Introduction
Diabetic cardiomyopathy (DCM), which occurs in type I and II diabetes, is a substantial risk factor for the subsequent development of heart failure and increased mortality (1). Its pathological substrate is characterized by early-onset diastolic dysfunction and late-onset systolic dysfunction in the absence of coronary artery disease, hypertension or significant valvular heart disease (2,3). The pathological mechanism of DCM may be due to metabolic disturbances, reactive hypertrophy, small vessel disease, autonomic dysfunction, insulin resistance and myocardial fibrosis. At present, conventional therapies for DCM include glycemic control and early administration of neurohormonal antagonists. DCM, however, has yet to be completely prevented (4,5). Therefore, the development of more effective therapeutic strategies for DCM is necessary.
Endothelial progenitor cells (EPCs) are a subset of bone marrow-derived stem cells present in peripheral circulation that have the potential to differentiate into functional and mature endothelial cells. Since their revolutionary discovery by Asahara et al (6) in 1997, EPCs have been demonstrated to be of importance in postnatal vasculogenesis through pivotal bioactivities, mobilization, homing, migration, differentiation and proliferation in angiovasculogenic tissues (7). However, decreased levels of circulating EPCs have been reported in a wide range of pathological conditions, such as chronic kidney disease (8), hypercholesterolemia (9), rheumatoid arthritis (10), obesity (11) and coronary artery disease (12). Furthermore, an altered status of circulating EPCs represents a diagnostic biomarker for endothelial dysfunction and individual diseases. Due to their role in enhacing angiogenesis, there has been a growing interest in using EPCs as therapeutic agents to supply the potent origin of neovascularization under pathological conditions (13–16). Endothelial dysfunction is a hallmark of diabetic vascular complications and reduction in circulating EPCs and functional impairment of cultured EPCs have been reported in type I and II diabetic patients (17,18). Recently, the ex vivo therapy of culture-expanded EPC transplantation has been proposed as a novel therapeutic strategy for diabetes mellitus and its associated vascular complications (19–21). However, the possibility of cellular therapy with EPCs for the treatment of DCM has yet to be investigated.
Based on recent advances in studies of EPCs in diabetes and the lack of treatment strategies that improve cardiac repair in diabetics, we hypothesized that EPCs might improve the cardiac function of diabetic rats. To test this hypothesis, a streptozotocin (STZ)-induced diabetic rat model was used. Subsequent to EPC isolation and proliferation in vitro, a series of experiments was conducted to determine the changes in morphology and other biological characteristics, and to assess whether transplantation of EPCs is to be considered a therapeutic candidate for the treatment of DCM.
Materials and methods
Animals and ethics statement
Male Sprague-Dawley rats at a postnatal age of 7–8 weeks (body weight, 200–220 g) were obtained from the Experimental Animal Center of the Mudanjiang Medical College. The rats were kept on straw bedding in cages under light/dark cycle conditions and were acclimatized to the laboratory conditions for 7 days prior to commencing the experiments. The animals were fed standard rodent diet and water ad libitum, unless otherwise indicated. The experiment protocol was approved by the Institutional Animal Care and Use Committee and was performed in accordance with the Guide for the Care and Use of Laboratory Animals (NIH Publication no. 85-23, National Academy Press, Washington, DC, revised 1996).
Isolation and culture of putative EPCs
EPCs were isolated from the femurs and tibias of donor rats, according to the method reported in previous studies (22,23). In brief, the animals were anesthetized with chloral hydrate (300 mg/kg) and sacrificed using cervical dislocation. Femur and tibias were excised from each rat under sterile conditions, while their bone marrow cavities were rinsed with phosphate-buffered saline (PBS). The rinsing solution was then subjected to density gradient centrifugation with Histopaque-1083 (Sigma, St. Louis, MO, USA). The obtained mononuclear cells were grown in M199 medium supplemented with 20% fetal bovine serum (FBS), 0.05 mg/ml bovine pituitary extract (Invitrogen Life Technologies, Carlsbad, CA, USA), antibiotics and 10 U/ml heparin on fibronectin-coated dishes. Seeded cells were cultured in a humidified incubator at 37°C in 5% CO2. After 10 days of culture, the cells were detached with trypsin-EDTA and passed through a cell strainer. To detect transplanted EPCs, cells were pre-labelled with a red fluorescent marker, CM-Dil (Invitrogen Life Technologies) following the manufacturer’s instructions.
