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Impairment in the number and function of CD34+/KDR+ circulating cells in diabetes and obesity with functional improvement after thymosin β4 treatment

Lee, Poay Sian Sabrinaa,b; Ye, Leic; Khoo, Eric Yin Haoa; Yeo, Tiong Chenga,b; Tan, Huay Cheema,b; Richards, Arthur Marka,b; Poh, Kian Keonga,b

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Cardiovascular Endocrinology: June 2016 - Volume 5 - Issue 2 - p 51-56
doi: 10.1097/XCE.0000000000000076
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Obesity is the fifth leading risk for worldwide mortality; at least 2.8 million adults die each year as a result of being obese 1. It is usually the result of a combination of genetic factors with an inappropriate choice of lifestyle. Obesity is closely associated with the development of type 2 diabetes and cardiovascular disease. An important mechanism by which obesity leads to type 2 diabetes is the development of insulin resistance 2. Initially, these obese individuals have normal fasting glucose levels with elevated plasma insulin, but become progressively more hyperglycemic and insulin resistant 3. Obese individuals diagnosed with type 2 diabetes almost invariably have endothelial dysfunction determined by vascular reactivity and/or by measurement of plasma markers of endothelial activation 4,5. Cells, coexpressing surface markers of hematopoietic stem cells, CD34 6 and kinase insert domain receptor (KDR) 7 have been shown to play a role in endothelial repair. Studies have also shown that the number and function of CD34+/KDR+ circulating cells are reduced in type 2 diabetic humans who are also obese 8,9.

Thymosin β4 (Tβ4) is a small, naturally occurring 43-amino acid peptide that belongs to a large family of small bioactive molecules 10 that are highly conserved, which could suggest fundamental biological importance 11. Being a protein known to have multiple biological properties essential to cardiac repair and regeneration, there has been increasing evidence suggesting the promising therapeutic effects of Tβ4 in treating endothelial dysfunction or cardiac infarction 12. Tβ4 has a chemoattractant property, especially on migration, and it is endothelial cell-specific 13. However, little is known about the effect of Tβ4 on the number and function of CD34+/KDR+ circulating cells.

We used male Zucker diabetic fatty (ZDF) rats as a genetic model of obese type 2 diabetes as they carry several characteristics that are in line with human obesity and type 2 diabetes in humans 14. Young ZDF rats show normal fasting glucose levels with slightly elevated plasma insulin levels and become progressively more hyperglycemic and insulin resistant as plasma insulin levels are decreased with pancreatic β-cell dysfunction 14. We hypothesize that Tβ4 can reduce endothelial dysfunction in diabetes and obesity. We aim to determine whether Tβ4 can improve the numbers and functions of CD34+/KDR+ circulating cells in a diabetic and obese animal model.



Animal experiments were approved by the Institutional Animal Care and Use Committee, National University of Singapore, and followed the Guide for the Care and Use of Laboratory Animals. Male ZDF (/Gmi-fa/fa) (n=20) rats and age-matched, Zucker lean (ZL) control (lean fa/+) (n=21) (Charles River Laboratories, Wilmington, Massachusetts, USA) were studied at 20 weeks of age weighing 400–650 g. They were housed individually in automated ventilated cages in environmentally controlled rooms (22–24°C; 50–70% relative humidity) under a 12 h light/dark cycle. All animals were maintained on Purina 5008 diet (LabDiet, St Louis, Missouri, USA) as recommended by the supplier. The composition of this diet by weight is 23% protein, 58.5% carbohydrate, and 6.5% fat.

Blood collection

Rats were anesthetized by isoflurane (4%) inhalation. For measurement of fasting glucose levels, a small sample of blood from overnight 12 h fasting was obtained through the tail vein and confirmed using an Accu-Chek Performa (Roche Diagnostics, Basel, Switzerland) blood glucose meter. Then, the rats were killed using an intraperitoneal injection of pentobarbital (30 mg/kg). Blood (∼8–10 ml/rat) was collected from cardiopuncture into an EDTA vacutainer tube. Blood was processed within 2 h of collection.

