Restoration of angiogenic capacity of diabetes-insulted mesenchymal stem cells by oxytocin
© Kim et al.; licensee BioMed Central Ltd. 2013
Received: 16 May 2013
Accepted: 29 August 2013
Published: 11 September 2013
Angiogenesis is the main therapeutic mechanism of cell therapy for cardiovascular diseases, but diabetes is reported to reduce the function and number of progenitor cells. Therefore, we studied the effect of streptozotocin-induced diabetes on the bone marrow-mesenchymal stem cell (MSC) function, and examined whether diabetes-impaired MSC could be rescued by pretreatment with oxytocin.
MSCs were isolated and cultured from diabetic (DM) or non-diabetic (non-DM) rat, and proliferation rate was compared. DM-MSC was pretreated with oxytocin and compared with non-DM-MSC. Angiogenic capacity was estimated by tube formation and Matrigel plug assay, and therapeutic efficacy was studied in rat myocardial infarction (MI) model.
The proliferation and angiogenic activity of DM-MSC were severely impaired but significantly improved by pretreatment with oxytocin. Krüppel-like factor 2 (KLF2), a critical angiogenic factor, was dramatically reduced in DM-MSC and significantly restored by oxytocin. In the Matrigel plug assay, vessel formation of DM-BMSCs was attenuated but was recovered by oxytocin. In rat MI model, DM-MSC injection did not ameliorate cardiac injury, whereas oxytocin-pretreated DM-MSC improved cardiac function and reduced fibrosis.
Our results show that diabetes influenced MSC by reducing angiogenic capacity and therapeutic potential. We demonstrate the striking effect of oxytocin on stem cell dysfunction and suggest the use of oxytocin as a priming reagent in autologous stem cell therapy.
KeywordsDiabetes Angiogenesis Stem cells Oxytocin Krüppel-like factor 2
Cell therapy with autologous bone marrow-mesenchymal stem cells (MSC) is a promising and safe modality with the potential for vascular regeneration in the treatment of ischemic diseases. MSC can differentiate into vascular lineage cells and can be directly incorporated into newly formed vessels . However, the initial clinical trials of stem cell therapy after myocardial infarction failed to reproduce the substantial benefits demonstrated in the preclinical animal studies, especially in elderly patients [2–5]. One possible reason for the conflicting results in cell therapy research is that the stem cells used in most of the animal studies were derived from young or healthy animals. Diabetes, obesity, or aging influence stem cell numbers and activities [6–11], thus the animal studies could not predict the outcomes of autologous stem cell therapy for a patient with diabetes or other risk factors [12, 13].
Diabetes is widely recognized to be an independent risk factor for coronary heart disease, stroke, peripheral arterial disease, cardiomyopathy, and congestive heart failure [14–17]. Furthermore, diabetes is closely associated with poor neovascularization after ischemia . Cells isolated from diabetic patients are significantly impaired in their ability to recover blood flow after ischemia compared with cells isolated from healthy donors . In addition, diabetic rats fail to induce neovascularization for recovery from hind limb ischemia, partly as the result of a dysfunction of endothelial progenitor cells . There is no doubt that diabetes is not the only factor associated with dysfunction of progenitor cells, but diabetes leads to significant cellular dysfunction such as poor migration, reduced proliferation, and poor vascular network formation . Diabetes-related changes in stem cells or progenitor cells may not only account for the reduced proliferation rate of these cells but also for their limited angiogenic potential .
Oxytocin is a neurohypophyseal hormone expressed in the hypothalamus. Previous studies revealed that oxytocin induces cardiomyogenesis of embryonic stem cells  and adult Sca-1 (+) stem cells , stimulates the migration of endothelial cells , and increases the engraftment  and cardiac differentiation potency  of umbilical cord blood-derived mesenchymal stem cells in infarcted myocardium.
Krüppel-like factor 2 (KLF2), which was first cloned by Lingrel and colleagues , is emerging as a master regulator of endothelial quiescence, anti-inflammatory and antithrombotic properties, and vascular tone by activating atheroprotective and inhibiting atherogenic transcription . Proangiogenic cells isolated from aged mice showed a lower level of KLF2 than do cells from young mice . Despite growing evidence of cellular dysfunction, however, the signaling pathways of stem cells in various physiological and pathological niches remain to be investigated.
