Selective inhibition of inositol hexakisphosphate kinases (IP6Ks) enhances mesenchymal stem cell engraftment and improves therapeutic efficacy for myocardial infarction
5-Diphosphoinositol pentakisphosphate (IP7), formed by a family of inositol hexakisphosphate kinases (IP6Ks), has been demonstrated to be a physiologic inhibitor of Akt. IP6K inhibition may increase Akt activation in mesenchymal stem cells (MSCs), resulting in enhanced cardiac protective effect after transplantation. The aim of this study was to investigate the role of IP6Ks for improving MSCs’ functional survival and cardiac protective effect after transplantation into infarcted mice hearts. Bone marrow-derived mesenchymal stem cells, isolated from dual-reporter firefly luciferase and enhanced green fluorescent protein positive (Fluc+–eGFP+) transgenic mice, were preconditioned with IP6Ks inhibitor TNP (0.5, 1, 5, and 10 μmol/L) for 2 h followed by 6 h of hypoxia and serum deprivation (H/SD) injury. TNP concentration dependently significantly decreased IP7 production with increased Akt phosphorylation. Moreover, TNP at 10 μmol/L significantly improved the viability and enhanced the paracrine effect of MSCs after H/SD. Furthermore, MSCs were transplanted into infarcted hearts with or without selective IP6Ks inhibition. Longitudinal in vivo bioluminescence imaging and immunofluorescent staining revealed that TNP pretreatment enhanced the survival of engrafted MSCs, which promoted the anti-apoptotic and pro-angiogenic efficacy of MSCs in vivo. Furthermore, MSC therapy with IP6Ks inhibition significantly decreased fibrosis and preserved heart function. This study demonstrates that inhibition of IP6Ks promotes MSCs engraftment and paracrine effect in infarcted hearts at least in part by down-regulating IP7 production and enhancing Akt activation, which might contribute to the preservation of myocardial function after MI.
KeywordsInositol phosphates Mesenchymal stem cells Myocardial infarction Apoptosis
Despite a wide range of therapeutic approaches, myocardial infarction (MI) continues to be a major cause of significant morbidity and mortality worldwide [3, 8].Stem cell therapy has emerged as a novel potential additional treatment of ischemic heart disease. Mesenchymal stem cells (MSCs) have been considered an optimal candidate because of their plasticity, ease of isolation and low immunogenicity. However, the strategy of stem cell transplantation is limited by the poor viability and function of donor cells [22, 33]. Therefore, optimizing approaches to augment engrafted cell survival and function is crucial to improving cell therapy for MI.
Genetically modified MSCs over-expressing the survival gene Akt (Akt-MSCs) were markedly resistant to hypoxic injury in vitro and in vivo . The improved survival of Akt-MSCs was associated with an enhanced cardioprotective effect. However, gene transfer approach is not suitable for the clinic because of some potential drawbacks, including insertional mutagenesis, high manufacturing costs, and possible tumorigenicity due to over-expression of Akt . Instead, a pharmaceutical approach involving temporary activation of Akt may be an attractive optimizing strategy to improve the viability of engrafted MSCs.
Inositol phosphates (IPs) are a diverse group of signaling molecules that are widely distributed in mammals . The 5-diphosphoinositol pentakisphosphate (IP7), formed by a family of inositol hexakisphosphate kinases (IP6Ks) including IP6K1, IP6K2, and IP6K3, serves multiple biological functions including inhibiting apoptosis and increasing insulin secretion [23, 24]. Thus far, IP7 has been demonstrated to be a physiologic inhibitor of Akt signaling . Furthermore, a purine analog [N6-(p-nitrobenzyl) purine, TNP] is identified as a reversible inhibitor of IP6Ks, which can concentrate dependently and selectively inhibit IP6K activity . Therefore, TNP provides a means to modulate cellular IP7 synthesis, potentially offering an innovative tool to regulate Akt activity. Accordingly, we hypothesized that IP6K inhibition by TNP could decrease IP7 production and increase Akt activation in MSCs, resulting in enhanced viability and cardiac protective effect after transplantation.