Peripheral blood mononuclear cell isolation

Mononuclear cells were isolated from rat peripheral blood by density gradient centrifugation using Ficoll-Paque Premium 1.084 (GE Healthcare, Little Chalfont, Buckinghamshire, UK). Briefly, Ficoll-Paque (10 ml) was added to a 50 ml Falcon tube. Blood (8–10 ml) was diluted with PBS without Ca/Mg at a ratio of 1 : 1 and then carefully layered on top of the Ficoll-Paque. Cells were centrifuged at 300g for 35 min at room temperature without the brake. The upper plasma layer was removed without disturbing the plasma-Ficoll-Paque and washed twice with PBS without Ca/Mg.

CFU-Hill assay

This assay was performed on the basis of the 5-day CFU-Hill assay using the CFU-Hill Liquid Medium Kit (StemCell Technologies, Vancouver, British Columbia, Canada). Mononuclear cells (2×106 cells) were plated together with CFU-Hill medium (2 ml) in each well of a BioCoat fibronectin-coated six-well plate (Becton Dickinson, Franklin Lakes, New Jersey, USA) onto each well. After 48 h, nonadherent cells from the six-well plate were then transferred to a BioCoat fibronectin-coated 24-well plate (Becton Dickinson) with the addition of Tβ4 (10 ng/ml) (ProSpec, Rehovot, Israel) and CFU-Hill colony formation was observed at day 5. Bright-field microscopy images were taken on days 0, 2, and 5.

Culture of CD34+/KDR+ circulating cells

Briefly, mononuclear cells (3×106 cells) were cultured on each well of a BioCoat fibronectin-coated 24-well plate in an endothelial growth media-2 bullet kit, which consists of endothelial basal medium supplemented with 0.5 ml single aliquots of rhEGF, vascular endothelial growth factor (VEGF), ascorbic acid, heparin, gentamicin+amphotericin-B (GA-1000), hydrocortisone, hFGF-B, R3-IGF-1, and 20% FBS (Lonza, Basel, Switzerland). Nonadherent cells were removed after day 4 and media were changed after 3 days in subsequent. Tβ4 (10 ng/ml) was added at day 14 and mature cells were determined at day 17 for migration and tubule formation experiments.

Flow cytometry

In brief, mononuclear cells (0.5×106 cells) was incubated initially with FcR blocking reagent (Miltenyi Biotec, Bergisch Gladbach, Germany) and then stained with mouse/rat CD34 affinity-purified polyclonal Ab, sheep IgG (R&D Systems, Minneapolis, Minnesota, USA), followed by anti-VEGF receptor 2 antibody (KDR/EIC) (Abcam, Cambridge, UK). Respective isotype antibodies served as controls. After incubation, cells were washed with PBS+2% FBS and fixed in PBS+0.5% formaldehyde. The FACSCalibur cytometer was set to acquire 500 000 events and analyses were carried out within the lymphocyte gate in accordance with the established protocol. Data were processed using the Becton Dickinson CellQuest Pro software (version 5.1). Cell phenotype was assessed as the percentage of CD34+/KDR+ per 1×106 mononuclear cells.

Migration and tubule formation assays

CD34+/KDR+ circulating cells harvested were detached using 1 mmol/l EDTA in PBS (pH 7.4), resuspended in 500 μl endothelial basal medium, and 1×104 cells were placed in the upper chamber of a sterile 6.5 mm Transwell with a 3.0 μm pore. The chamber was then placed in a 24-well plate containing the medium and vascular endothelial growth factor (50 ng/ml). After 24 h incubation at 37°C, the lower side of the filter was washed with PBS and fixed with 2% formaldehyde. For quantification analysis, cell nuclei were stained with DAPI. Cells that migrated into the lower chamber were counted manually in four random microscopic fields. For the tubule formation assay, an ECM gel was layered into 96-well plates for 1 h at 37°C. CD34+/KDR+ circulating cells (1×104) were plated with vascular endothelial growth factor (50 ng/ml), followed by incubation at 37°C for 8 h. Incorporated cells were counted from four random microscope fields per each rat.