Flow cytometry
To confirm that the isolated EPCs maintain their phenotypic characteristics after culture, flow cytometry was performed on freshly isolated cells (at day 0), as well as after 10 days of culture. Briefly, after being washed with PBS, cells were incubated for 30 min at 4°C with mouse anti-rat antibodies for CD31 and CD34 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and rabbit anti-rat antibodies for CD133 and vascular endothelial growth factor receptor 2 (VEGFR2) (Abnova, Taipei, Taiwan), followed by anti-mouse or anti-rabbit fluorescein isothiocyanate (FITC)-labeled secondary antibodies. Finally, flow cytometry was performed with a FACSCalibur cytometer (BD Biosciences, San Jose, CA, USA) and data were analyzed using CellQuest software.
Induction of diabetes
A rat DCM model was induced as described previously (24). In brief, 30 rats were injected intraperitoneally with 60 mg/kg body weight of STZ (Sigma) prepared in 0.1 ml citrate buffer (0.1 mmol/l, pH 4.5) to induce diabetic conditions, while 8 rats received an equal volume of vehicle and served as a non-diabetic group. Seven days subsequent to STZ injection, blood samples were collected from the tail vein after 12 h of fasting. Nineteen rats reached the diabetic rat standard of a fasting blood glucose level >300 mg/dl, while the normal value in the control rats injected with vehicle ranged from 80 to 120 mg/dl. Eight weeks subsequent to vehicle and STZ injection, 16 diabetic rats remained, while the control group survived. The remaining animals were used for additional experiments.
EPC transplantation
Sixteen diabetic rats were randomized into the EPC and DCM groups (n=8 per group). Rats in the EPC group were transplanted intravenously through the tail vein ∼1×106 EPCs (in 0.5 ml culture medium). Rats in the control and DCM groups received an equal volume of culture medium at the same time point. The rats were sacrificed at 4 weeks subsequent to EPC transplantation. Prior to being sacrificed, blood was drawn to detect the level of fasting blood glucose.
Echocardiographic evaluation
Prior to being sacrificed, each rat was anesthetized with chloral hydrate (300 mg/kg). A catheter (PE-50; Becton-Dickinson, Parsippany, NJ, USA) was inserted into the right carotid artery and then advanced into the left ventricular (LV) chamber to measure the following indices: heart rate (HR), LV systolic blood pressure (LVSP), LV end diastolic blood pressure (LVEDP), maximum rising rate of LV pressure (LV+dp/dtmax) and minimum declining rate of LV pressure (LV-dp/dtmin), as delineated previously (25). The rectal temperature was maintained at 36–38°C using a heating pad throughout the procedure. Rats were sacrificed immediately after echocardiographic evaluation. The hearts were harvested and cut into 2 sections, 1 was immersed in 10% formalin solution for Masson’s trichrome staining, while the other in liquid nitrogen for western blot analysis.
Masson’s trichrome staining
The middle of the LV was fixed in 10% neutral-buffered formalin overnight, embedded in paraffin and sectioned to a thickness of 5 μm. The sections were stained with Masson’s trichrome staining to measure interstitial fibrosis. Interstitial collagen was quantified at a final magnification of ×200 with a microscope (BA400 Binocular Microscope; Motic, Xiamen, China) connected to a video camera. The resulting image was processed on a computer image-analysis system. The content of interstitial collagen (expressed as the fractional area of the entire cross section) was averaged on 9 fields selected across the wall thickness in the septum and the free wall.
Protein extraction and western blot analysis
Western blot analysis was performed as previously described (24). The hearts were lysed in radioimmunoprecipitation (RIPA) assay buffer (Beyotime Institute of Biotechnology, Haimen, China). The heart tissue homogenates were clarified by centrifugation and protein concentrations of the lysates were determined using a bovine serum albumin standard line. Equal amounts of protein were boiled at 100°C for 10 min and chilled on ice, subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis, and then electrotransferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5% non-fat dry milk (w/v) and then incubated overnight at 4°C with rabbit anti-caspase-3, rabbit anti-Bcl-2, mouse anti-Bax, rabbit anti-collagen I, rabbit anti-manganese superoxide dismutase (MnSOD) and rabbit anti-p67phox (dilution 1:1000; Abcam, Cambridge, MA, USA), followed by their respective horseradish peroxidase-conjugated secondary antibodies. After extensive washing, the band detection was revealed by an ECL plus chemiluminescence kit (Millipore).
Statistical analysis
Data are presented as the means ± standard deviation (SD). Statistical differences in the groups were evaluated by one-way analysis of variance, while the least-significant difference (LSD) test was used for multiple comparisons. Graphs were plotted and statistical calculations were performed using SigmaPlot 12.0 (Systat Software, Inc., San Jose, CA, USA). P<0.05 was considered to indicate statistically significant differences.
