Introduction
Male erectile dysfunction (ED) poses a serious threat to the quality of life, particularly in older men, causing extreme morbidity among elderly and diabetic patients. ED caused by diabetes is a refractory symptom. The incidence of diabetes mellitus-induced ED (DMED) is 19.0-86.3%, which is three times the rate of ED in non-diabetic patients (1,2). The mechanism of DMED is not entirely clear, but it is associated with problems in blood vessels, nerves, neurotransmitters, and the endocrine system, among others (3-5). Vascular lesions caused by diabetes may induce atheromatous plaque formation in the cavernous arteries of the penis, resulting in reduced blood flow and structural or functional destruction of arterioles and sinusoidal endothelial cells in the cavernous body, further affecting erectile function. Phosphodiesterase 5 inhibitors, which are often used to treat ED in the general population, have almost no therapeutic effect on DMED (6). There is thus a clear need for a new approach to DMED treatment.
Adipose-derived mesenchymal stem cells (ADSCs) are multipotent adult stem cells derived from adipose tissue (7). They have the advantages of easy extraction, simple culture and rapid proliferation, and they have become an important type of seed cell for use in regenerative medicine (8). Some scientists have used ADSCs to treat diabetic varicose veins by reprogramming them into endothelial cells that promote capillary formation. In addition, ADSCs have anti-inflammatory properties and can repair nerve damage (9,10). ADSC transplantation therapy is widely used to treat diseases such as myocardial ischemia. Studies have shown that ADSCs can reduce myocardial infarction area, promote angiogenesis and reverse ischemic injury disruption of cardiac remodeling (11,12). ADSCs have also been used to treat a variety of animal models of erectile dysfunction, including DMED models, penile cavernous nerve injury models, penile induration models, and penile cavernous radiation injury models (13-16).
Bone marrow mesenchymal stem cells (BMSCs) are a type of pluripotent stem cells originating from the mesoderm. They are able to differentiate into bone, cartilage and fat (17). Autologous bone marrow transplantation can avoid the complications caused by transplant rejection, but research on and application of this technique are costly (18). Bivalacqua et al (19) cultured BMSCs in vitro, transfected them with angiogenic endothelial nitric oxide synthase (eNOS), and injected these BMSCs into the corpora cavernosa of aged male rats. Histological analysis revealed that BMSCs survived at least 21 days in the corporal tissue and did not cause an obvious inflammatory response. Studies have demonstrated that BMSCs can improve erectile function in older rats (20,21). However, BMSCs are difficult to obtain, and the limited number of available BMSCs cannot meet the clinical demand.
Selecting the optimal type of stem cells to treat DMED is crucial. To date, there is little research comparing the therapeutic effects of ADSCs and BMSCs on diabetic erectile dysfunction. In the present study, a rat model of diabetes was established, the rats were treated with ADSCs and BMSCs, and the therapeutic effects of both types of stem cells in the treatment of DMED were evaluated.
Materials and methods
Reagents
CD34, CD45, CD73, CD90 and CD105 antibodies were purchased from BD Biosciences. Streptozotocin, apomorphine (APO) and vitamin C were purchased from Sigma-Aldrich; Merck KGaA. Anti-eNOS and β-actin antibodies were purchased from Cell Signaling Technology, Inc. Modified Lillie-Mayer hematoxylin staining solution, ethanol eosin staining solution and Sirius Red staining solution were purchased from Reagan.
Male Sprague-Dawley (SD) rats were provided by Guangdong Pharmaceutical University. All animal experiments were approved by the Animal Ethics Committee of Guangdong Pharmaceutical University.
Isolation and culture of rat ADSCs and BMSCs
Male 6-week-old SD rats were anesthetized, sacrificed by cervical dislocation and soaked in iodophor for 2 min, followed by 75% alcohol for 5 min. Following removal of the epididymal fat, type 1 collagenase was used to digest and separate ADSCs.
To isolate BMSCs, the femur, tibia, and fibula were separated, and the medullary cavity was rinsed with 0.9% saline solution until the rinsing liquid was clear and the bones were whitish. The rinsing liquid was centrifuged, the supernatant was discarded, and the serum-containing medium was added to resuspend cells.