Statistical analysis

Data are expressed as mean±SD. Comparisons of the number and function of CD34+/KDR+ circulating cells between the ZL and ZDF rats at baseline were performed using unpaired t-tests. The effect of Tβ4was analyzed using one-way analysis of variance. All statistical data were calculated using GraphPad Prism 5.0 for Windows (GraphPad Software, La Jolla, California, USA). Statistical significance was assumed if the P value was less than 0.05.


Zucker lean and Zucker diabetic fatty rats at baseline

Forty-one rats (ZDF=21; ZL=20) were used for this study. ZDF rats were heavier (body weight: 641±37 g) compared with ZL rats (body weight: 433±33 g) at the start of the study. Blood glucose concentrations were 9.4±1.4 and 21.2±3.2 mmol/l in ZL control rats and ZDF rats, respectively (P<0.0001) (Fig. 1). Endothelial progenitor cells (EPCs) for ZDF rats had significantly higher dysfunction in terms of colony-forming unit (P=0.001), percentage of CD34+/KDR+ (P<0.0001), number of migrated cells (P=0.004), and mean branch length per mm2 (P=0.01) compared with the controls (Fig. 1).

Fig. 1
Fig. 1:
Baseline body weight, blood glucose levels, and indices for the number and functionality of CD34+/KDR+ circulating cells for ZL and ZDF rats. KDR, kinase insert domain receptor; ZDF, Zucker diabetic fatty; ZL, Zucker lean.

Effect of thymosin β4 on the number and function of CD34+/KDR+ circulating cells

There was no significant change in the number of Tβ4-treated CD34+/KDR+ circulating cells from ZDF rats (P=0.57) (Fig. 2a). The colony-forming unit ability of ZDF rats also did not improve with Tβ4 (P=0.36) (Fig. 2b). However, there were significant improvements in migration and tubule formation, between pre-Tβ4 treatment and post-Tβ4 treatment on CD34+/KDR+ circulating cells isolated from ZDF rats. After Tβ4 was administered, the CD34+/KDR+ circulating cells from ZDF rats showed an average increase of 73% in the number of migrated cells (P<0.0001) (Fig. 2c). Comparison between post-treatment ZDF rats and ZL rats showed that the number of migrated cells for post-treatment ZDF rats was comparable with that of ZL rats (P=0.08). A 31% increase in tubule formation was also observed in ZDF rats after Tβ4 treatment (P=0.003). Tubule formation ability of ZDF rats after treatment was restored to levels similar to that of ZL rats (P=0.73) (Fig. 2d).

Fig. 2
Fig. 2:
Effect of thymosin β4 (Tβ4) (10 ng/ml) on number (a), colony-forming capacity (b), migratory (c), and tubule formation activities (d) of CD34+/KDR+ circulating cells for ZDF rats. P<0.05 (statistically significant). KDR, kinase insert domain receptor; ZDF, Zucker diabetic fatty; ZL, Zucker lean.

Correlation between glucose level, body weight, and number and function of CD34+/KDR+ circulating cells

Serum glucose was correlated negatively with CD34+ and KDR+ cells (r=−0.6360; P<0.0001; Fig. 3). There were also trends toward negative correlations between body weight and CD34+ and KDR+ cells, colony-forming units, and migrated EPCs (Table 1).

Fig. 3
Fig. 3:
Serum glucose is negatively correlated with the number of CD34+/KDR+ circulating cells for ZL and ZDF rats. Dotted lines represent the 95% confidence band of the best fitted line; the straight line represents linear correlation analysis. P<0.05 (statistically significant). KDR, kinase insert domain receptor; ZDF, Zucker diabetic fatty; ZL, Zucker lean.
Table 1
Table 1:
Effect of body weight on the number and function of CD34+/KDR+ circulating cells for ZL and ZDF rats


We reported that the number and function of CD34+/KDR+ circulating cells isolated from ZDF rats are significantly reduced compared with ZL rats and showed for the first time that Tβ4 treatment improves cell migration and tubule formation in ZDF rats.