Detailed methodology is available in the Online Data Supplement.
Fluc+–eGFP+ transgenic mice [Tg(fluc–egfp)] were bred on a C57BL/6a background to stably express both firefly luciferase Fluc and enhanced green fluorescence protein (eGFP) in all tissues and organs. The experiments were approved by the Fourth Military Medical University Committee on Animal Care.
Hypoxia/serum deprivation injury
MSCs were stimulated with hypoxia/serum deprivation (H/SD) injury as described previously . Briefly, after being replaced in Hanks buffer, MSCs were exposed to hypoxia (94 % N2, 5 %CO2, and 1 % O2) in an anaerobic system (Thermo Forma) at 37 °C for 6 h. In the control group, MSCs were maintained at normoxia (95 % air, 5 % CO2) for equivalent periods.
Radiolabeling and analysis of inositol polyphosphates
To test the IP6Ks activity and cellular concentration of IP7 in MSCs, we monitored inositol polyphosphate levels by high-performance liquid chromatography (HPLC) after labeling with [3H]inositol . In brief, MSCs were seeded onto 6-well culture plates at 1.0 × 104 cells/cm2. After washing with inositol-free medium, cells were cultured with 100 μCi/ml [3H]inositol in DMEM supplemented with 10 % FBS for 72 h. The cells were then scraped after being lysed with 0.5 mol/L trichloroacetic acid. The supernatant was collected and prepared for HPLC analysis. Left ventricles of hearts were dissected and lysed at 4 °C in buffer containing Tris-buffered saline, 0.1 % Triton X-100 (1 mM, Sigma), 4 % glycerol, EDTA (1 mM, Sigma) and protease inhibitor PMSF (1 mM, Roche Molecular Biochemicals). The insoluble material was removed by centrifugation at 6,000×g, and the supernatants were diluted in SDS sample buffer. Finally, the inositol phosphates were eluted by Strong anion exchange high-performance liquid chromatography (SAX-HPLC) with an optimal gradient.
In vitro assessment of TNP's effects on MSCs after H/SD injury
MSCs were stimulated with H/SD injury for 6 h after incubation with different concentrations of TNP (0.5, 1, 5, and 10 μmol/L). The cell viability of MSCs was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and bioluminescence imaging (BLI) using the IVIS Kinetic system as described [5, 35]. MSC apoptosis was determined by flow cytometer assay using an Annexin V-FITC/PI kit (Merck) according to the manufacturer’s instructions. Caspase-3 activity was measured using a caspase-3 assay kit (Abcam) according to the manufacturer’s instructions. The expressions of proteins were evaluated by Western blotting analysis following the standard protocol. The concentrations of VEGF, FGF2, IGF-1, and HGF secreted by MSCs were determined by enzyme-linked immunosorbent assay (ELISA, Life Technologies) .
Myocardial infarction mice model and MSC transplantation
Myocardial infarction (MI) was accomplished by ligation of the left anterior descending (LAD) artery . In brief, mice were intubated and ventilated with 50 % oxygen in room air. A left thoracotomy was performed and the pericardium was opened after anesthesia (2 % isoflurane and oxygen). LAD was permanently ligated with a 6.0 suture. The ligation was deemed successful when the anterior wall of the LV turned pale and characteristic ECG changes were recorded. MSCs, cultured in inositol-free medium, were administrated with 10 μmol/L TNP in DMSO for 24 h at 37 °C. For control treatment, 3 μl of DMSO was added to MSCs. The DMSO concentration was 0.1 % of the total volume of medium in all cases. After TNP pre-treatment, the culture medium was aspirated off, and cells were washed with inositol-free medium for three times before collection. TNP pre-treated MSCs (1 × 106) or MSCs (1 × 106) were injected directly into the peri-infarcted areas (multiple injections within the presumed infarct and the border zone) immediately after MI.