Results
Characterization of bone marrow-derived EPCs
After a 10-day culture, the isolated cells were observed as bone marrow-derived EPC colony-forming units, exhibiting endothelium-specific cord-like structures. Flow cytometry showed that endothelial lineage-specific markers such as CD31, CD34, CD133 and VEGFR2 significantly increased in 10 days subsequent to plating (Fig. 1). The expression changes in these factors are characteristic of those of EPCs (26).
In vivo tracking of EPCs in myocardial tissues
The trafficking of EPCs into the myocardial tissues of STZ-induced diabetic rats was monitored using double staining of CM-Dil and 4′,6-diamidino-2-phenylindole (DAPI). As shown in Fig. 2, numerous CM-Dil-labeled EPCs showing red fluorescence adoptively transferred into the myocardial tissues of STZ-induced diabetic rats, indicating that EPCs had the potential to migrate into the myocardium in the STZ-induced diabetic model at 4 weeks after transplantation.
EPC transplantation improved myocardial function in diabetic rats
Twelve weeks after the induction of diabetes, rat cardiac function was evaluated by invasive hemodynamic measurements. The metabolic and hemodynamic parameters of the animals are presented in Table I. A lower LVSP and higher LVEDP were observed in the DCM compared to the control group. STZ-diabetic animals showed significant impairments in the contraction (+dp/dt) and relaxation rate (−dp/dt), compared to the control animals, indicating that the systolic and diastolic functions in the diabetic rat heart were significantly impaired. Four weeks subsequent to EPC transplantation, the above-mentioned hemodynamic abnormalities were notably attenuated. However, there were no significant differences in the heart rate in the 3 groups. In addition, plasma glucose concentrations in diabetic rats exceeded 16.7 mmol/l, however, these concentrations decreased significantly subsequent to EPC transplantation.
EPC transplantation attenuated myocardial interstitial fibrosis in diabetic rats
Myocardial fibrosis is a pathological hallmark in the development of DCM. Masson’s trichrome staining was used to examine whether EPC transplantation affected the myocardial fibrosis in a STZ-induced diabetic rat model. Obvious interstitial and perivascular fibrosis were detected in the myocardium of the DCM group compared with the control group (Fig. 3A–C). Quantification of the fibrosis area showed a statistically significant increase of fibrosis in the DCM group, whereas EPC transplantation markedly attenuated fibrosis progression in the diabetic hearts (P<0.01) (Fig. 3D). Moreover, western blot analysis showed that the type I collagen expression was markedly upregulated in the DCM group, but inhibited by the EPC transplantation (P<0.01) (Fig. 3E–F). These results suggest that EPC transplantation has the potential to attenuate diabetes-induced myocardial interstitial fibrosis.
EPC transplantation protected cardiomyocytes against apoptosis in diabetic rats
Cardiomyocyte apoptosis is a critical process in the pathogenesis of DCM. The expression of apoptotic markers, such as Bcl-2, Bax and caspase-3, was examined by western blot analysis in order to investigate whether or not transplantation of EPCs improved myocardial function by protecting cardiomyocytes against apoptosis. Bcl-2 was found to be downregulated, whereas Bax and caspase-3 were upregulated in the DCM group (Fig. 4). These changes, however, were markedly attenuated by the administration of EPCs. Since Bcl-2 is characterized by anti-apoptosis, while Bax and caspase-3 by pro-apoptosis, our results suggest that transplantation of EPCs has the potential to protect cardiomyocytes against apoptosis.
EPC transplantation prevented oxidative stress in diabetic rats
The effects of EPC transplantation on the endogenous pro-oxidants and antioxidants, including p67phox and MnSOD, were determined by western blot analysis. The results showed an elevated expression of p67phox in dietetic hearts, suggesting that an endogenous pro-oxidant system was triggered under diabetic conditions, which may further aggravate oxidative stress. EPC transplantation markedly attenuated the p67phox expression (Fig. 5). However, a statistically significant decrease in the MnSOD expression in STZ-induced diabetic hearts was observed, compared to control hearts. Transplantation of EPCs resulted in a statistically significant increase in the MnSOD expression in the DCM group. These findings demonstrate that transplantation of EPC exerts its protective effects by targeting the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and mitochondrial redox enzymes.
Discussion
In this study, we have shown that transplantation of EPCs significantly improved cardiac function and attenuated cardiac fibrosis in a rat model of STZ-induced diabetes, as evidenced by echocardiography, Masson’s trichrome staining and the analysis of type I collagen expression. Moreover, EPC implantation effectively prevented STZ-induced cardiac apoptosis and oxidative stress, indicating the possible mechanisms whereby EPC transplantation exerts its cardioprotective effects against myocardial fibrosis and improves diastolic dysfunction in diabetic rats. The present study is known to be the first to report the potential therapeutic effects of EPC transplantation in an animal model of DCM.
