutres.2015.12.009 0271-5317/© 2016 Elsevier Inc. All rights reserved.
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rescue mice from lethal sepsis [5]. TGFBIp also acts as a lethal mediator in conditions such as sepsis, in which serum TGFBIp levels are substantially increased [3,5]. Once released into the extracellular milieu, TGFBIp can bind to cell surface receptors, such as integrins αvβ3 and αvβ5, in human endothelial cells [6]. Polyozellus multiplex (PM; Thelephoraceae) is a wild mushroom in Korea and Japan and is usually harvested in early autumn for food. The chemical components of PM, polyozellin (POZ) [7], thelephoric acid [8,9], polyozellic acid [8], kynapcin-9 [8], kynapcin-12 [10], kynapcin-13 [11], kynapcin-24 [12], and kynapcin-28 [11], have been isolated and characterized. Moreover, PM shows a variety of biological activities such as anticancer properties [13], inhibitory effects on proryl endopeptidase activity [7], and lipid peroxidation [14]. In HT-29 intestinal epithelial cells derived from human, POZ had an inhibitory effect on interleukin-8 and matrix metalloproteinase-7 production mediated by tumor necrosis factor α [15]. In addition, POZ has demonstrated antioxidative [16] and prolyl endopeptidase inhibitory activities [7] in a cell-free system. However, to the best of our knowledge, the effects of POZ on TGFBIp-mediated septic responses have not been studied yet. Based on previous reports, which demonstrated the potential effects of TGFBIp on vascular inflammatory responses [3,5] and antioxidant activities of POZ [16], we hypothesized that treatment with POZ would suppress TGFBIp- or cecal ligation and puncture (CLP)–induced septic responses in human endothelial cells and mice. To test our hypothesis, we used human endothelial cells and male C57BL/6 mice. The in vitro experimental design with exposure of human umbilical vein endothelial cells (HUVECs) to lipopolysaccharide (LPS) enables us to determine the effects of POZ on the secretion of TGFBIp protein and expression of TGFBIp messenger RNA (mRNA) and its receptors (integrin αvβ3 and αvβ5). To investigate the antiseptic effects of POZ in TGFBIpmediated septic responses, the following experimental techniques were used: hyperpermeability assessment, F-actin staining, assessing expression of cell adhesion molecules (CAMs), and determining adhesion and migration of leukocytes to HUVECs. For the in vivo assessment, CLP-induced septic mice were used because this model more closely resembles human sepsis [17]. In the CLP-induced septic model, we also tested the effects of POZ on lethality, pulmonary injury, and several organ injury markers.
2.
Methods and materials
2.1.
Reagents
whole blood (15 mL) obtained by venipuncture from 5 healthy volunteers and maintained as previously described [20].
2.3.
2.2.
Cell culture
Primary HUVECs were obtained from Cambrex Bio Science (Charles City, IA, USA) and maintained as described previously [6,18,19]. All experiments were carried out with HUVECs at passages 3 to 5. Human neutrophils were freshly isolated from
Animals and husbandry
Male C57BL/6 mice (6-7 weeks old, weighing 27 g), purchased from Orient Bio Co (Sungnam, Republic of Korea), were used after a 12-day acclimatization period. Animals were housed 5 per polycarbonate cage under controlled temperature (20°C-25°C) and humidity (40%-45%) and a 12:12-hour light/dark cycle. Animals received a normal rodent pellet diet and water ad libitum during acclimatization. Mice were euthanized by CO2 anesthesia followed by cervical dislocation [21]. All animals were treated in accordance with the Guidelines for the Care and Use of Laboratory Animals issued by Kyungpook National University (institutional review board no. KNU 2014-13).
2.4.
Cecal ligation and puncture
For induction of sepsis, male mice were anesthetized with 2% isoflurane (Forane; JW Pharmaceutical, Seoul, South Korea) in oxygen delivered via a small rodent gas anesthesia machine (RC2; Vetequip, Pleasanton, CA, USA), first in a breathing chamber and then via a facemask. They were allowed to breath spontaneously during the procedure. The CLP-induced sepsis model was prepared as previously described [6]. This protocol was approved by the Animal Care Committee at Kyungpook National University prior to conducting the study (institutional review board no. KNU 2012-13).