The potential disadvantage of autologous stem cells is that patients with diabetes may have a decline in number and regenerative capacity of bone marrow and circulating stem cells; thus, patients who have diabetes may have not only damaged myocardium but also a lessened capacity for repair.
In this study, we aim to compare bone marrow-MSCs obtained from non-diabetic and diabetic rats, and examine whether diabetes-insulted MSC could be rescued by pretreatment with oxytocin.
Proliferation is reduced in DM-MSC and recovered by oxytocin
Animal characteristics after induction of diabetes
n = 10
n = 24
442.50 ± 20.43
461.47 ± 41.37
94.83 ± 8.70
92.47 ± 6.47
n = 10
n = 17
558.33 ± 22.51
349.00 ± 67.96*
118.50 ± 9.27
571.58 ± 57.64*
1.47 ± 0.01
1.16 ± 0.21*
45.49 ± 0.78
44.60 ± 1.67
HW (g)/TL (mm)
32.28 ± 0.35
25.99 ± 4.17*
Tube formation and KLF2 are reduced in DM-MSC and recovered by oxytocin
To confirm whether the KLF2 is a mediator of oxytocin in DM-MSC, knockdown of KLF2 was induced by siRNA transfection. Oxytocin failed to induce both KLF2 mRNA and tube formation in KLF2 siRNA-transfected DM-MSC (Figure 3D). This result suggested that oxytocin might act on angiogenic potential of DM-MSC through KLF2 induction.
To examine whether KLF2 induction by oxytocin is specific to MSC, KLF2 plasmid DNA was transfected to 293T cells. Oxytocin treatment did not show any significant induction of KLF2 protein in 293T cells (Figure 3E).
Impaired angiogenic capacity of DM-MSC is restored by oxytocin
Introducing exogenous KLF2 DNA into DM-MSC contributes to restoration of angiogenic activity of DM-MSC
Restored therapeutic effects in myocardial infarction (MI) by OT-DM-MSC
MSC are one of the most actively studied stem cell sources for treating various cardiovascular diseases. Autologous MSC application is clinically available without ethical issues or immunological problems. The use of autologous MSC, however, is affected by factors such as aging or systemic diseases, which may contribute to the functional impairment of stem cells. Diabetes is one of the risk factors for cardiovascular diseases, and type 1 diabetes is a disorder characterized by hyperglycemia and a proinflammatory state [29, 30]. Our results showed that diabetes impairs the neovascularization of bone marrow-derived MSC, and these findings are consistent with previous reports [31, 32]. Several reports demonstrated diabetes exerted a detrimental effect on stem cells or progenitor cells. Endothelial progenitor cells obtained from type 1 diabetes patients  or streptozotocin-induced diabetes mice  showed the significant reduction of circulating cell number and cellular function. Prolonged exposure to high glucose condition has drastic effects on the differentiation potential, proliferation capacity, and cell survival of adipose tissue-derived MSC . To overcome, we searched for a priming reagent to restore the angiogenesis activity of DM-MSC, and we found a transient treatment of DM-MSC with oxytocin for 24 hours improved tube formation capacity.
Oxytocin, a neurohypophyseal nonapeptide, modulates social recognition, emotion, and the female reproductive system . In previous studies, we provided evidence for beneficial roles of oxytocin in umbilical cord blood-derived mesenchymal stem cells [22, 23].
We next explored which effecter was controlled by oxytocin to restore the angiogenic potential. A previous report showed that cell number is reduced and KLF2 expression is repressed in proangiogenic cells as a result of senescence . KLF2 is a well-known zinc-finger transcriptional regulator that is involved in endothelial development, functional regulation, and angiogenesis [25, 35, 36]. On the other hand, KLF2 inhibited function and expression of hypoxia-inducible factor α (HIF1-α) in hypoxia-mediated endothelia angiogenesis . This discrepancy might be resulted from different inducers of angiogenesis. KLF2 might be required for development of physiological or therapeutic angiogenesis, and regulation of inflammation or hypoxia-induced angiogenesis. In this study, we focused on intrinsic angiogenic potential of MSC rather than on pathological lesion. We found that KLF2 was highly expressed in normal MSC, whereas it was significantly repressed in DM-MSC. Our data and those of a previous report  show that down-regulated KLF2 is closely associated with aggravation of the angiogenic potential of stem/progenitor cells. In addition to angiogenesis, oxytocin-treated DM-MSC successfully induced as well anti-inflammatory IL-10 as angiogenic cytokines such as VEGF and HGF in injected infarct lesion.