Identification of ADSCs and BMSCs by flow cytometry
ADSCs and BMSCs were washed once with 0.01 mol/l phosphate-buffered saline (PBS) and mixed with CD34, CD45, CD73, CD90 and CD105 monoclonal antibodies at a working concentration of 1:100. The resultant solutions were incubated at 4°C for 30 min and washed twice with 0.01 mol/l PBS, after which time ADSCs and BMSCs were detected by flow cytometry.
Establishment and stem cell treatments of diabetic rat models
After 1 week of adaptive feeding, 40 male SD rats were randomly divided into two groups (control group, n=10; and diabetes group, n=30). The rats in the control group (Ctrl) were provided with a normal diet, while the diabetic rats were fed a high-fat, high-sugar diet. One month later, the diabetic rats were intraperitoneally injected with streptozotocin (40 mg/kg) (22). Blood glucose was monitored starting 3 days after streptozotocin injection. The animals were considered as diabetic when the glucose levels were >16.7 mM for 3 consecutive days (reference range, 5-8 mM).
The 30 diabetic rats were randomly divided into three groups (n=10 per group): One group was treated with ADSCs (AD group), one with BMSCs (BM group), and one with PBS alone (PBS group).
Injection sera were created by resuspending ADSCs and BMSCs (1x107 cells) in 1 ml of PBS. Rats were anesthetized with pentobarbital (50 mg/kg, i.p.), and injected with the cell suspensions at the mid-penile corpus cavernosum (23). The AD group rats were injected with 100 µl of ADSC suspension (1x106 cells), the BM group rats were injected with 100 µl of BMSC suspension, and animals in the PBS group were injected with 100 µl of PBS. The control group did not receive any treatment.
APO-induced erection
APO was used to induce penile erections in rats before and at 4 weeks after stem cell transplantation. The rats were placed in a dark room under observation, kept calm, and allowed to adapt to the environment for 10 min. They were then subcutaneously injected with APO (100 µg/kg) in the back of the neck, and the number of erections in 30 min was recorded, as measured by intracavernous pressure (ICP) and mean arterial pressure (MAP). Healthy 14-week-old rats were used as controls.
Measurement of ICP and MAP
ICP and MAP were measured as previously described (24). The stimulus parameters were 20 Hz, a pulse width of 0.2 m sec, 1.5 mA, and a duration of 50 sec. The ratio of maximum ICP (mmHg) to MAP (mmHg) was calculated to normalize variations in systemic blood pressure.
Immunofluorescence analysis
After the completion of the APO experiment, five animals from each group were randomly selected, anesthetized and sacrificed by cervical dislocation. Half of the penile tissue from each subject (10 µm) was dehydrated in 30% sucrose and then frozen. After the sections were completely dried, immunofluorescence staining for eNOS was performed, using the penile tissue of normal rats as a control. At least five random fields per section were examined, and the semi-quantitative evaluations were analyzed with Image-Pro Plus software, version 6.0 (Media Cybernetics, Inc.).
Western blot analysis
The remaining half of the penile tissue from each subject was combined with an appropriate amount of pre-cooled RIPA lysis buffer (Fude) and homogenized at 4°C to extract proteins from the tissue. The extracted proteins were denatured and used to detect the expression of eNOS by western blotting. The protein expression levels of β-actin served as the housekeeping control. Images were captured with Tanon-5200 and quantified using Gel-pro 6.0 software.
Hematoxylin and eosin (H&E) staining and Sirius Red staining
The remaining 5 animals in each group were sacrificed by cervical dislocation, and the middle section of penile tissue was removed. Half of this tissue from each subject was fixed and dehydrated. Paraffin-embedded sections were stained with H&E or Sirius Red. The numbers of blood vessels and the levels of fibrosis in each group were compared. Images were captured using an Olympus microscope (Olympus Corporation). At least five random fields per section were examined, and semi-quantitative evaluations were performed with Image-Pro Plus software, version 6.0.
Transmission electron microscopy
The remaining half of the penile tissue from each subject was fixed in 2% glutaraldehyde. The tissue was washed with PBS, followed by 1% citric acid for 2 h at room temperature. The tissue was then dehydrated, embedded in paraffin, and cut into ultrathin sections. After double staining tissue samples with uranyl acetate and lead citrate, the morphology of endothelial cells and smooth muscle cells in the cavernous tissue was observed under a transmission electron microscope.