2.5.
Cell viability assay
3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) was used as an indicator of cell viability as previously described [22]. Cells were grown in 96-well plates at a density of 5 × 103 cells/well. After 24 hours, cells were washed with fresh medium, followed by treatment with POZ. After an incubation period of 48 hours, cells were washed and 100 μL of MTT (1 mg/ mL) was added, followed by incubation for 4 hours. Finally, dimethyl sulfoxide (150 μL) was added in order to solubilize the formazan salt formed, and the amount of formazan salt was determined by measuring the optical density at 540 nm using a microplate reader (Tecan Austria GmbH, Grödig, Austria).
2.6.
Polyozellin was isolated from the methanolic extract of PM by performing column chromatography, as in our previous study [7,16]. Evans blue and crystal violet were obtained from Sigma (St Louis, MO, USA). Vybrant DiD (used at 5 μM) was obtained from Invitrogen (Carlsbad, CA, USA). TGFBIp protein was purified as described previously [6].
381
Enzyme-linked immunosorbent assay for TGFBIp
TGFBIp concentrations in cell culture media or mouse serum were determined by competitive enzyme-linked immunosorbent assay (ELISA), as described previously [3,6]. Ninety-six– well plastic flat microtiter plates (Corning, NY, USA) were coated with TGFBIp protein in 20 mM carbonate-bicarbonate buffer (pH 9.6) with 0.02% sodium azide overnight at 4°C. The plates were rinsed 3 times with phosphate-buffered saline (PBS)–0.05% Tween 20 (PBS-T) and kept at 4°C. Lyophilized culture media was preincubated with anti-TGFBIp antibodies (diluted 1:2000 in PBS-T) in 96-well plastic round microtiter plates for 90 minutes at 37°C. Preincubated samples were transferred to a precoated plate and incubated for 30 minutes at room temperature. The plates were rinsed 3 times with PBS-T and incubated for 90 minutes at room temperature with
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peroxidase-conjugated antirabbit IgG antibodies (diluted 1:2000 in PBS-T; Amersham Pharmacia Biotech, Uppsala, Sweden). The plates were rinsed 3 times with PBS-T and incubated for 60 minutes at room temperature in the dark with 200 μL substrate solution (100 μg/mL o-phenylenediamine and 0.003% H2O2). After stopping the reaction with 50 μL of 8 N H2SO4, the absorbance was read at 490 nm.
2.7.
antimouse IgG antibodies (Sigma) for 1 hour. The cells were washed again 3 times and developed using ophenylenediamene substrate (Sigma). Colorimetric analysis was performed by measuring absorbance at 490 nm. All measurements were performed in triplicate wells. The same experimental procedures were used for monitoring the cell surface expression of αvβ3 and αvβ5 using specific antibodies obtained from EMD Millipore (Billerica, MA, USA).
Permeability assay in vitro 2.10.
Permeability was quantitated by spectrophotometric measurement of the flux of Evans blue–bound albumin across functional HUVEC monolayers using a modified 2-compartment chamber model, as previously described [6]. Briefly, HUVECs were plated (5 × 104 cells/well) in transwells with a pore size of 3 μm and a diameter of 12 mm for 3 days. The confluent monolayers were treated with TGFBIp (5 μg/mL for 6 hours) followed by incubation with POZ for 6 hours.
2.8.
