Hetrombopag, Athrombopoietinreceptor Agonist, Protected Cardiomyocytessurvival from Oxidative
Hetrombopag, AThrombopoietinReceptor Agonist, Protected CardiomyocytesSurvival from Oxidative Stress Damage As An Enhancer of Stem Cells
Nannan Zhou1, Jianchun Wang1, Xiaodong Li1, Yong Zhao1,Yuanyuan Sun1,ChengweiZou2[*]
1 Department of Geriatric Cardiology, Shandong Provincial Hospital Affiliated to Shandong University, Shandong University, Jinan, P.R. China
2 Department of Cardiovascular Surgery, Shandong Provincial Hospital Affiliated to Shandong University, Shandong University, Jinan, P.R.China
Online Supplementary material:
1.Materials and methods
All studies were reviewed and approved by the Medical Ethics Committee Shandong Provincial Hospital Affiliated to Shandong University, and were carried out in accordance with the Declaration of Helsinki.All rats received care in compliance with the principles of laboratory animal care and the Guide for Care and Use of Laboratory Animals of the National Institutes of Health.
1.1Reagents
(Z)-5-(3-(2-(1-(bicyclo[4.2.0]octa-1,3,5-trien-3-yl)-3-methyl-5-oxo-1H-pyrazol-4(5H)-ylidene)hydrazinyl)-2-hydroxyphenyl)furan-2-carboxylic acid (hetrombopag)and eltrombopag were synthesized by our team, as previously described in patentsWO-2009092276and WO-0189457. Cytokines including rhTPO (R&D Systems, Minneapolis, MN, USA), recombinant murine interleukin-3 (rm IL-3) (R&D Systems, Minneapolis, MN, USA), recombinant human flt-3 ligand (rhFL)(Amgen, Thousand Oaks, CA) and recombinant human colony-stimulating factor (rhCSF) (Amgen, Thousand Oaks, CA) were obtained as indicated.
1.2Cell lines and culture
Murine myeloid 32D clone 3 cellsandInterleukin-3 (IL-3) dependent murine BaF3 cellsused in this study were purchased fromAmerican Type Culture Collection (Manassas, VA, USA).32D cells were stably transfected with a human TPO receptor (c-mpl) expression plasmid or pcDNA3.1 control vector to yield 32D-mpl and 32D-pCDNA3.1 cell lines [1-2]. BaF3-hTPOR was established to transfect human c-mpl, and the BaF3 parental control line had no transfected human TPO receptor[3]. 32D-mpl and 32D-pCDNA3.1 cell lines were maintained inRPMI-1640 medium (Invitrogen, Carlsbad, CA, USA)with 2 mM L-glutamine (adjusted to contain 1.5g/L sodium bicarbonate, 4.5g/L glucose, 10 mM HEPES, 1.0 mM sodium pyruvate, and 2.5 ng/ml rmIL-3) supplemented with 10% heat-inactivated fetal bovine serum (JRH Biosciences, Lenexa, KS, USA).BaF3 cells and their transfectant were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 50 units/ml penicillin/streptomycin (Invitrogen), and WEHI conditioned media (BD Biosciences, San Jose, CA, USA). Cells were cultured at 37°Cin a humidified 5% CO2environment.
1.3Proliferation assay
Cells were seeded into a 96-well plate at a concentration of 2×105cells/ml and treated with different concentrations of drugs. Transfected TPO receptor-expressing BaF3 cells were incubated with hetrombopag or eltrombopag, both dissolved in dimethylsulfoxide (DMSO), or with rhTPOat the indicated concentration for approximately 22 h. Non-transfected BaF3 cells were incubated with hetrombopag, eltrombopag, rhTPO, or recombinant murine IL-3 (rmIL-3) at the indicated concentration for approximately 34 h. 32D-mpl and 32D-pCDNA3.1 cells were incubated with hetrombopag, eltrombopag, rhTPO, or recombinant murine IL-3 (rmIL-3) at the indicated concentration for approximately 72 h. Cell proliferation was assayed using WST-1/1-methoxy PMS reagent (cell counting kit,Dojin, Kumamoto, Japan) according to instructions from the manufacturer. A450/A650 was measured using a microplate reader (SPECTRAmax 340PC, Sunnyvale, CA, USA) immediately and 2 h after addition of WST-1/1-methoxy PMS reagent. Proliferation activity of the compound was calculated as the percentage of maximum proliferative activity of rhTPO for TPO receptor-expressing BaF3 cells and 32D-mpl cells.
