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Auto-mobilized adult hematopoietic stem cells advance neovasculature in diabetic retinopathy of mice

TIAN, Bei; LI, Xiao-xin; SHEN, Li; ZHAO, Min; YU, Wen-zhen

doi: 10.3760/cma.j.issn.0366-6999.2010.16.020
Original article
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Background Hematopoietic stem cells (HSCs) can be used to deliver functionally active angiostatic molecules to the retinal vasculature by targeting active astrocytes and may be useful in targeting pre-angiogenic retinal lesions. We sought to determine whether HSC mobilization can ameliorate early diabetic retinopathy in mice.

Methods Mice were devided into four groups: normal mice control group, normal mice HSC-mobilized group, diabetic mice control group and diabetic mice HSC mobilized group. Murine stem cell growth factor (murine SCF) and recombined human granulocyte colony stimulating factor (rhG-csf) were administered to the mice with diabetes and without diabetes for continuous 5 days to induce autologous HSCs mobilization, and subcutaneous injection of physiological saline was used as control. Immunohistochemical double staining was conducted with anti-mouse rat CD31 monoclonal antibody and anti-BrdU rat antibody.

Results Marked HSCs clearly increased after SCF plus G-csf-mobilization. Non-mobilized diabetic mice showed more HSCs than normal mice (P=0.032), and peripheral blood significantly increased in both diabetic and normal mice (P=0.000). Diabetic mice showed more CD31 positive capillary vessels (P=0.000) and accelerated endothelial cell regeneration. Only diabetic HSC-mobilized mice expressed both BrdU and CD31 antigens in the endothelial cells of new capillaries.

Conclusion Auto-mobilized adult hematopoietic stem cells advance neovasculature in diabetic retinopathy of mice.

Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University; Beijing Ophthalmology & Visual Science Key Laboratory, Beijing 100730, China (Tian B)

Department of Ophthalmology, Peking University People's Hospital, Beijing 100044, China (Li XX, Shen L, Zhao M and Yu WZ)

Correspondence to: Prof. LI Xiao-xin, Department of Ophthalmology, Peking University People's Hospital, Beijing 100044, China (Tel: 86–10–58269659. Tax: 86–10–58269514. Email: drlixiaoxin@yahoo.com.cn)

(Received January 3, 2010) Edited by PAN Cheng

Hematopoietic stem cells (HSCs) are endowed with two properties: self-renewal and capacity for hematopoietic differentiation. These abilities make HSCs clinically useful in therapeutic transplantation for diseases such as leukemia and lymphoma. HSCs can be used to deliver functionally active angiostatic molecules to the retinal vasculature by targeting active astrocytes1 and may be useful in targeting pre-angiogenic retinal lesions, as in diabetic retinopathy.2,3 Diabetic retinopathy is the most common ocular complication leading to blindness in diabetes. In diabetic retinopathy, the breakdown of the internal blood-retina barrier causes abnormal expression of a series of related factors (e.g. VEGF, ang-2), endothelial cell damage and pathological neovascularization.4,5 For this reason, many studies have focused on reducing the breakdown of the blood-retina barrier in the proliferative stage in order to inhibit neovascularization. Although developments have allowed treatment of proliferative diabetic retinopathy with the neutralizing antibody of VEGF, ang-2, and other agents, no effective treatment exists to control the pathological change from non-proliferative to proliferative stage.6,7 The object of the present study was to describe changes in endothelial progenitor cells (EPC) in the retinas of diabetic mice after mobilization of HSC by treatment with murine and human growth factors.

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METHODS

Animals

The animals in this study were 15 weeks old female polygenic-inheritance diabetic (KK ray) mice and normal mice (c57BL/6) (Chinese Academy of Medical Sciences, Beijing, China). Mice with blood glucose >16.6 mmol/L for three continuous days were included. The two groups of mice were randomly divided into treatment and control groups. The mice in the treatment group were given 200 μg·kg-1·d-1 of murine stem cell growth factor (SCF) and 50 μg•-1d-1 of recombined human granulocyte colony stimulating factor (rhG-csf) via subcutaneous injection for 5 days. The control group was given saline injections of the same dosage.8 BrdU (Sigma-Aldrich, USA) was given fourteen days after autologous stem cell mobilization via intraperitoneal injection everyday. Thirteen days later, the animals were sacrificed for related studies.9 All procedures were conducted according to the Association for Research in Vision and Ophthalmology (ARVO) statement for the Use of Animals in Ophthalmic and Vision Research.

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Flow cytometric analysis of mouse PB

Before mobilization and seven days after continuous combined subcutaneous injection of SCF and G-csf,7 200 μl peripheral blood was collected with 1% heparin (BD, Becton Dickinson, USA) and stored at 4°C, 5 μl anti-CD34-PE (BD) and 5 μl anti-sca1-FITC (BD) were added to 106 cells, and incubated for 20 minutes at room temperature in a dark place. Pharmingen's PharM LyseTM (BD) was used to completely lyse the red blood cells. Cells were washed three times with phosphate buffer solution (PBS) containing 5% fetal bovine serum (FBS). Then 0.5 ml was added to the test machine. Data analysis was performed using fluorescence-activated cell sorting (FACS) Calibur Cellquest software for flow cytometer (BD).