Immunofluorescence staining
HUVECs were grown to confluence on glass coverslips coated with 0.05% poly-L-lysine in complete media containing 10% fetal bovine serum and maintained for 48 hours. Cells were then stimulated with TGFBIp (5 μg/mL) for 16 hours with or without 6 hours POZ (10 or 20 μM). For cytoskeletal staining [23], the cells were fixed in 4% formaldehyde in PBS (vol/vol) for 15 minutes at room temperature, permeabilized in 0.05% Triton X-100 in PBS for 15 minutes, and blocked in blocking buffer (5% bovine serum albumin in PBS) overnight at 4°C. Then, the cells were incubated with F-actin–labeled fluorescein phalloidin (F 432; Molecular Probes, Invitrogen) and visualized by confocal microscopy at a ×630 magnification (TCS-Sp5; Leica Microsystem, Wetzlar, Germany).
2.9.
Migration assay in vitro
Migration assays were performed in transwell plates with a diameter of 6.5 mm, with filters having a pore size of 8 μm. HUVECs (6 × 104) were cultured for 3 days in order to obtain confluent endothelial monolayers. Before the addition of human neutrophils to the upper compartment, cell monolayers were treated with TGFBIp (5 μg/mL for 6 hours) followed by treatment with POZ for 6 hours. Cells in the upper chamber of the filter were aspirated, and nonmigrating cells on top of the filter were removed using a cotton swab. Human neutrophils on the lower side of the filter were fixed with 8% glutaraldehyde and stained with 0.25% crystal violet in 20% methanol (wt/vol). Each experiment was repeated in duplicate wells, and within each well, 9 randomly selected high-power microscopic fields (×200) were counted and expressed as a migration index.
2.11.
Cell-cell adhesion assay
Adherence of human neutrophils to endothelial cells was evaluated by fluorescent labeling of human neutrophils as previously described [25–27]. Briefly, purified human neutrophils (1.5 × 106 cells/mL, 200 μL/well) were labeled with Vybrant DiD dye and then added to washed and stimulated HUVECs. HUVEC monolayers were treated with TGFBIp (5 μg/mL) for 6 hours followed by treatment with POZ for 6 hours.
Expression of CAMs and receptor expression 2.12.
Expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and E-selectin on HUVECs was determined by whole-cell ELISA, as described previously [6,24]. Briefly, confluent monolayers of HUVECs were treated with TGFBIp (5 μg/mL) for 6 hours followed by POZ (10 or 20 μM) for another 6 hours (Table). The medium was removed, and cells were washed with PBS and fixed with 50 μL of 1% paraformaldehyde for 15 minutes at room temperature. After washing, 100 μL of mouse antihuman monoclonal antibodies (VCAM-1, ICAM-1, and E-selectin; Temecula, CA, USA; 1:50 each) were applied. After 1 hour (37°C, 5% CO2), the cells were washed 3 times, followed by application of 100 μL of 1:2000 peroxidase-conjugated
Table – POZ content of the experimental diets fed to mice Dose (μM)
Dose (μg/mouse)
Dose (μg/kg)
10 μM 20 μM
8.8 17.5
325.9 648.1
The average weight of mouse is 27 g, and the average blood volume is 2 mL.
In vivo permeability and leukocyte migration assay
For the in vivo study, male mice were anesthetized with 2% isoflurane (Forane; JW Pharmaceutical) in oxygen delivered via a small rodent gas anesthesia machine (RC2; Vetequip), first in a breathing chamber and then via a facemask. They were allowed to breath spontaneously during the procedure. Mice were treated with TGFBIp (0.1 mg/kg, intravenously) for 6 hours followed by treatment with POZ (8.8 or 17.5 μg/mouse) for 6 hours. For the in vivo permeability assay [23], 1% Evans blue dye solution in normal saline was injected intravenously into each mouse. Thirty minutes later, mice were euthanized and peritoneal exudates were collected by washing cavities with 5 mL of normal saline and centrifuging at 200g for 10 minutes. Absorbance of supernatants was read at 650 nm. Vascular permeabilities are expressed as μg of dye/mouse that leaked into the peritoneal cavity and were determined using a standard curve, as previously described [28,29]. For assessment of leukocyte migration [23], mice were euthanized after 6 hours and peritoneal cavities were washed with 5 mL of normal saline. Samples (20 μL) of peritoneal fluids obtained were mixed with 0.38 mL of Turk solution (0.01% crystal violet in 3% acetic acid), and the numbers of leukocytes were counted under a light microscope.