In vivo evaluation of MSC engraftment
Bioluminescence imaging (BLI) was performed to track engrafted MSCs using an IVIS® Kinetic system (Caliper, Hopkinton, MA, USA) as described previously . The mice were anesthetized with 2 % isoflurane. After intraperitoneal injection with d-luciferin (375 mg/kg body weight), recipient mice were anesthetized and imaged for 1 min on days 2, 3, 5, 7, 10, 14, 21 until sacrificed. Peak signals (photons/s/cm2/sr) from a fixed region of interest (ROI) were analyzed using Living Image® 4.0 software (Caliper, MA, USA). The survival of MSCs was also confirmed by GFP immunofluorescent stain.
Histological analysis of apoptosis and angiogenesis
Apoptosis in the heart at 48 h after MSCs transplantation was determined by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay as previously described . Fast green/Sirius red stain was performed to detect fibrosis in cardiac muscle in 4 weeks post-procedure. Fibrosis was evaluated by measuring the collagen area as a proportion of the total left ventricular area using the Imaging Pro Plus software. The capillary density was determined by CD31 immunohistochemistry. Vessels in the peri-infarct zone were counted in randomly chosen high-power fields (HPFs, magnification ×400). The results are expressed as vessels per HPF. Immunofluorescence staining of CD31 was performed to visualize CD31-positive vessels. Cell nuclei were stained with DAPI. Sections were imaged using confocal microscope (FluoView-FV1000, Olympus, Japan).
Evaluation of heart function
Echocardiography studies were performed under anesthesia with 2 % isoflurane at 2 days, 7 days, and weekly until sacrificed at 4 weeks post-operation using a 30-MHz transducer on a Vevo® 2100 ultrasound system (VisualSonics, CA). The left ventricular end-systolic volume (LVESV) and left ventricular end-diastolic volume (LVEDV) were measured to calculate left ventricular ejection fraction (LVEF). The left ventricular end-systolic diameter (LVESD) and left ventricular end-diastolic diameter (LVEDD) were measured to calculate left ventricular fractional shortening (FS). For evaluating the LV fractional shortening (FS), endocardial borders were outlined at end-diastole and end-systole, using a left ventricular short-axis view, and FS (%) was calculated by (LV end-diastolic diameter−LV end-systolic diameter)/LV end-diastolic diameter × 100 %. We also evaluated the fractional area shortening and left ventricular anterior wall thickness systolic (LVAWTs). The LV internal area (LVIA) was measured without including the papillary muscles. Both were recorded in end-systole and end-diastole. The fractional area shortening (FAS) was calculated as [(LVIAED − LVIAES)/LVIAED] × 100 %. Heart rates of mice were controlled above 400/min throughout the echocardiography performance.
The results are presented as mean ± SEM. Statistics were calculated using Prism 5.0 (GraphPad Software Inc, San Diego, CA, USA). Linear regression analysis was performed to determine the correlation between two variables. Statistical comparisons for different groups were performed using either the Student’s t test or one-way ANOVA. p values < 0.05 were considered statistically significant.
Characterization of MSCsFluc+GFP+
MSCsFluc+GFP+ were isolated from reporter transgenic mice [Tg(fluc–egfp)] that constitutively express both Fluc and eGFP. Flow cytometry results revealed that MSCsFluc+GFP+ were uniformly positive for MSC markers CD29, CD90, CD44, and negative for CD31, CD45, CD34, SMA, c-Kit (supplemental Fig. 1A). MSCsFluc+GFP+ exhibited fibroblast-like morphology and expressed GFP (supplemental Fig. 1B). The multipotency of MSCsFluc+GFP+ was confirmed by the capacity to differentiate into adipogenic and osteogenic lineages, as assessed by oil red O staining and alizarin red S staining, respectively (supplemental Fig. 1B). Representative bioluminescence images demonstrated a robust linear correlation between the number of MSCsFluc+GFP+ and average Fluc radiance (r2 = 0.98; supplemental Fig. 1C), indicating that BLI of Fluc was a reliable tool to monitor viable transplanted MSCsFluc+GFP+ quantitatively in vivo.