The most important limitation in this study is that we did not address the exact mechanism of KLF2 induction by oxytocin.
Taken together, these results show that DM-MSC showed impaired angiogenesis and reduced KLF2, which were restored by oxytocin treatment. Stem cell therapy with autologous MSC in persons with risk factors such as diabetes, aging, or systemic disorders is expected to be less effective than stem cell therapy in the preclinical animal experiments. For therapeutic application, therefore, the future challenge is to establish a safe procedure for patients with risk factors to normalize the function of endogenous stem cells before autologous stem cell application. We suggest that strong potential exists for translating the results of the present study to human trials, which would be very beneficial to patients who are at risk of cardiovascular disease in the setting of diabetes.
In the present study, we evaluated a putative relation between diabetes and poor angiogenesis of stem cells in the myocardial infarction model. Our data showed that the cellular function of MSC might be disturbed by exposure to the diabetic niche. In addition, we studied oxytocin and KLF2 in relation to the restoration of the angiogenic potential of DM-MSC.
This study was approved by the Chonnam National University Institutional Animal Care and Use Committee (CNU AICUC-H-2010-11). Twelve-week-old adult male Sprague–Dawley rats (Jung Ang, Korea) received intraperitoneal injections of streptozotocin (65 mg/kg, Sigma, USA) dissolved in 50 mM sodium citrate buffer (pH 4.5) to induce type 1 diabetes. Age-matched controls were injected with an equivalent volume of saline. Fasting blood glucose levels were measured in tail veins, and rats with a blood glucose level > 250 mg/dl were considered to be diabetic and were included in the study.
Culture of MSC and 293T cells
MSCs were isolated and cultured as described in previous reports [38–40]. MSCs were obtained from the tibia and femur under sterile conditions by using a syringe to flush the cavity out with warmed phosphate-buffered saline (PBS), collected by centrifugation, and resuspended with DMEM with 10% fetal bovine serum. Cells were plated into culture dishes, and nonadherent cells were removed by changing the medium after 72 hours. All cells used in this study were from the third and fourth passages of MSC. To identify stemness, adipogenic, chondrogenic, and osteogenic differentiations were performed in monolayer culture of MSC by using Stem Cell Differentiation Kits (Life technologies, USA). Briefly, MSC were cultured in growth medium or differentiation medium. After 7 days, cells were fixed in formalin and stained with Oil Red O (Sigm-Aldrich, USA), Safranin O (Sigm-Aldrich, USA) and Alizarin Red S (Sigm-Aldrich, USA) staining to determine the adipogenesis, chondrogenesis and osteogenesis, respectively.
Oxytocin (100 nM, Sigma-Aldrich, USA), curcumin (Sigma-Aldrich, USA), carvedilol (kindly provided by Chong Kun Dang Pharm., Korea), and rosuvastatin calcium (kindly provided by AstraZeneca Korea) were added to the growth medium for 24 hours for the experiments. 293T cells were purchased from the ATCC (USA) and maintained in Dulbecco’s Modified Eagle’s Medium (Invitrogen, USA) supplemented with 10% fetal bovine serum (Invitrogen, USA).
Proliferation assay of MSCs
Cells were plated on 48-well plate and the proliferation rate was measured with WST-1 (Roche Applied Science, USA). Briefly, WST-1 reagent was added at each time points, and the absorbance was measured at 490nm after incubation for 2 hours.
In vitro angiogenesis assay
Tube formation was assayed by using an in vitro angiogenesis assay kit (Chemicon, USA). Cells (1 × 104) were plated onto matrix gel-coated 96-well plates and were cultured in DMEM without serum. Tube formation was monitored and photographed by using an inverted microscope (Olympus CRX41, Japan), and images were analyzed by using Image-Pro software (Media Cybernetics, Inc., USA). Angiogenic activity was quantified by measuring tube length and tube area. Total tube length in four fields per well was averaged, and three wells were used to produce one value per condition.