Circulating EPCs commonly coexpressed several surface markers such as CD34, CD133, and KDR 15. In this study, CD34+/KDR+ circulating cells present in the peripheral rat blood were selected. It has also been observed that the CD34+/KDR+ circulating cell count decreased when total burden of cardiovascular risk factors increased, particularly in diabetic patients 16. Moreover, spindle-shaped EPCs were also first observed in CD34+ mononuclear blood cells and CD34+/KDR+ cells express endothelial-specific markers, including VE-cadherin and E-selectin 17. Subsequently, multiple studies have documented that CD34+ and KDR+ cells derived from peripheral blood, bone marrow, and umbilical cord blood are enriched for endothelial lineage potential 18.

Male ZDF rats chosen for this study were genetically modified at the leptin receptor, resulting in rats with high circulating leptin levels carrying the diabetic lineage 19. In addition, the recommended Purina 5008 diet maintained high blood insulin levels for a longer period of time. An increased duration of diabetes in the ZDF rats was shown to heighten the rate of derangement of glucose metabolism, which may subsequently affect cardiac contractile function 20. We needed ZDF rats 20 weeks of age as cardiac function was shown to be significantly depressed after that 21. ZL counterparts were used as controls.

Recently, Tβ4 has emerged as a potential candidate for protein-based cardiovascular therapy 22. It can be found extracellularly as well as in the majority of the circulating cells such as platelets and macrophages, with the exception of erythrocytes 23. It is also expressed in the developing endothelium 24. Tβ4 has been shown to be able to diffuse freely into tissues to promote angiogenesis, cell proliferation, and cell migration 25, and support cardiac regeneration by inhibiting myocardial and endothelial cell death after myocardial infarction 26. In addition, Tβ4 was found to be capable of promoting vessel formation and collateral growth not just during development but also from adult epicardium, enhancing cardiomyocyte survival 27. Furthermore, Tβ4 can promote angiogenesis by stimulating endothelial cell differentiation and migration 28. We aimed to investigate the effect of Tβ4 on functions and number of CD34+/KDR+ circulating cells in diabetes and obesity.

We found that Tβ4 is effective in significantly improving the number of migrated cells isolated from ZDF rats. Qiu et al. 29 have reported that a low dose of 10 ng/ml significantly improved migration of human EPCs. Consistent with this, we also observed that a low dose of 10 ng/ml Tβ4 is effective in improving EPC migration in the setting of diabetes and obesity. We did not find the incremental value of Tβ4 at higher doses, but a human study showed that maximum migration occurred when 1000 ng/ml of Tβ4 was used 29.

Tβ4 is also mediated angiogenesis by stimulating factors such as the synthesis of VEGF 10. In the presence of Tβ4, VEGF expression was increased and Tβ4’s angiogenic properties could be further observed when overexpression of Tβ4 was associated with the stimulation of blood vessel formation 30. In addition, Tβ4 enhanced angiogenesis and wound repair in both normal and aged rodents 31. This effect also occurred in HUVECs that were transfected with Tβ4 32. Our results showed that EPCs isolated from ZDF rats showed a 31% increase in the mean branch length after Tβ4 was administered, consistent with the previous literature on the angiogenic properties of Tβ4.

We found a modest relationship between high blood glucose levels and a decrease in the number of CD34+/KDR+ circulating cells (r=−0.64). Other parameters were not statistically significant, although they showed consistent trends. Thus, it may appear that increased blood glucose level is an important contributor toward the number of CD34+/KDR+ circulating cells in the peripheral blood. In contrast, there was no significant correlation observed between body weight and cell number and function. These may be confounded by the possibility of the obesity paradox phenomenon, where obesity may be beneficial and protective in preservation of EPC number and function compared with normal weight. However, all parameters were consistent with some correlations of almost −0.5.

Tβ4 significantly increased the migratory ability of CD34+/KDR+ circulating cells and improved their tubule formation capacity in ZDF rats. These values appeared to be normalized to levels comparable with those of ZL control rats. Future studies on the in-vivo effect of Tβ4 on CD34+/KDR+ circulating cells are warranted.


This work was supported by a grant (NMRC/NIG/1038/2010) from the National Medical Research Council, Singapore.

Conflicts of interest

There are no conflicts of interest.


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CD34+/KDR+ circulating cells; diabetes; obesity; thymosin β4

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