Statistical analysis
All analyses were performed with the GraphPad Prism 4.0 software (GraphPad Software, Inc.). Data are expressed as the mean ± standard deviation and then analyzed using one-way ANOVA followed by Tukey's multiple comparisons test. P-values <0.05 were considered to indicate statistically significant differences.
Results
Identification of ADSCs and BMSCs by flow cytometry
To determine whether the isolated cells were MSCs, cell surface marker expression was examined. The isolated cells expressed the known MSC markers CD73, CD90 and CD105, but not the hematopoietic and endothelial markers CD34 and CD45 (Fig. 1). Flow cytometry results revealed that the positive rates of CD73, CD90 and CD105 expression in ADSCs were 98.88, 99.34 and 95.07%, respectively, while the positive rates of CD34 and CD45 were 0.42 and 0.04%, respectively. The positive rates of CD73, CD90, and CD105 in BMSCs were 99.76, 99.91 and 94.14%, respectively, and the positive rates of CD34 and CD45 were 0.84 and 0.66%, respectively.
Characteristics of the animals
The mean body weight of the diabetic rats was significantly lower (P<0.01) compared with that of the normal and ADSC-treated diabetic rats (Table I and Fig. 2A). No significant differences in body weight were observed between the diabetic and the AD or BM groups. The fasting blood glucose levels were significantly higher (P<0.01) in diabetic rats compared with controls (Table I and Fig. 2B). No significant differences in blood glucose levels were observed between the diabetic group and the AD or BM groups.
Effect of ADSCs and BMSCs on erectile function
The ICP/MAP ratios and total ICP values for all groups are presented in Fig. 2C and Table I. The ICP/MAP ratios of diabetic rats were lower compared with those of normal rats (P<0.01), a phenomenon that was reversed through treatment with ADSCs (P<0.01) or BMSCs (P<0.05). The ICP/MAP ratios of ADSC-treated diabetic rats were higher compared with those of BMSC-treated diabetic rats. The total ICP values produced by stimulation of the cavernous nerve were lower in diabetic rats compared with normal rats (P<0.01), but returned to normal levels following treatment with ADSCs (P<0.01) or BMSCs (P<0.05). The total ICP values of ADSC-treated diabetic rats were higher compared with those of BMSC-treated diabetic rats.
ADSCs improve the expression of eNOS in the penile tissue of diabetic rats
The expression of eNOS in the penile tissues of the four groups was determined by western blot analysis. As shown in Fig. 3, the expression of eNOS was significantly lower in the diabetic group compared with that in the control group (P<0.05). The expression of eNOS was higher in the AD and BM groups compared with that in the diabetic group (P<0.05), and slightly higher in the AD group compared with that in the BM group.
eNOS expression in the penile tissue was also compared across groups by immunofluorescence analysis, which yielded results similar to those of western blotting: The expression of eNOS was significantly lower in the diabetic group compared with that in the control group (P<0.05), higher in the AD and BM groups compared with that in diabetic rats (P<0.05), and slightly higher in ADSC-treated rats compared with BMSC-treated rats (Fig. 4).
ADSC treatment promotes revascularization in the corpus cavernosum of diabetic rats
H&E staining was used to observe the number of blood vessels in the corpus cavernosum. As shown in Fig. 5, diabetic rats had significantly fewer blood vessels compared with healthy controls (P<0.01). This effect was reversed by treatment with ADSCs (P<0.01) or BMSCs (P<0.05). The number of blood vessels in ADSC-treated diabetic rats was higher compared with that in BMSC-treated diabetic rats.
ADSC treatment reduces collagen content in the corpus cavernosum of diabetic rats
Collagen fiber content in the penile tissue of diabetic rats was investigated using Sirius Red staining. As shown in Fig. 6, the penile tissue of diabetic rats exhibited a significantly increased collagen content, which was reversed by treatment with ADSCs (P<0.01) or BMSCs (P<0.05). The collagen fiber content in ADSC-treated diabetic rats was lower compared with that in BMSC-treated diabetic rats.