Reverse transcriptase-polymerase chain reaction (RT-PCR)
To compare the mRNA expression level of KLF2, RT-PCR was done as previously described . Primers were designed from Bioneer (Daejeon, Korea) and the sequences were as follows: human KLF2 forward, caagacctacaccaagagttcgca, reverse, tacatgtgccgtttcatgtgcagc; rat KLF2 forward, ttgcagctacaccaactgcg, reverse, tgtcgcttcatgtgcagagc; rat glyceraldehydes 3-phosphate dehydrogenase (GAPDH) forward, ggccaaggtcatccatga, reverse, tcagtgagcccaggatg. Band densities were estimated using the Scion image 4.02 software (Scion Corporation, USA).
Western blot analysis
Cells were lysed and analyzed as previously described  with blotting with KLF2 antibody (Novus Biologicals, USA) and flag antibody(Sigma-Aldrich, USA). β-actin (Sigma, USA) was used as a loading control. Band densities were estimated using the Scion Image 4.02 software (Scion Corporation, USA).
Plasmid DNA or siRNA transfection
KLF2 plasmid DNA was purchased from OriGene Technologies (Rockville, USA). MSC were transfected with KLF2 plasmid DNA or gWIZ mammalian expression vector (Genlantis, USA) by using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s instruction. Overexpression of KLF2 was confirmed by RT-PCR and Western blot at 1 day and 2 days after transfection, respectively. To study the effect of oxytocin on KLF2 expression, KLF2 plasmid DNA was transfected to 293T cells for 1 day, and then cells were treated with oxytocin or PBS for 1 day. To knock down KLF2, KLF2 siRNA and scrambled siRNA (Bioneer, Korea) were transfected to DM-MSC with Lipofectamine 2000. After 2 days, siRNA transfected cells were treated with oxytocin (100 nM) for 24 hours.
Matrigel plug assay
PBS or 2 × 105 cells were mixed with phenol-red-free Matrigel (Matrigel™ Basement Membrane Matrix High Concentration, BD Biosciences, USA), and subcutaneously injected into 7-week-old male Balb/c athymic nude mice. After 2 weeks, the Matrigel plugs were harvested and processed for analysis. To estimate the degree of vascularization, H&E-stained digital images were analyzed by measuring the erythrocyte-filled area and expressing that as a percentage of the total area of Matrigel.
Rat model of myocardial infarction (MI)
Male Sprague–Dawley rats (weighing 200–230 g, Jung Ang Animals, Korea) were used to induced MI previously described . After 7 days, rats were randomly divided into 4 groups (n=5 each group) and anesthetized for reoperation. To visualize the injected MSCs for immunohistochemical examination, cells were infected with lenti-GFP virus for 48 hours and washed out with PBS before injection. DM-MSCs were pretreated with PBS or oxytocin (100 nM) for 24 hours before injection. Rats were injected with PBS alone, non-DM-MSC, DM-MSC, or oxytocin-pretreated DM-MSCs (OT-DM-MSC) into the peri-infarct area. MSCs (5 × 105 diluted in 100 μL of PBS) were directly injected into the peri-infarct area. Finally, the heart was repositioned in the chest, and the chest was closed. The animals remained in a supervised setting until they were fully conscious.
After 2 weeks, echocardiography was performed as previously described .
For immunohistochemical analysis, slides were stained as previously described  with primary antibodies against von Willebrand factor (vWF, Biomedicals, Switzerland, 1:100), GFP (Abcam, USA), VEGF (Santa Cruz, USA), HGF (Santa Cruz, USA), and IL-10 (Abcam, USA).
All data are presented as means ± SDs. P values were calculated by using the unpaired Student’s t-test. For analysis of the in vivo ischemia experiments, the Scheffe's test was performed for multiple comparisons after ANOVA between the groups. A P < 0.05 was considered statistically significant.
Mesenchymal stem cell
Rüppel-like factor 2.
This study was supported by a grant of the National Research Foundation of Korea Grant funded by the Korean Government (MEST), Republic of Korea (2010–0020261), and the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (HI12C0199).
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