1.4Western blotting
Human blood was drawn from healthy volunteers with a one-tenth volume of 3.8% sodium citrate added as an anticoagulant. Animal blood was drawn with a one-tenth volume of 3.8% sodium citrate as an anticoagulant. Platelet-rich plasma was prepared as previously described[3-4]. Platelets were resuspended in Tyrode's-HEPES buffer (pH 7.35), and were stimulated with hetrombopag or rhTPO at 37 °C for 15 min. The rat cardiac myocytes from each group in H2O2-induced damage experiment were washed twice with PBS. For western blot analysis, cells were lysed with RIPA buffer on ice for 30 minutes. Antibodies against STAT5, phospho-STAT5 were obtained from Cell Signaling Technology (Beverly, MA, USA). Total lysates were separated by electrophoresis using a 10 ~ 15% SDS-PAGE gel under reducing conditions and transferred to a sheet of polyvinylidenedifluoride membrane (Millipore, Bedford, MA, USA). The membranewith blotted protein was blocked for 1 h with blocking buffercontaining 5% nonfat dry milk and 0.05% Tween-20 in Trisbufferedsaline (TBS-T), followed by incubation with antibodies (diluted 1:1,000) in blocking buffer overnight at 4°C. Then, themembrane was washed three times with TBS-T for 30 min andincubated at room temperature for 1 h with diluted (1:2,000)secondary horseradish peroxidase-conjugated IgG(Santa Cruz Biotechnology, Inc.). Blots were developed using an ECL kit.
1.5 Protocol for heart infarction and cytokine determinations
Acquisition of cryopreservedhuman UCB MNCs:Cryopreserved (-196°C) mononuclear fractions of Human UCBwere obtained from DV Biologics (DV Biologics LLC, UK). Afterthawed at 37°C,these cryopreservedhuman UCB MNCs weretransferred into a centrifuge tube containing Isolyte S, pH 7.4 (B. Braun Medical Inc., Bethlehem Pennsylvania). The cells were extensively washed, centrifuged at 1,500 rpm for 7 minutes, the supernatant discarded, and the viability was >90%determined by Trypan blue dye exclusion technique.These cryopreservedhuman UCB MNCscontained approximately 1.3% CD34+ cells as determined by flow cytometry.
Heart infarction:Forty-four male Sprague-Dawley rats, weighing between 250 and 300 g, were anesthetized with 4%isoflurane, and coronary artery ligation andcytokine determinations were performed as previously described [4]. In brief, the pericardium was opened and the left coronary artery was permanently ligated with3~0 silk suture. The heart rate and arterial oxygen saturation were continuouslymonitored during the surgery in all rats.Infarction of the anterior wall of the left ventricle was confirmed by the presence of discoloration of the anterior myocardial wall and decreased wall motion after coronary artery ligation. Rats were given either 0.5 mLIsolytealone, or ex vivo expanded UCB MNCs (cell numbers from1×104to1× 106) orcryopreserved UCB MNCs(cell numbers from1×104to1× 106), in 0.5 mLIsolyte by injecting directly into the apex of the left ventricle within 2 h after the coronary artery ligation. The chest in each rat was then closed in three layers with 3 ~ 0 Vicryl and 6 ~ 0 Prolene suture, and each rat was allowed to recover. Buprenorphine (0.3mg/kg) was given for post-operative analgesia every 6 hours. Immunosuppressive therapy was not given to any rat.
Cytokine determinations: The hearts were then extracted from the rats at 12 h (n = 4) after myocardial infarction, and the ventricles dissected free from the heart. The ventricular tissue was placed in 750 μL of lysis buffer (20 mMTris, pH 7.5, 0.3 M NaCL, 2% sodium deoxycholate, 2% TX-100), plus a Protease Inhibitor Cocktail (Roche) and homogenized until there was no visible tissue. The homogenate was placed on a rocker plate for 2 hours at 4°C and then centrifuged at 12,000 RPM for 30 minutes at 4°C. The protein concentration of the supernatant was then determined by the Bradford Assay and bovine serum albumin was used as a standard. 50 μg of protein from each ventricular supernatant was added to 2 mL of blocking buffer (RayBiotech, Atlanta, GA) and each solution was placed on a separate cytokine array membrane (RayBiotech) in a plastic tray and incubated for 3 hours at room temperature on a rocker plate. The solution was then aspirated and each membrane incubated for 12 hours at 4°C with a mixture of biotinylated cytokine primary antibodies specific for tumor necrosis factor-alpha (TNF-α), monocytechemoattractantprotein (MCP), fractalkine, ciliaryneurotrophic factor (CNTF), macrophage inflammatory protein (MIP) and interferon-gamma (IFN-γ) (RayBiotech) on a rocker plate. The membranes were rinsed and HRP conjugated secondary antibodies were added to each membrane. The membranes were incubated at room temperature for 2 hours, subjected to an enhanced chemiluminescence detection kit (Amersham) for 60 seconds, and exposed on radiographic film (Amersham) for 90 seconds. The blots on the radiographic film were scanned and the protein densities determined with ImagePro image analysis software (Media Cybernetics, Bethesda, MD). All measurements were performed in duplicate, and the results for each cytokine were then normalized to positive controls present on each membrane. Mean ± SEM values were then determined.
1.6Statistics
Data were presented as the mean±SEM.Multiple comparisons were analyzed by one-way ANOVA followed by Bonferroni'spost-hoc test. Differences were considered statistically significant at p < 0.05.