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2.13. Hematoxylin and eosin staining and histopathologic examination Male C57BL/6 mice underwent CLP and were administered POZ (8.8 or 17.5 μg/mouse) intravenously at 12 hours and 50 hours after CLP (n = 5). Mice were euthanized 96 hours after CLP. To analyze the phenotypic changes in the mice lungs [23], lung samples were removed from each mouse, washed 3 times in PBS (pH 7.4) to remove remaining blood, and fixed in 4% formaldehyde solution (Junsei, Tokyo, Japan) in PBS (pH 7.4) for 20 hours at 4°C. After fixation, the samples were dehydrated through a series of ethanol, embedded in paraffin, sectioned at 4-μm thickness, and placed on a slide. The slides were deparaffinized in a 60°C oven, rehydrated, and stained with hematoxylin (Sigma). To remove overstaining, the slides were quickly dipped 3 times in 0.3% acid alcohol and counterstained with eosin (Sigma). The excess stain was removed using a series of ethanol and xylene, and coverslips were placed on the slides. Light microscopic analyses of lung specimens were performed by blinded observation to evaluate pulmonary architecture, tissue edema, and infiltration of the inflammatory cells as previously defined [30]. The results were classified into 4 grades, where grade 1 represented normal histopathology; grade 2 indicated minimal neutrophil leukocyte infiltration; grade 3 represented moderate neutrophil leukocyte infiltration, perivascular edema formation, and partial destruction of pulmonary architecture; and, finally, grade 4 included dense neutrophil leukocyte infiltration, abscess formation, and complete destruction of pulmonary architecture.
2.14.
Measurements of organ injury markers
Plasma levels of aspartate transaminase (AST), alanine transaminase (ALT), blood urea nitrogen (BUN), and creatinine were measured using commercial assay kits (Pointe Scientific, Lincoln Park, MI, USA).
2.15.
Statistical analyses
All experiments were performed independently at least 3 times. Values are expressed as means ± SEM. The statistical significance of differences between test groups was evaluated by 1-way analysis of variance and Tukey post hoc test. Kaplan-Meier survival analysis was performed for evaluation of overall survival rates. A power analysis was performed using “R” software (Bell Laboratories, Murray Hill, NJ). SPSS for Windows, version 16.0 (SPSS, Chicago, IL, USA), was used to perform statistical analysis, and statistical significance was accepted for P values less than .05.
3.
Results and discussion
3.1.
Effects of POZ on LPS and CLP-mediated release of TGFBIp
Our previous study demonstrated that stimulation of TGFBIp release by LPS from human endothelial cells and 100 ng/mL LPS is sufficient to induce release of TGFBIp [5,6]. Similarly, in the current study, 100 ng/mL LPS stimulated the release of TGFBIp by HUVECs (Fig. 1A). To investigate the effects of POZ on LPS-mediated release of TGFBIp, HUVECs were stimulated with 100 ng/mL LPS for 1 hour, followed by treatment with
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increasing concentrations of POZ for 6 hours. As shown in Fig. 1A, POZ inhibited the release of TGFBIp in HUVECs, with an optimal effective concentration greater than 5 μM. However, in the absence of LPS pretreatment, POZ did not affect TGFBIp release (Fig. 1A). In order to confirm these effects in vivo, CLP-induced septic mice were used because this model more closely resembles human sepsis than LPSinduced endotoxemia [17]. As shown in Fig. 1B, treatment with POZ resulted in marked inhibition of CLP-induced release of TGFBIp. The average circulating blood volume for mice is 72 mL/kg [31]. Because the average weight of the mice used in the study was 27 g and the average blood volume was 2 mL, the amount of POZ (8.8 or 17.5 μg/mouse) injected yielded