Bone marrow (BM)-derived mesenchymal stem cells (MSCs) represent the leading candidate cell for tissue regeneration in the ischemic myocardium. However, the poor survival of stem cells transplanted into the ischemic myocardium presents a major obstacle in stem cell-based therapy. C1q tumor necrosis factor-related protein 3 (CTRP3) is a newly identified adipokine, similar to adiponectin, with beneficial effects on metabolic regulation. It has been shown to enhance the survival of cardiomyocytes during ischemia, while its expression is reduced following ischemia. In the present study, we examined the hypothesis that CTRP3 may enhance the survival of MSCs during exposure to hypoxia/serum deprivation (SD), and attempted to elucidate the underlying mechanisms. MSCs were obtained from rat bone marrow and cultured. Apoptosis was induced by hypoxia/SD for up to 24 h and the apoptotic rates were assessed by flow cytometry. MSC proliferation was measured using a Cell Counting kit-8 assay. The expression levels of Akt, Bcl-2, Bax, cytochrome
Ischemia/reperfusion injury remains a leading cause of mortality in developed countries, despite significant advances in the medical treatment of heart failure (
C1q tumor necrosis factor-related proteins (CTRPs) are members of the highly conserved family of adiponectins. Each of the 10 known members (CTRP1-CTRP10) consists of 4 distinct domains, including an N-terminal signal peptide, a short variable domain, a collagen-like domain and a C-terminal C1q-like globular domain (
Male Sprague-Dawley rats, weighing 60–80 g, were handled in accordance with the US National Institutes of Health published guidelines. All procedures were approved by the Institutional Animal Care and Use Committee of Harbin Medical University. The study was conducted in compliance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH, revised in 1996).
Dulbecco's modified Eagle's medium (DMEM)/F12 and fetal bovine serum (FBS) were purchased from HyClone Laboratories (Logan, UT, USA). The Annexin V-FITC apoptosis detection kit, and anti-CD44, anti-CD29 and anti-CD90 antibodies were obtained from BD Biosciences (San Diego, CA, USA). Anti-CD34 and anti-CD45 antibodies were obtained from eBioscience (San Diego, CA, USA). Rabbit monoclonal antibodies against Akt, phospho-Akt (Tyr308, Ser473), caspase-3, Bax, Bcl-2 and cytochrome
BM-MSCs were isolated from the femurs and tibias of Sprague-Dawley rats, as previously described (
The characteristics of the MSCs were determined by immunophenotyping. Cells were harvested, washed with phosphate-buffered saline (PBS), and labeled with phycoerythrin (PE)-conjugated anti-CD45 and anti-CD90 antibodies, and fluorescein isothiocyanate (FITC)-labeled anti-CD44, anti-CD29 and anti-CD34 antibodies. Labeled cells were assayed by flow cytometry and analyzed using FACSDiva Pro software (BD Biosciences).
The
To investigate the involvement of the PI3K/Akt pathway, the cells were pre-incubated with the PI3K/Akt inhibitor, LY294002 (25 μM), in complete medium for 90 min prior to exposure to hypoxia/SD. CTRP3 was added in the presence of the inhibitor during exposure to hypoxia/SD.
Apoptotic rates were estimated by detecting phosphatidylserine on the cell plasma membrane using the fluorescent dye [propidium iodide (PI)] with the Annexin V-FITC apoptosis detection kit (BD Biosciences), according to the manufacturer's instructions. In brief, cells were harvested and washed in ice-cold PBS, resuspended in 300 μl of binding buffer and incubated with 5 μl of Annexin V-FITC solution for 30 min at 4°C in the dark, followed by further incubation with 5 μl PI for 5 min. Cells were then analyzed immediately by bivariate flow cytometry using a BD FACSCanto cytometer equipped with FACSDiva Pro software. Approximately 1–5×105 cells were analyzed in each sample.
Cell proliferation was assessed using a CCK-8 assay kit according to the manufacturer's instructions. Cells were incubated with CCK-8 solution in 96-well plates for 1 h at 37°C. The absorbance of each well was quantified at 450 nm.
The potential toxic effects of CTRP3 in cultured MSCs were examined at various concentrations. The MSCs were incubated in culture medium supplemented with CTRP3 for 24 h under hypoxia/SD culture conditions, and for 3 days under normal culture conditions. Trypan blue was added to the medium and the cells were incubated at 37°C for 15 min. Trypan blue-positive cells were then counted under a phase-contrast microscope. Five fields were randomly selected for each dish and at least 3 dishes were counted for each concentration.
Western blot analysis was carried out as previously described (
Mitochondrial membrane potential was determined using the JC-1 mitochondrial membrane potential assay kit. Briefly, the MSCs were seeded in 6-well plates. Following treatment, the cells were washed twice with PBS, and 1 ml staining dye/well was added (culture medium:JC-1 working dye, 1:1) and incubated at 37°C for 20 min. The cells were then washed twice with cold JC-1 staining buffer and examined under a fluorescence microscope.