Although in recent years an excessive number of studies have been available on EPCs, the definition of EPCs has yet to be determined due to the lack of unique cell surface molecules that specifically recognize EPCs (27,28). Currently, the most widely accepted definition is the co-expression of the surface markers CD31, CD133, CD34 and VEGFR2 (13). The characteristics of EPCs delineated in the present study are consistent with the common features of EPCs reported in previous studies. In addition, the co-expression of these markers also characterizes the isolated EPCs at a specific maturation stage (14). EPCs have been reported to ameliorate acute myocardial infarction and diabetic erectile dysfunction by promoting angiogenesis (29–30). However, few data are available regarding the role of EPCs in other diabetic complications, particularly in DCM. In the present study, EPC administration markedly improved cardiac function, while fluorescent-labeled EPCs were identified in cardiac tissues by immunofluorescence analysis, suggesting that EPCs act via a direct transdifferentiation into mature cardiomyocytes. These results corresponded to those drawn by Badorff et al (31), showing that EPCs differentiate into cardiomyocytes when co-cultured with neonatal rat cardiomyocytes. Notably, the discovery rate of EPCs was very low in the cardiac tissues of STZ-induced diabetic rats, which further supports the generally accepted concept that apart from direct cell fusion, EPCs may exert a cardioprotective benefit via their paracrine effects (32).
Increased myocardial interstitial fibrosis is a hallmark pathologic feature of DCM and has been considered to be responsible for the diastolic dysfunction occurring in this condition (33). In the present study, 12-week diabetic condition was found to induce interstitial fibrosis in the left ventricle of STZ rats, as demonstrated by morphologic data and the analysis of type I collagen expression. However, EPC administration significantly reduced left ventricular interstitial fibrosis in diabetic rats. These results are consistent with recent findings showing that EPC administration prevents renal interstitial fibrosis in a murine chronic renal failure model (34), as well as in a mouse model of unilateral ureteral obstruction (35). In addition, Liu et al (36) have demonstrated that transplanted EPCs ameliorate carbon tetrachloride-induced liver fibrosis in rats. These results, along with results drawn from previous studies, suggest an anti-fibrotic effect of EPCs under different pathologic conditions. Nonetheless, the detailed mechanism involved in the anti-fibrotic effects of EPC transplantation needs to be further elucidated.
Hyperglycemia-induced apoptosis is an early event in the pathophysiology of DCM (37). Caspase-3 is considered a primary executioner of apoptosis. In the present study, caspase-3 was observed to have been activated in STZ-induced diabetic rat hearts. The Bcl-2 family is thought to be the critical factor in the apoptotic signaling pathways, and the relative ratio of anti- and pro-apoptotic proteins is crucial for the determination of cell survival or death (38). In this study, the hearts in the DCM group were found to have exhibited an increased Bax expression and a decreased Bcl-2 expression at the protein level. However, these changes were markedly reversed by EPC administration, suggesting that transplantation of EPCs might have the potential to protect cardiomyocytes from apoptotic death in diabetic conditions. These results are consistent with previous ones demonstrating that several cytokines, released by peripheral blood or bone marrow-derived EPC, are potent inhibitors of apoptosis (39–40). Furthermore, a recent study by Sen et al (41) showed that autologous transplantation of the genetic modification of EPCs also inhibited cardiac apoptosis in a rat model of myocardial infarction. Collectively, results in the current study confirmed previous findings demonstrating that cardiomyocytes apoptosis is associated with the progression of DCM, and that EPC implantation has the potential to prevent cardiomyocytes apoptosis in diabetic conditions.
STZ treatment induces systemic generation of reactive oxygen species (ROS) by activating NADPH oxidase, thus leading to cardiac dysfunction (42). To further investigate the mechanisms by which EPCs exert their cardioprotective effects, the expression of NADPH oxidase subunit p67phox and mitochondrial ROS-eliminating enzyme MnSOD was examined. The results showed that EPC transplantation attenuated the increased p67phox expression and the decreased MnSOD expression that were induced by STZ injection. These findings indicate that EPC transplantation ameliorated diabetic cardiac damage by targeting the redox signaling pathways.
In summary, transplantation of bone-marrow EPCs improved cardiac function, protected cardiomyocytes against apoptosis and reduced the extent of myocardial interstitial fibrosis in a STZ-induced diabetic rat model. Additional experiments showed that this cardioprotective effect may be associated with the antioxidative properties of EPCs. Thus, EPC transplantation may be a novel therapeutic approach for the prevention of diabetic myocardial complications.