a maximum concentration of 10 or 20 μM in the peripheral blood. To determine the molecular mechanism by which POZ inhibited the release of LPS-mediated TGFBIp, we tested the effects of POZ on the transcriptional regulation of TGFBIp by LPS in HUVECs. Thus, we measured the effect of POZ on LPS-induced TGFBIp mRNA levels using real-time quantitative reverse transcriptase polymerase chain reaction. As shown in Fig. 1C, LPS induced an increase in the expression levels of TGFBIp mRNA and treatment with POZ resulted in decreased expression levels of LPS-induced TGFBIp mRNA. Next, we investigated the effects of POZ on expression of the TGFBIp receptors, integrins αvβ3 and αvβ5, in HUVECs [6]. As shown in Fig. 1C, treatment with LPS resulted in a more than 4fold increase in expression of αvβ5 in HUVECs and treatment with POZ resulted in significantly inhibited expression of αvβ5. However, consistent with previous findings [6], the expression of integrin αvβ3 was not changed by LPS [6] or POZ (Fig. 1D). Therefore, the inhibitory effects of POZ on the release of TGFBIp were mediated by suppression of TGFBIp receptor (integrin αvβ5). To assess the cytotoxicity of POZ, cell viability assays were performed in HUVECs treated with POZ for 24 hours. At concentrations up to 50 μM, POZ did not affect cell viability (Fig. 1E). High plasma concentrations of TGFBIp in patients with sepsis are known to be related to the severity of sepsis [6], and pharmacologic inhibition of TGFBIp is known to improve survival in animal models of sepsis [5]. Therefore, prevention of CLP-induced release of TGFBIp by POZ suggests the potential for use of POZ in the treatment of vascular inflammatory diseases.
3.2. Effect of POZ on TGFBIp-mediated vascular barrier disruption A permeability assay was performed to determine the effects of POZ on the barrier integrity of HUVECs. Treatment with 20 μM POZ alone did not alter barrier integrity (Fig. 2A). In contrast, TGFBIp is known to cause cleavage and disruption of endothelial barrier integrity [5,6]. Thus, HUVECs were treated with various concentrations of POZ for 6 hours after the addition of TGFBIp (5 μg/mL). As shown in Fig. 2A, treatment with POZ resulted in a dose-dependent decrease in TGFBIp-mediated disruption of barrier integrity. TGFBIp-mediated vascular permeability in mice was assessed in order to confirm this vascular barrier protective effect in vivo. As shown in Fig. 2B, treatment with POZ resulted in markedly inhibited peritoneal leakage of dye induced by TGFBIp. Cytoskeletal proteins are important for the maintenance of cell integrity and shape [32]. In addition, redistribution of
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Fig. 1 – Effects of POZ on release of TGFBIp and expression of receptors. A, HUVECs were treated with the indicated concentrations of POZ for 6 hours after stimulation with LPS (100 ng/mL, 1 hour), and TGFBIp release was measured by ELISA. B, Male C57BL/6 mice that underwent CLP were administered POZ at 10 or 20 μM/mouse each intravenously 12 hours after CLP (n = 5). Mice were euthanized 24 hours after CLP. Serum TGFBIp levels were measured by ELISA. C, The same as panel A, except that real-time qRT-PCR analysis was performed using specific primers for TGFBIp and actin, as described in the Materials and Methods section. D, Confluent HUVECs were activated with LPS (100 ng/mL, 3 hours), followed by incubation with POZ for 6 hours. Expression of αvβ3 (white bar) and αvβ5 (black bar) was determined by cell-based ELISA. E, Effect of POZ on cellular viability was measured by MTT assay. D = 0.2% DMSO is the vehicle control. Values are expressed as means ± SEM (triplicate, n = 5/group). *P < .05 vs LPS alone (A, C, D) or CLP alone (B). DMSO indicates dimethyl sulfoxide; qRT-PCR, quantitative reverse transcriptase polymerase chain reaction.
