This study conforms to the principles outlined in the Declaration of Helsinki. Ethical approval was received from the Ethics Committee of the Faculty of Medicine of Menoufia University and Menoufia University Teaching Hospital (Egypt). Informed written consent was obtained from all patients.
Isolation and culture of human mesenchymal stem cells isolated from Wharton’s Jelly (hWMSCs)
Umbilical cords were aseptically collected from 10 healthy full-term pregnancies, at Menoufia University Teaching Hospital (Egypt). hWMSCs were isolated as described previously with trivial modifications. Umbilical cords were cut into 2 to 3 mm pieces and vessels were stripped manually from those cord segments. Wharton’s Jelly was digested with collagenase 10 mg/mL (37°C for 4 hours). Mesenchymal cells were recovered, centrifuged (1000 g for 30 minutes) and suspended in fresh Dulbecco's Modified Eagle's (DMEM) medium including cord blood serum (100µl/ml) , L-glutamine (100 μg/mL), penicillin (100 U/ml), streptomycin (100 μg/ml streptomycin) and fungizone (0.25 μg/mL). Cultures were maintained in a humidified incubator with 5% CO2 at 37°C for 10 days, until hWMSCs colonies were observed. Adherent cells were detached using a trypsin-EDTA solution .
Cell characterization by flow cytometry was carried out as previously described . Briefly, hWMSCs in culture were trypsinized (0.25% trypsin/1 mM EDTA) and then incubated in the dark for 30 minutes with CD44 and CD34 monoclonal antibodies (R&D System, USA) conjugated with phycoerythrin (PE). 100 μl of cell suspension was added to 10 μl of CD44-PE and CD34-PE. After incubation cells were gated out on CD44 and CD34 expression an argon laser FACS Calibur (Becton Dickinson, USA), at the Laboratory of Clinical Pathology Department, Menoufia University Teaching hospitals.
Endothelial differentiation was performed as described previously with some modifications . hWMSCs were incubated in 35 mm plates in primary culture medium supplemented with 50 ng/ml vascular endothelial growth factor (VEGF, R&D System, USA). Endothelial phenotype was confirmed through flow-cytometric analysis of surface markers CD34-PE as described earlier..
Animals and experimental design
30 male Wistar albino rats were purchased from a local experimental animals providing facility and recruited for the present study. All experimental procedures were conducted in adherence to the Guiding Principles in the Use and Care of Animals published by the National Institutes of Health (NIH Publication No 85–23, Revised 1996). Animal care and use was approved by Ethics Committee, Menoufia Faculty of Medicine, Egypt. Animals were kept for 10 days prior to the start of the study to allow proper acclimatization. The animals were fed standard laboratory chow and allowed free access to water in an air-conditioned room with a 12 h light-dark cycle. Diabetes mellitus was confirmed forty eight hours after streptozotocin injection by measuring fasting blood glucose level. Rats with fasting blood glucose levels ≥ 200 mg/dl were considered diabetic. Rats were then divided randomly into the following groups (10 rats per group):
1-control group: 18–20 months old, weighing 350–400 g received a single intraperitoneal injection of isotonic saline. Six weeks later, they received a single intravenous injection of isotonic saline via tail vein.
2- Aged group: 18–20 months old, weighing 350–400 g, received a single intraperitoneal injection of 50 mg/kg streptozotocin (Sigma-Aldrich, St Louis, MO, USA), and 6 weeks later they also received a single intravenous injection of isotonic saline via tail vein.
3-Aged diabetic + mesenchymal cell-derived endothelial cells group: (18–20 months old, weighing 350–400 g, received single intraperitoneal injection of 50 mg/kg streptozotocin, and 6 weeks later they received a single intravenous injection of 2*106 ECs in 0.5 ml isotonic saline via tail vein.
Measurement of systolic blood pressure
Systolic blood pressure was determined in the rats by means of a rat-tail pressure detecting equipment (Harvard apparatus Ltd, Aden Berge, England) connected to a pneumatic transducer (Harvard U.K.). Changes in pressure were recorded via a physiograph (MK III-S, Narco BioSystem, USA).
Evaluation of aortic pulse wave velocity (PWV) and renal artery resistance (RAR) was done using pulsed doppler flowmeter (Hadeco, Hayashi Denki Co. Ltd., Japan). Briefly, rats were anesthetized via intraperitoneal injection of sodium thiopental (STP, 60 mg/kg), and a midline abdominal incision was made exposing the aorta and the left renal arteries. The chest wall was also opened by extending the midline incision to facilitate blood sample collection and excision of the aorta for the tissue homogenate preparation and PCR experiments. The tip of the probe was filled with coupling gel, and then the probe was placed over the blood vessel until a stable record could be recorded.