Hypoxia increased IP6Ks activity and IP7 production
TNP enhanced the viability of MSCsFluc+GFP+ after H/SD injury
TNP increased Akt activity and growth factors secretions in MSCsFluc+GFP+ after H/SD injury
It has been shown that MSCs contribute to cardiac repair and regeneration at least in part by paracrine mechanism. Therefore, we evaluated the effect of TNP on cytokine secretion in MSCsFluc+GFP+ with or without exposure to H/SD. After 6 h H/SD injury, only VEGF (1.1 ± 0.1 × 103 vs. 0.5 ± 0.1 × 103 pg/ml in normoxia condition, p < 0.05) and bFGF (26.6 ± 2.5 vs. 12.1 ± 1.6 pg/ml in normoxia condition, p < 0.05) increased in control groups. Furthermore, TNP administration at 10 μmol/L significantly increased VEGF (3.4 ± 0.3 × 103 vs. 1.3 ± 0.1 × 103 pg/ml, p < 0.05), bFGF (88 ± 5.1 vs. 50 ± 4.8 pg/ml, p < 0.05), IGF-1 (192 ± 4.1 vs. 140 ± 7.8 pg/ml, p < 0.05), and HGF (100 ± 5.7 vs. 49 ± 3.3 pg/ml, p < 0.05) secreted by MSCsFluc+GFP+ after H/SD (Fig. 3d–g).
TNP enhanced the retention of engrafted MSCs
TNP facilitated MSC-mediated pro-angiogenic and anti-apoptotic effects
MSCs pretreated with TNP reduced fibrosis and preserved heart functional recovery after MI
In the present study, we found that IP6K inhibition exerted a protective effect on the survival of MSCs under hypoxia, which was associated with decreased production of IP7 and increased Akt activity. Intra-myocardial injection of bone marrow-derived MSCs pretreated with IP6ks inhibitor exerted even greater cardioprotection against ischemic injury. Overall, this study demonstrates that transplantation of MSCs with IP6K inhibition during acute phase of MI is an effective strategy for cardiac protection.
Although both experimental and clinical studies of MSC-based therapy for MI have reached an exciting and promising stage, previous studies revealed only marginal improvements in cardiac function after the transplantation of MSCs into infarcted hearts . Poor viability of donor cells is thought to be a major limitation for MSCs’ clinical application. Our previous studies have demonstrated a high level of MSCs death between day 3 and 7 after implantation . In the present study, we tracked the engrafted MSCs by BLI and also found similar acute cell death within 1 week after transplantation into the infarcted heart. Meanwhile, transplantation with MSCs alone could not prevent cardiomyocytes death and improve cardiac function significantly, which was consistent with the results of Yang et al. [31, 32]. Many factors are believed to contribute to the high losses of MSCs after transplantation. The noxious milieu in ischemic myocardium that lacks nutrients and oxygen, coupled with enhanced inflammatory response, may play important roles here [5, 12]. Moreover, donor MSCs seem more sensitive to hypoxic and inflammatory injury due to the stressful procedure of cell preparation and transplantation . Therefore, reinforcement of the viability of MSCs in hypoxic conditions after transplantation is crucial for improving the efficiency of cell therapy.