the actin cytoskeleton, detachment of cells, and loss of cellcell contact due to cytokine stimulation are all associated with an increased endothelial monolayer permeability [33,34]. Therefore, we next examined the effects of POZ on actin cytoskeletal arrangement in HUVECs by immunofluorescence staining of HUVEC monolayers with F-actin–labeled fluorescein phalloidin. Control HUVECs exhibited a random distribution of F-actin throughout the cells, with some localization of actin filament bundles at the cell boundaries (Fig. 2C). Barrier disruption in HUVECs induced by TGFBIp treatment (5 μg/mL) was accompanied by the formation of paracellular gaps (shown by arrows). In addition, treatment
with POZ (10 or 20 μM) inhibited the formation of TGFBIpinduced paracellular gaps with the formation of dense F-actin rings (Fig. 2C). These results suggest that POZ treatment inhibited the TGFBIp-mediated morphologic changes and gap formation in endothelial cells, which are associated with F-actin redistribution, thereby increasing vascular barrier integrity. Sepsis inducers, such as high-mobility group box 1 and LPS, are known to induce proinflammatory responses by promoting phosphorylation of p38 MAPK [35–39]. Therefore, we determined whether TGFBIp could also enhance the phosphorylation of p38 MAPK and, if so, whether POZ
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Fig. 2 – Effects of POZ on TGFBIp-mediated permeability in vitro and in vivo. A, The effects of posttreatment with different concentrations of POZ for 6 hours on the barrier disruptions caused by TGFBIp (5 μg/mL, 6 hours) were monitored by measuring the flux of Evans blue bound albumin across HUVECs. B, The effects of POZ at 10 or 20 μM/mouse on TGFBIpinduced (0.1 mg/kg, intravenously) vascular permeability in mice were examined by measuring the amount of Evans blue in peritoneal washings (expressed μg/mouse, n = 5). C, Staining for F-actin. HUVEC monolayers grown on glass coverslips were stimulated with TGFBIp for 1 hour, treated with POZ for 6 hours, and stained for F-actin. Arrows indicate intercellular gaps. D, HUVECs were activated with TGFBIp (5 μg/mL, 6 hours), followed by treatment with different concentrations of POZ for 6 hours. The effects of POZ on TGFBIp-mediated expression of phospho p38 were determined by ELISA. Results are expressed as the means ± SEM of at least 3 independent experiments (triplicate, n = 5/group). *P < .05 vs TGFBIp alone.
inhibited TGFBIp-induced activation of p38 MAPK in HUVECs. As shown in Fig. 2D, TGFBIp induced the activation of p38 MAPK, which was significantly inhibited by treatment with POZ. These findings demonstrate inhibition of TGFBIp-mediated endothelial disruption and maintenance of human endothelial cell barrier integrity by POZ in mice treated with TGFBIp.
3.3. Effects of POZ on TGFBIp-mediated CAM expression, neutrophil adhesion, and migration Several studies have reported that TGFBIp enhanced the expression of CAMs, such as ICAM-1, VCAM-1, and E-selectin, on the surfaces of human cells, thereby promoting adhesion and migration of leukocytes across the endothelium to sites of inflammation [3,6,40,41]. According to our findings, TGFBIp induced up-regulation of the surface expression of VCAM-1, ICAM-1, and E-selectin (Fig. 3A), and POZ inhibited this effect, suggesting that the inhibitory effects of POZ on the expression of CAMs are mediated via attenuation of the TGFBIp signaling pathway by POZ. In addition, elevated expression of CAMs corresponded well with enhanced binding of human neutrophils to TGFBIp-activated endothelial cells, followed by their migration. In addition, treatment with POZ resulted in down-
regulation of human neutrophils adherence and their subsequent migration across activated endothelial cells in a concentration-dependent manner (Fig. 3B, C). These results suggest that POZ not only inhibits endotoxin-mediated release of TGFBIp in endothelial cells but also down-regulates the proinflammatory signaling effect caused by the release of TGFBIp, thereby inhibiting amplification of inflammatory pathways by nuclear cytokines. To confirm this effect in vivo, we examined TGFBIp-induced migration of leukocytes in mice. TGFBIp was found to stimulate migration of leukocytes into the peritoneal cavities of mice, and treatment with POZ resulted in a significant reduction of peritoneal leukocyte counts (Fig. 3D). Experiments on CAMs are widely performed in vitro for study of regulation of the interactions between leukocytes and endothelial cells [42,43]. In the current study, treatment with POZ resulted in down-regulation of TGFBIpinduced levels of VCAM-1, ICAM-1, and E-selectin, suggesting that POZ inhibits the adhesion and migration of leukocytes to inflamed endothelium.