Blood sample collection
At the end of the experiments, all rats were fasted overnight. Rats were then anaesthetised using STP (as mentioned vid supra). Blood samples were collected via cardiac puncture, left to clot for 10-15 minutes and then centrifuged at 3000 rpm for another 15 minutes. Serum samples were stored at -20 °C for subsequent analysis of different biomarkers. All rats were then scarified by cervical dislocation.
Preparation of aortic tissue homogenate
A midline incision was made to open the chest and abdominal walls and expose the aorta. The aorta was excised and cross-chopped into fine slices using a surgical scalpel. Rat aorta was homogenized in ice cold phosphate buffer (pH 7.4), suspended in chilled 0.25 M sucrose solution, and rapidly blotted on a filter paper. Mincing and homogenization of tissue were performed to release soluble proteins in ice-cold Tris hydrochloride buffer (10mM, pH 7.4). Tissue homogenate was centrifuged at 7000 rpm for 20 minutes and the supernatant was collected and stored at -20 °C for subsequent estimation of NO.
Serum levels of interleukin 6 (IL-6), tumour necrosis factor alpha (TNF-α) (Quantikine® ELISA, R&D Systems Inc., MN, USA), endothelin-1 (ET-1) (Immuno-Biological Laboratories Co. Ltd., Takasaki-Shi, Gunma, Japan), angiotensin II (AGN-II) (Sigma-Aldrich, St Louis, MO, USA), and advanced glycation end products (AGEs) (MyBioSource, USA) were determined by quantitative sandwich enzyme immunoassay technique using an automatic optical reader (SUNRISE Touchscreen, TECHAN, Salzburg, Austria). Serum levels of malondialdehyde (MDA) (QuantiChrom™, BioAssay Systems, USA), and reactive oxygen species (ROS) (Elabscience®, USA) were determined by routine kinetic and fixed rate colorimetric methods on a Jenway Genova autoanalyser (UK). Tissue level of nitric oxide (NO) (QuantiChrom™, BioAssay Systems, USA) was determined by quantitative sandwich enzyme immunoassay technique.
Quantification of eNOS mRNA
The thoracic aorta was isolated and cut into 12 mm segments. Total RNA was extracted from rat aorta using TRI reagent (Sigma-Aldrich, UK). Extracted RNAs were reverse transcribed using the high capacity RNA-to-cDNA kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. Real-time RT-PCR was performed using a Biosystem 7300 (Applied Biosystems, CA, USA). To quantify changes in gene expression, the comparative Ct method was used to calculate the relative-fold changes normalized relative to the housekeeping gene GAPDH. The gene specific primers for eNOS were: 5′-ACT GCG TCG CTT CAT TAG GT-3′ (forward) and 5′-TAG GCA AGC GCT TTA CCA CT-3′ (reverse), while primers for GAPDH were: 5′-TGG GTG TGA ACC ACA AGA AA-3′ (forward) and 5′-GTG GCA GTG ATG ACA TGG AC-3′ (reverse) for rat GAPDH. Results are shown as the mean of three samples, with each sample assayed in duplicate.
Haematoxylin and Eosin (H&E) stain
Specimen from both thoracic aortas and cardiac muscles of the aged diabetic ECs treated and non-treated rats were fixed in 10% formol saline for 4-6 days. The specimens were thoroughly washed in tap water and then dehydrated in graded ethanol solutions. After that, the specimens were cleared in xylene depending on the size of the specimen (guided by inspection at five minutes intervals). The specimens were soaked in soft paraffin wax at 60 °C for two hours, and then in hard paraffin wax at room temperature. Next, tissue blocks were cut into 5 micron-thick sections using rotator microtome. Tissue sections were dipped in a warm water-bath, picked up on clean slides, and placed on hot plate for 2 minutes. Finally, tissue sections were stained with haematoxylin and eosin.
Antihuman primary antibody to CD31 (Human CD31/PECAM-1 Antibody, R&D Systems, USA) was used for the immunostaining experiments. The tissue sections were incubated with the anti-CD31 antibody overnight at 4°C. The binding of the primary antibody was observed using a commercial avidinbiotin-peroxidase detection system recommended by the manufacturer (DAKO, Carpenteria, USA). Finally, the slides were stained with diaminobenzene (DAB).
Results are expressed as mean ± standard deviation (SD). Kolmogorov-Smirnov test was performed on all data sets to ensure normal distribution (p > 0.5). Analyses of Variances (ANOVA) with Tukey’s honesty significant difference (HSD) tests were used for statistical analysis using Origin® software. The probability of chance (p values) values < 0.05 were considered significant.