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Histological analysis

After 4% paraformaldehyde cardiac perfusion was conducted, mice eyeballs were excised. Using 4% paraformaldehyde for specimen fixing, 3 μm serial sections were made after paraffin embedding. Three sections were randomly selected from each eyeball. Periodic acid-schiff (PAS) stains were performed so as to count the number of capillaries with PAS+ cells in the ambitus of the retina. Immunohistochemical stain using anti-mouse rat CD31 (PECAM-1) monoclonal antibodies (BD) as the marker of endothelial progenitor cells was performed as previously described.10,11 Three sections were randomly selected from each eyeball, and the capillary vessel density at the equatorial retina was calculated according to the number of CD31 positive vessels observed in ten microscopic fields. Immunohistochemical double staining was conducted with anti-mouse rat CD31 (PECAM-1) monoclonal antibody (BD) and anti-BrdU rat antibody (AbD Serotec, Kidlington, UK), using BrdU as a marker for new vessels as previously described.12

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Statistical analysis

In this study, measured data were analyzed to indicate mean ± standard deviation (SD), t test was used to analyze paired data. Univariate analysis of variance (ANOVA) test was used to analyze the data using SPSS 11.5 (SPSS Inc., Chicago, USA). Differences were considered statistically significant at P <0.05.

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RESULTS

Identification of periphery blood HSCs

The number of CD34-/low and Sca1+ cells is shown in Table 1. After SCF plus G-CSF-mobilized, PB primitive CD34-/low or Sca1- cells clearly increased (Figure 1). ANOVA results showed the number of CD34-/low and Sca1+ cells in non-mobilized diabetic mice was higher than in normal mice (P=0.032), and mobilization by SCF and G-csf significantly increased their number in periphery blood of both diabetic and normal mice (P=0.000). Diabetes had no influence on HSC mobilization in periphery blood (P=0.720).

Table 1

Table 1

Figure 1.

Figure 1.

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Pathological change in retinal tissues with autologous HSCs mobilization

Figure 2A shows that no capillary vessel proliferation was found in PAS stained retina of normal mice of 19 weeks old under light microscopy although obvious abnormal capillary vessel proliferation blockage and basement membrane thickening were found in the retina of diabetic mice of the same age (Figure 2B). In immuno- histochemical sections where CD31 marked blood vessels (n=10 sections), the numbers of CD31 positive capillary vessel endothelial cells in each (×40) microscopic field in each group are shown in Table 2. Diabetic mice had more CD31 positive capillary vessels in the retina than normal mice (P=0.000). The difference between the autologous stem cell mobilization group and the control group was statistically significant (P=0.000). The number of CD31 positive capillary vessels in diabetic mice increased after autologous stem cell mobilization (P=0.000). In pathological sections, we found CD31 stains of the retinal blood vessels of mice injected with physiological saline were negative or weakly positive (Figure 2C). However, the number of capillaries with CD31+ cells increased in HSC-mobilized mice (Figure 2D). In this study, BrdU and CD31 were used for retinal immunohistochemical double stain after autologous mobilization, and only diabetic HSC-mobilized mice expressed the two antigens in the endothelial cells of new capillary vessels (Figure 2E). This indicates that stem cell mobilization can accelerate capillary vessel regeneration in the retina of diabetic mice (Table 2, Figure 2F).

Figure 2.

Figure 2.

Table 2

Table 2

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DISCUSSION

In this study, CD34-/low and Sca1+ were used as markers for early HSCs.3,13 The number of CD34-/low and Sca1+ cells in diabetic mice without mobilization was higher than in normal mice, indicating that the damage to vascular endothelial cells caused by diabetes activated HSCs. Combined use of SCF and G-csf could successfully mobilize both diabetic and normal mice to multiple the number of HSCs in periphery blood, indicating that diabetes had no influence on the mobilization of HSCs in periphery blood. CD31 was the marker for EPC.14 The KK mouse, an accepted model for diabetic retinopathy,15 demonstrates microangiopathies associated with diabetes and changes in the eye associated with pre-proliferative disease. In this study, we found the number of capillaries with CD31+ cells obviously increased in HSCs in KK mouse. This result demonstrates HSCs can promote hyperplasia in the EPC of diabetic mice. In normal mice with HSCs-mobilized, CD31+ cells increased over the control group, but were less than in diabetic mice control groups. This result demonstrates that, in hyperglycemia, more HSCs can differentiate to EPCs. It remains to be seen whether hyperglycemic damage to the vessel activates self renovation and mobilizes HSCs in peripheral blood.

Abnormal expression of factors related to angiogenesis (e.g. VEGF, ang-2, and others) and breakdown of the internal blood-retina barrier play an important role in the development of this disease. Other studies have shown fewer EPC in the peripheral blood of Types I and II diabetic patients, and decreased differentiation, adhesion and chemotaxis. The hypothesis has been put forward that diabetic microcirculation pathological changes result from EPC dysfunction.16,17 Many studies have proven that HSCs migrate from the bone marrow to peripheral blood, are differentiated into circulating EPCs (CEPs), go to damaged organs, then are differentiated into EPCs, developing into healthy vascular endothelial cells and forming vessels with normal functions during physiological angiogenesis.3,18 Seminal research by Otani et al13 proved the feasibility of treating ischemic diabetic retinopathy by repairing retinal blood vessels with HSCs. In this study, autologous mobilization increased the number of retinal capillaries with CD31 marker. Brdu was used as tracer for formation of new retinal blood vessels after autologous HSCs mobilization in diabetic mice. However, the retinal blood vessel may take one of two paths: to become a normal functional vessel, where the endodermis cell has tight, integrated connections; or to become an abnormal vessel which does not function normally. This study only proved that HSCs can advance vessel regrowth. Whether these vessels support improvements in diabetic retinopathy requires further study.

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Acknowledgements:

We thank Dr. LI Liao-qing for preparation of histologic samples, and Prof. LIU Xue-zong, Dr. ZHANG Jun and Dr. LU Ai-li for assistance.

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    Keywords:

    adult, hematopoietic stem cells; diabetic retinopathy; stem cells

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