3.4.
Protective effect of POZ in CLP-induced septic mortality
Sepsis is a systemic response to serious infection and has a poor prognosis when it is associated with organ dysfunction,
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Fig. 3 – Effects of POZ on TGFBIp-mediated proinflammatory responses. A-C, HUVECs were stimulated with TGFBIp (5 μg/mL) for 6 hours, followed by treatment with POZ for 6 hours. TGFBIp-mediated (A) expression of VCAM-1 (white bar), ICAM-1 (gray bar), and E-selectin (black bar) in HUVECs; (B) adherence of human neutrophils to HUVEC monolayers; and (C) migration of human neutrophils through HUVEC monolayers were analyzed. D, The effects of posttreatment with POZ at 10 or 20 μM/mouse on leukocyte migration into the peritoneal cavities of mice caused by TGFBIp (0.1 mg/kg, intravenously) were analyzed. Values are expressed as means ± SEM (triplicate, n = 5/group). *P < .05 vs TGFBIp.
hypoperfusion, or hypotension [39,44]. Based on the abovedescribed findings, we hypothesized that treatment with POZ would result in reduced mortality in our CLP-induced sepsis mouse model. To investigate the question of whether POZ protects mice from CLP-induced sepsis lethality, POZ was administered to mice after CLP. Twenty-four hours after the operation, animals manifested signs of sepsis such as shivering, bristled hair, and weakness. Administration of POZ (8.8 or 17.5 μg/mouse) 12 hours after CLP did not prevent CLP-induced death (data not shown); therefore, it was administered 2 times (once 12 hours after CLP and once 50 hours after CLP), which resulted in an increase in the survival rate from 30% to 50%, according to the Kaplan-Meier survival analysis (P < .0001; Fig. 4A). This marked survival benefit achieved by administration of POZ suggests that suppression of TGFBIp release and of TGFBIp-mediated inflammatory responses provides a therapeutic strategy for management of sepsis and septic shock.
3.5. Protective effect of POZ against CLP-induced pulmonary and tissue injury To confirm the protective effects of POZ on CLP-induced death, we determined the effects of POZ on CLP-induced pulmonary injury. There were no significant differences between lungs of sham and sham + POZ in light microscopic observations (data not shown). In the CLP group, interstitial edema with massive infiltration of the inflammatory cells into the
interstitium and alveolar spaces were observed, and the pulmonary architecture was severely damaged (Fig. 4B, C). These morphologic changes were less pronounced in the CLP + POZ group (Fig. 4B, C). Systemic inflammation during sepsis frequently causes multiple-organ failure, in which the liver and kidneys are major target organs [45]. Cecal ligation and puncture resulted in significant increases in the plasma levels of ALT and AST (markers of hepatic injury; Fig. 4D), and creatinine and BUN (markers of renal injury; Fig. 4E, F), which were reduced by POZ. Another important marker of tissue injury, lactate dehydrogenase, was also reduced by POZ in CLP-operated mice (Fig. 4G). In this study, we tested the hypothesis that treatment with POZ would suppress TGFBIp- or CLP-induced septic responses in human endothelial cells and mice. The results showed that POZ suppressed LPS-induced secretion and mRNA expression of TGFBIp or CLP-induced TGFBIp secretion. Polyozellin inhibited TGFBIp-mediated hyperpermeability, activation of p38, up-regulation of CAM expression, and adhesion and migration of leukocytes to HUVECs. Finally, POZ protected against CLP-induced lethality and organ damage. Therefore, the data are in accordance with our hypothesis that POZ can protect against TGFBIp- or CLP-induced septic responses in human endothelial cells and mice. The molecular mechanism of the anti-inflammatory effects of POZ against the TGFBIp-mediated septic response may be mediated by the suppression of TGFBIp release and transcriptional suppression of TGFBIp mRNA (Fig. 1A-C), the