Akt, a PH domain containing serine/threonine kinase, regulates survival signal, growth factor production, and protein synthesis through the phosphorylation of multiple substrates . The Bcl-2 family, regulated by Akt activity, makes up the key regulators of apoptosis [1, 25]. Moreover, previous studies [15, 18, 29] also have proved that increased Akt activation resulted in decreased expression of Bax and increased expression of Bcl-2, suggesting that Akt activation was negatively correlated with the expressions of pro-apoptosis proteins, Bax. In the present study, we found that the phosphorylation of Akt was decreased by hypoxia injury, associated with an increased Bax/Bcl-2 ratio. However, these effects were abolished by TNP, indicating that IP6Ks inhibition protected the impaired activation of Akt induced by H/SD injury. Therefore, protection of impaired Akt activation is seen as an attractive therapeutic target to optimize MSCs therapy for ischemic heart disease. Various strategies have been adopted to increase the Akt activation of engrafted MSCs. Mangi et al.  found that the genetically modified MSCs (Akt-MSCs) exhibited significantly enhanced intramyocardial retention and exerted a potent myocardial protection effect by paracrine mechanisms. Haider et al.  also revealed that increasing Akt activation by over-expressing insulin-like growth factor (IGF)-1 promoted MSCs survival and enhanced their therapeutic efficacy. However, the potential tumorigenicity risk and higher manufacturing cost might hamper genetically engineered cell therapy for the clinic. An alternative approach of gene delivery is the pretreatment with small molecules to up-regulate donor cells Akt activity.
Inositol phosphates (IPs) are a group of signaling molecules that are widely distributed in all eukaryotic cells . IP6 can be further metabolized to generate 5-diphosphoinositol pentakisphosphate (IP7) by a family of three IP6 kinases (IP6Ks) . Although the physiological effects of IPs remain poorly characterized, IP7 appears to be involved in cell apoptosis . In the present study, we found increased apoptotic MSCs with significantly enhanced IP7 generation after hypoxia injury. Moreover, the IP6K inhibition by TNP was also associated with decreased apoptosis, increased viability and paracrine effect of MSCs, indicating that IP7 may be involved in the hypoxia injury of MSCs.
IP7 could inhibit Akt signaling by binding at the PH domain of Akt to block phosphorylation both in vitro and in vivo . In the present study, we demonstrated that blocking IP6Ks by TNP decreased IP7 synthesis and increased phosphorylation of Akt at T308 and S473 in MSCs, indicating the down-regulation of IP7 expression by IP6K inhibition enhanced the activation of Akt in MSCs. Furthermore, we documented that blocking IP6Ks using a pharmacological inhibitor TNP improved the survival of MSCs in the ischemic heart. Brunner et al.  found boosted migration of MSCs from bone marrow to peripheral blood and heart reduced apoptosis of cardiomyocytes and improved cardiac function significantly. Our research also proved that transplantation with TNP pretreated MSCs was effective at preventing apoptosis of cells in the heart and myocardial fibrosis, leading to improved LV function.
Although there is controversy on the precise mechanisms underlying these therapeutic effects associated with engrafted stem cells [14, 19], a growing body of evidence supports the hypothesis that paracrine mechanisms play a more essential role in stem cell-mediated tissue protection instead of cardiac differentiation . Moreover, many bioactive factors secreted by MSCs, such as VEGF, bFGF, HGF, and IGF-1, promote neovascularization and inhibit host cardiomyocyte death . However, the effect of hypoxia injury on the paracrine effects of MSCs is still not clear. In the current study, we found that the secretions of VEGF and bFGF in MSCs under hypoxic condition were increased compared with normal groups. However, it seems a contradiction that hypoxia significantly decreased IGF-1 and HGF in MSCs. We presumed that the low-grade augmentation of the VEGF and bFGF secretions maybe a compensatory response of MSCs to hypoxic injury [28, 30]. Meanwhile, we found that IP6Ks inhibition significantly increased VEGF, bFGF, IGF-1, and HGF secreted by MSCs after hypoxia injury. Taken together, our results demonstrated that TNP administration protected the impaired paracrine effects of MSCs under hypoxia.
Although our study bears clinical relevance, there are some limitations. First, physiological functions of IP7 have not been extensively characterized. Further studies defining the exact mechanism(s) are needed. Second, the H/SD model is an artificial experimental model that cannot fully simulate the in vivo ischemic and inflammatory environment.
This work was supported by National Nature Science Foundation of China (No. 81325009, 81270168, 81090274, 81227901), National Basic Research Program of China (2012CB518101), Program for Changjiang Scholars and Innovative Research Team in University (IRT1053), China’s Ministry of Science and Technology 863 Program (2012AA02A603).
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