2.Results
2.1Hetrombopag molecular structure and characterization
Hetrombopagbelongs to the biarylhydrazone class of compounds, with an empirical formula of C23H18N4O5and a molecular weight of approximately 430.13 D. Hetrombopag promoted the proliferation of human TPO receptor expressing BaF3 and 32D-mpl cell linesin a concentration-dependentmanner (Fig. 1A and 1B).The EC50values of hetrombopag were 1.2 nM (95% confidence intervals: 0.90 ~ 1.65nM) and 0.78 nM (95% confidence intervals: 0.54 ~ 1.14nM) for these two cells respectively, and the corresponding values of eltrombopag were 13.59nM(95% confidence intervals: 9.86 ~ 18.75nM) and 9.39 nM (95% confidence intervals: 7.24 ~ 12.17 nM). The maximum proliferative activity of hetrombopag was equivalent to that of rhTPO onBaF3/hTPOR cells, butgreater than that of rhTPO on32D-mpl cell lines (Fig. 1). Like eltrombopag or rhTPO, hetrombopag didn't promote proliferation of parental BaF3 cellsor 32D cells without expressing human TPO receptor (Fig. 1C and 1D), which indicated that the activity of this compound was dependent on the TPO receptor.rmIL-3stimulated proliferationof parental BaF3 cells and 32D cells without expressing human TPO receptor as a positive control (Fig. 1C and 1D).
Fig.1. Dose-dependent proliferation of BaF3/hTPOR and 32D-mpl cells induced by hetrombopag, eltrombopag and rhTPO(A and B). Hetrombopag, eltrombopag and rhTPOinduced proliferation of BaF3/hTPOR and 32D-mpl cells but notparental BaF3 cellsor 32D cells without expressing human TPO receptor (C and D).Dataare presented as mean ± SEM (n = 3).**p < 0.01vsrhTPO.
2.2Species specificity of hetrombopag
Given that most small molecular TPO receptor agonists including AS1670542 [3], AKR-501 [5], eltrombopag[6], generally exhibited species-specificity,we hypothesized that hetrobopag should exist species-specificity. The TPO receptor is expressed on the surface of platelets, and TPO induces signal transduction, including STAT5 [3, 5]. The species specificity of hetrombopag was examined by demonstrating the STAT5 activation in platelets of a number of species. As expected, hetrombopaginduced tyrosine phosphorylation of STAT5 in human blood platelets,but not inbeagle dog, rat, mice, and cynomolgus monkey platelets (Fig 2).In contrast,rhTPOinduced tyrosine phosphorylation of STAT5 in all species tested.
Fig 2.Tyrosine phosphorylation of STAT5 in platelets of different species stimulated by hetrombopag or rhTPO. Platelets from human, cynomolgus monkey,beagle dog, mice and rat were stimulated with hetrombopag or rhTPO. The lysates were separated usingSDS-PAGE gels.Blots were developed using an ECL kit.
2.3 Cytotoxic effects of hetrombopag on RAW 264.7 cells.
Fig 3. Cytotoxic effects of hetrombopag on RAW 264.7 cells. RAW264.7 cells (1.0 ×105cells/well) were treated with various concentration of hetrombopag (3, 10 and 30 μM) or LPS (10 μg/ml), and cells were incubated for 20h. After incubation, the cell viability were measured by MTT assay. The cell viability was defined as the % of non-stimulated cells, and the datawere expressed as mean ± SEM (n = 3). ***p < 0.001 in comparison with Control.
As shown in Fig 3,hetrombopagat the concentration of 3 ~ 30 μMhad no influence on cell viability of RAW 264.7 cells.
2.4 Myocardial cytokine release
Studies revealed that myocardial injury could cause the expression of inflamatory cytokines in the myocardium during the early stages of acute infarction, and the concentrations of TNF-α, MCP-1, fractalkine, CNTF, MIP and INF-γin the myocardium would be most pronounced 12 hours after the onset of the acute myocardial infarction [7-9]. And therefore, myocardial inflammatory cytokines concentrations at 12 h after myocardial infarction were detected.
Fig 4. Inflammatory cytokines release in Isolyte alone, cryopreserved human UCB MNCs or ex vivo expanded human UCB MNCs-treated rat hearts at 12h after myocardial infarction. A) TNF-a; B) MCP-1; C)fractalkine; D) MIP-1; E) CNTF;F) INF-γ.Results for each cytokine were normalized to positive controls present on each membrane. Mean ± SEM values were then determined.*p < 0.05, **p < 0.01 in comparison with control (Isolyte), and #p < 0.05, ##p < 0.01in comparison with the same number of cryopreserved human UCB MNCs.
As shown in Fig 4 of supplementary materials, ex vivo expanded human UCB MNCs can limit inflamatory cytokines release in the myocardium after acute LV infarction, and statistical differences were observed when the number of cells injected were greater than 1 × 104. Besides, ex vivo expanded human UCB MNCs were more effective than cryopreserved human UCB MNCs in inhibiting inflamatory cytokine release at the same number of cells, and statistical differences were observed at 1 ×104, 5× 104 and 1 ×105 groups. 1×105ex vivo expanded human UCB MNCs nearly achieve the maximal effect, and the number of cells increased from 105 to 106 couldn’t further reduce inflamatory cytokines. While the number of cryopreserved human UCB MNCs required for maximal effect was ten-fold increases and close to 106.
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Competing Interests: The authors have declared that no competing interests exist.