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Fig. 4 – Effects of POZ on lethality or pulmonary injury after CLP. A, Male C57BL/6 mice (n = 20) were administered POZ (10 μM/mouse, □; 20 μM/mouse, ■) at 12 hours and 50 hours after CLP. Animal survival was monitored every 6 hours after CLP for 132 hours. Control CLP mice (●) and sham-operated mice (○) were administered sterile saline (n = 20). The Kaplan-Meier survival analysis was used for determination of overall survival rates vs CLP-treated mice. (B) The same as panel A, except that mice were euthanized 96 hours after CLP. Histopathologic scores of the lung tissue were recorded as described in the Materials and Methods section. *P < .05 vs CLP. C, Photomicrographs of lung tissues (hematoxylin and eosin staining, ×200). Sham group (grade 1); CLP group (grade 3); right, CLP + POZ (10 or 20 μM/mouse; grade 2). Illustrations indicate representative images from 3 independent experiments. D-G, The same as panels B and C, except that mice were bled and euthanized. Levels of AST (D), ALT (D), creatinine (E), BUN (F), and lactate dehydrogenase (G) in plasma were measured. Values are expressed as means ± SEM (triplicate, n = 20/group). *P < .05 vs CLP. LDH indicates lactate dehydrogenase. expressions of TGFBIp receptor (integrin αvβ5; Fig. 1D), and TGFBIp-mediated hyperpermeability (Fig. 2A-C) via suppression of the activation of p38 (Fig. 2D). Furthermore, the inhibitory mechanism of POZ on the interaction between leukocytes and endothelial cells is mediated by the inhibition of the expression of CAMs such as VCAM, ICAM, and Eselectin (Fig. 3). The possible main target of POZ in TGFBIpmediated septic response could be the interactions between released TGFBIp and its receptor (integrin αvβ5), because the binding of the ligand (TGFBIp) to its receptor (αvβ5) mediated
severe vascular inflammatory responses downstream such as hyperpermeabililty, adhesion, and migration of leukocytes toward endothelial cells [5,6]. Based on the current findings, POZ has potential as a therapeutic agent for severe vascular inflammatory diseases, such as sepsis and septic shock. The main limitation of this study was the inability to determine the pharmacokinetics of POZ. After POZ is injected intravenously, it undergoes absorption, distribution, metabolism, and excretion, which describes the disposition of a
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pharmaceutical compound within an organism. These 4 criteria all influence the POZ levels, kinetics, and the overall performance of the drug. Thus, additional work will be required to elucidate the pharmacokinetic properties of POZ in vivo. Our results demonstrate that POZ inhibits both LPS and CLP-mediated release of TGFBIp, expression of TGFBIp receptor (integrin αvβ5), and TGFBIp-mediated barrier disruption through increases in barrier integrity and inhibition of CAM expression. In addition, POZ reduces human neutrophil adhesion and migration toward HUVECs. These barrier protective effects of POZ were confirmed in a mouse model, in which treatment with POZ resulted in a reduction in TGFBIp-induced mortality. Our findings indicate that POZ merits use as a potential therapeutic agent for severe vascular inflammatory diseases, such as sepsis and septic shock.
Authorship B.J. and J-S.B. designed the study; B.J. and E-J.Y. conducted the research; B.J., E-J.Y., and J-S.B. analyzed data; and J-S.B. wrote the manuscript. All authors read and approved the final manuscript.
Acknowledgment This research was supported by the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Science, ICT & Future Planning (2014R1A2A1A11049526) and by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute), funded by the Ministry of Health & Welfare, Republic of Korea (Grant No. HI15C0001).
Conflict of interest statement The authors declare no conflicts of interest.
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