Data are expressed as the means ± standard deviation (SD). Differences among groups were tested by a one-way ANOVA. Comparisons between groups were evaluated using the Student's t-test. A value of P<0.05 was considered to indicate a statistically significant difference.
MSCs were uniformly spindle-like. FACS analysis revealed that the majority of cells from passage 3 expressed the common MSC surface markers, CD29 (98.98±0.55%), CD90 (99.06±0.63%) and CD44 (25.30±5.80%), but were found negative for CD34 (0.97±0.03%) and CD45 (1.38±1.30%) (
In preliminary experiments, the maximal induction of early apoptosis by hypoxia/SD in the MSCs occurred at 24 h. We investigated whether CTRP3 protects MSCs from this process. MSCs were exposed to increasing concentrations of CTRP3 (3–3,000 ng/ml) followed by exposure to hypoxia/SD for 24 h, and cell apoptosis was determined by FACS analysis. CTRP3 (3–3,000 ng/ml) effectively blocked the apoptotic process, and the ratio of apoptotic cells decreased with a pronounced effect at 300 ng/ml [treated cells, 2.60±0.23 vs. apoptotic control (untreated cells), 6.95±0.37] (
Caspase-3 is a key mediator of apoptosis. We further evaluated the anti-apoptotic effects of CTRP3 by western blot analysis using a caspase-3 monoclonal antibody, and confirmed that CRTP3 significantly inhibited the cleavage of caspase-3 in a concentration-dependent manner following exposure to hypoxia/SD (apoptotic control, 6.17±0.17 vs. treated cells, 2.63±0.34) (
To the best of our knowledge, the potential toxicity of CTRP3 to MSCs at the tested concentrations has not been previously addressed. We therefore examined the effects of CTRP3 on MSC viability. Trypan blue assays indicated that up to 3,000 ng/ml of CTRP3 had no adverse effects on the viability of MSCs (
Apoptosis occurred over several hours, and we therefore measured cell proliferation, which could interfere with the measurements of the apoptotic process. The cell proliferation rate, as assessed by CCK-8 assay, showed no significant change during the 3 days of treatment with 300 ng/ml CTRP3, either under normal culture or under 24-h hypoxia/SD culture conditions (
The PI3K/Akt signaling pathway is considered to be important in promoting survival in numerous cell types (
To determine whether CTRP3 exerts its anti-apoptotic effects by inhibiting the activation of the mitochondrial pathway under hypoxia/SD conditions, we examined its effects on the Bcl-2/Bax ratio, the release of cytochrome
Autologous MSCs can be easily prepared from adult patients and are immunologically safe. They therefore offer great advantages for regenerating and repopulating the injured myocardium and restoring its function when transplanted into ischemic or infarcted hearts (
CTRP3 is a key member of the family of adipokines and has broad functions, not only in adipokine secretion and metabolism, but also in inflammation, cell proliferation, differentiation, apoptosis and cardiac protection (
In preliminary experiments, early apoptosis in MSCs induced by hypoxia/SD peaked at 24 h. Using the same model, we found that CTRP3 protected MSCs from apoptosis in a concentration-dependent manner. However, CTRP3 only inhibited the early hypoxia/SD-induced apoptosis of MSCs, and had no effect on late apoptosis or necrosis. CTRP3 had no apparent effect on MSC proliferation, thus eliminating the potential interference between the effects of proliferation and apoptosis.
In the present study, we examined the role of the PI3K/Akt pathway in MSC survival and in modulating the anti-apoptotic effects of CTRP3. PI3K/Akt is an important pro-survival pathway, and the phosphorylation of Akt plays a major role in anti-apoptotic functions in various types of cells (
A previous study demonstrated that hypoxia/SD induces apoptosis in MSCs through the mitochondrial apoptotic pathway (
In conclusion, the results of the present study suggest that CTRP3 promotes MSC survival under conditions mimicking the ischemic myocardium. CTRP3 protects MSCs from hypoxia/SD-induced mitochondrial apoptosis through the PI3K/Akt signaling pathway. These findings highlight a potential novel therapeutic strategy for protecting MSCs from apoptosis, and form the basis for the future clinical exploitation of CTRP3 and MSCs in cardiac regeneration therapies.
We thank Dr Wei Liu for her expert assistance with the experimental design and excellent technical assistance, and Dr Bo Sun for her assistance with the FACS analysis. Dr Wei Liu and Dr Bo Sun are members of the Key Laboratory of Myocardial Ischemia Mechanism and Treatment (Harbin Medical University), Ministry of Education. This study was supported by grants from the Key Laboratory of Myocardial Ischemia, Harbin Medical University, Ministry of Education (no. KF201315).