ADSC treatment ameliorates cell impairment in penile tissue, as observed on transmission electron microscopy
The ultrastructural characteristics of penile tissue cells were observed by transmission electron microscopy. In the corpora cavernosa of diabetic rats, endothelial cells and smooth muscle cells were damaged (Fig. 7), with injuries to the plasma membrane, concentrated cytoplasm and condensed nuclei. Stem cell treatment ameliorated cellular impairments induced by diabetes; this effect was more obvious in the ADSC-treated group compared with that in the BMSC-treated group.
Discussion
There have been major advances in ED research over the past decade (25), but DMED remains difficult to treat. Stem cell therapy has been proposed as a viable treatment strategy, and some clinical and preclinical trials have been conducted in a few related areas (26,27). A small number of studies have examined the use of ADSCs and BMSCs in the treatment of DMED (28-30) but, to the best of our knowledge, there is currently no report comparing the effects of ADSCs and BMSCs on DMED. Therefore, in the present study, a diabetic rat model was established by streptozocin induction, and ADSCs and BMSCs were transplanted into the penile tissue. The effects of ADSC and BMSC treatment were then compared, and the mechanism of action of each type of stem cell was investigated.
ADSCs and BMSCs were successfully isolated, cultured in vitro, and their identity was confirmed by flow cytometry. Diabetic rats were treated with ADSCs and BMSCs, and their erectile function following treatment was determined by their ICP/MAP ratio. The results demonstrated that ADSC treatment restored the erectile function of diabetic rats.
Several studies have reported that ED is induced by inadequate relaxation of the corpus cavernosum with defects in NO production (31). NO is formed from the conversion of L-arginine by NOS, which exists in three isoforms: Endothelial (eNOS), neuronal (nNOS), and inducible (iNOS). eNOS expression has been identified both in the cavernosal endothelium and in smooth muscle cells (32). Endothelium-generated NO appears to be crucial for maintaining an erection (33). Therefore, in the present study, the expression of eNOS in the penile tissues was examined. According to both western blot and immunofluorescence analyses, ADSC treatment increased the expression of eNOS in diabetic rats, and eNOS expression was slightly higher in the ADSC-treated group compared with that in the BMSC group. Accordingly, H&E staining revealed that ADSC treatment increased the number of blood vessels in the penile tissue of diabetic rats. These findings suggest that ADSC treatment was more effective compared with BMSC treatment in promoting revascularization of the corpus cavernosum in diabetic rats.
In addition, the ultrastructural characteristics of endothelial and smooth muscle cells in each group were observed under a transmission electron microscope. Cellular morphological characteristics suggested that ADSCs were more effective compared with BMSCs in ameliorating diabetes-induced impairment of endothelial and smooth muscle cells in penile tissue.
A mounting number of studies have demonstrated that ADSC treatment can promote revascularization, which suggests that ADSCs are a promising approach to the treatment of diseases such as critical limb ischemia (11), ischemic cardiomyopathy (34) and myocardial infarction (35). ADSCs are adult stem cells isolated from adipose tissue that are capable of self-renewal and multi-directional differentiation (36). They have low immunogenicity and can be easily collected with minimal risk. In the field of regenerative medicine, ADSCs are widely used in tissue repair and regeneration. All these characteristics make ADSCs the most suitable type of stem cell for use in the treatment of ED (37). ADSC transplantation has achieved encouraging results in multiple ED models, including DMED (38,39). There is increasing evidence that the therapeutic effects of ADSCs on ED may be mediated through paracrine indirect effects of growth factors or cytokines, rather than by direct differentiation into specific cell types (40), a subject which requires further study.
Several studies have indicated that BMSC treatment may also promote revascularization (41,42). However, in the present study, ADSCs promoted angiogenesis more effectively compared with BMSCs, which is consistent with the earlier findings of Wu et al (43). To the best of our knowledge, this is the first study to directly compare the therapeutic effects of ADSCs and BMSCs on ED in diabetic rats.
However, this was a preliminary study that mainly focuses on comparing the therapeutic effects of ADSCs and BMSCs on DMED. On this basis, more in-depth mechanism research is still needed, and experiments for protein or non-coding RNA are required as the next step.
In conclusion, treatment with ADSCs significantly restored erectile function in diabetic rats by improving eNOS expression, promoting revascularization, and reversing cellular functional impairment. ADSC treatment was found to be more effective compared with BMSC treatment in restoring erectile function in diabetic rats. These findings may provide a new approach to the treatment of DMED.