Cell surface markers of mesenchymal stem cells (MSCs). Surface marker molecules expressed in passage 3 MSCs were (A) analyzed by flow cytometry and (B) quantified by the image analysis of positive cells.
C1q tumor necrosis factor-related protein 3 (CTRP3) protects mesenchymal stem cells (MSCs) against hypoxia/serum deprivation (SD)-induced apoptosis. CTRP3 (3–3,000 ng/ml) was added at the time point of exposure to hypoxia/SD and was maintained in the incubation medium throughout the hypoxia/SD treatment period. Apoptosis was measured by flow cytometry. (A) Representative plots from 3 FACScan flow cytometric analyses of apoptotic cells following Annexin V and PI staining. (B) Fold changes of cell apoptosis relative to control cells. Each column represents the mean ± SD of 3 independent experiments. (C) Western blot analysis using the caspase-3 antibody to monitor the cleavage of caspase-3 induced by hypoxia/SD, and the inhibition of the cleavage of caspase-3 by various concentrations of CTRP3. The blot is representative of 3 independent experiments. (D) Fold changes of cleavage of caspase-3 relative to β-actin. Each column represents the mean ± SD of 3 independent experiments. (A and C) Cont., control, (B and D) *P<0.05 vs. control; ▲P<0.05 vs. hypoxia/SD; ○P<0.05 vs. hypoxia/SD + CTRP3 (300 ng/ml). Cl. caspase-3, cleaved caspase-3. Control, non-ischemic controls; CTRP 0, apoptotic controls (untreated cells).
Effects of C1q tumor necrosis factor-related protein 3 (CTRP3) on cell viability and proliferation. CTRP3 was added to the culture medium to determine its effects on cell viability and the proliferation of mesenchymal stem cells (MSCs). (A and B) Trypan blue staining experiments indicated that up to 3,000 ng/ml of CTRP3 had no toxic effects on cultured MSCs. Each column in (B) represents the mean ± SD of 3 independent experiments. (C and D) Proliferation growth curves of MSCs were measured by CCK-8 assays. CTRP3 (300 ng/ml) had no significant effect on the cell proliferation rate during the 3 days of treatment (C) under normal or (D) under 24-h hypoxia/SD culture conditions. Each data point represents the mean ± SD of 3 independent experiments.
C1q tumor necrosis factor-related protein 3 (CTRP3) protects mesenchymal stem cells (MSCs) from hypoxia/serum deprivation (SD)-induced apoptosis through the phosphoinositide 3-kinase (PI3K)/Akt pathway. (A and B) MSCs were subjected to hypoxia/SD and stimulated with CTRP3 (300 ng/ml) for the indicated time periods of time. The expression levels of (A) Akt and phospho-Akt (S473), and (B) Akt and phospho-Akt (T308) were analyzed by western blot analysis. Fold changes were estimated relative to Akt. Each data point represents the mean ± SD of 3 independent experiments. (C and D) Representative western blots of (C) Akt and phospho-Akt [(Ser(S)473], and (D) Akt and phospho-Akt [Tyr(T)308] in cells cultured under hypoxia/SD conditions treated with CTRP3 (300 ng/ml) and pre-treated with LY294002 (a PI3K inhibitor, 25 μM). Fold changes were estimated relative to Akt. Each data column represents the mean ± SD of 3 independent experiments. To determine the role of the PI3K/Akt pathway in the anti-apoptotic effects of CTRP3, the cells were pre-treated with LY294002 (25 μM) for 90 min in complete medium prior to exposure to hypoxia/SD. CTRP3 (300 ng/ml) was added in the presence of the inhibitor at the time point of exposure to hypoxia/SD. (E and F) All drugs were maintained in the incubation medium throughout the hypoxia/SD treatment period. Apoptosis was quantified by flow cytometry. Data are presented as fold changes, estimated relative to the corresponding control cells. Each column represents the mean ± SD of 3 independent experiments. (C, D and F) *P<0.05 vs. control; ▲P<0.05 vs. hypoxia/SD + CTRP3 (300 ng/ml).
C1q tumor necrosis factor-related protein 3 (CTRP3) exerts anti-apoptotic effects by inhibiting mitochondrial dysfunction. Mesenchymal stem cells (MSCs) were treated with hypoxia/serum deprivation (SD) for 24 h. In a parallel experiment, cells were pre-treated with LY294002 [a phosphoinositide 3-kinase (PI3K) inhibitor, 25 μM] for 90 min prior to exposure to hypoxia/SD for 24 h. CTRP3 (300 ng/ml) was added at the time point of exposure to hypoxia/SD. All drugs were maintained in the incubation medium throughout the hypoxia/SD treatment period. (A) Bcl-2/Bax, (B) cytochrome