Galectin-3 Enhances Proliferation and Angiogenesis of Endothelial Cells Differentiated from Bone Marrow Mesenchymal Stem Cells

Galectin-3 Enhances Proliferation and Angiogenesis of Endothelial Cells Differentiated from Bone Marrow Mesenchymal Stem Cells S.Y. Wan, T.F. Zhang, and Y. Ding ABSTRACT Purpose. To observe the effects of galectin-3 on proliferation and angiogenesis of endothelial cells differentiated from bone marrow mesenchymal stem (MSCs). Methods. Cultured MSCs were isolated from bone marrow of Sprague–Dawley rats and purified by gradient centrifugation with lymphocytes separation medium. Cells of passage 3 were differentiated into endothelial cels by vascular endothelial growth factor and basic fibroblast growth factor. These cells were identified as endothelial cells by immunohistochemistry staining and electronic microscopy after 14 days. The cells were cultivated with the galectin-3 at the concentrations of 0.1, 1, and 5 ␮g/mL for 24 hours. The proliferation of endothelial cells were measured by 3-(4,5-methylth-iazol-2-yl)-diphenyltetrazolium bromide (MTT) and the cell cycle was investigated by using flow cytometry. The functionality of angiogensis was observed when the cells appeared tube formation in presence of glacetin-3. Results. The proliferation activity, analyzed by MTT method, in the galectin-3 groups (1 and 5 ␮g/mL) were 0.3002 ⫾ 0.0159 and 0.3514 ⫾ 0.0133, respectively, which were significantly greater than that in the control group (0.2339 ⫾ 0.0041; P ⬍ .05). Flow cytometry detection showed that S phase cells (%) are 29.42 ⫾ 0.45, 34.56 ⫾ 0.82, and 52.58 ⫾ 2.84 in groups of 0.1, 1, and 5 ␮g/mL, respectively, and G2M phase cells increased from 4.88 ⫾ 1.12 to 5.26 ⫾ 0.45 with the concentrations of 1 and 5 ␮g/mL, respectively, which demonstrated significant difference compared with the control group (P ⬍ .05). The tubular network formation was lengthened significantly compared with the control group (P ⬍ .05). Conclusion. Galectin-3 can promote the proliferation and angiogenesis of endothelial cells differentiated from bone marrow mesenchymal stem cells. NGIOGENESIS IS a crucial process comprising a series of cellular events that lead to neovascularization, and includes embryonic development, wound healing, and tumor growth. There is a cascade of events, emanating from endothelial cell proliferation, survival, migration, and maturation to form capillary tubes.1,2 Angiogenesis is defined as new blood vessel formation from existing vessels through the generation of endothelial cells.3 The proliferation of endothelial cells play an important role in angiogenesis, and rapid proliferation and growth of endothelial cells make its function implement promptly. Optimum healing of tissue damage requires a well-orchestrated integration of the complex biological and molecular events of cell migration and proliferation, as well as extracellular matrix deposition, angiogenesis, and remodeling.4

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Endothelial cell morphogenesis is a carbohydratedependent process; it is neutralized by specific sugars and antibodies. The carbohydrate recognition event on the From the Department of Surgery (S.Y.W., Y.D.), The Second Affiliated Hospital of Anhui Medical University, Hefei, and the Department of Surgery (T.F.Z.), Lu’An Affiliated Hospital of Anhui Medical University, Lu’An, Anhui Province, China. S.Y. Wan and T.F. Zhang contributed equally to this work. Supported in part by the Central Research Laboratory of the First Affiliated of Anhui Medical University and Department of Health of Anhui Province fund. Address reprint requests to Sheng-Yun Wan, Department of Surgery, The Second Affiliated Hospital of Anhui Medical University, 678 Furong Avenue, Hefei 230601, Anhui Province, China. E-mail: [email protected]

© 2011 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710

0041-1345/–see front matter doi:10.1016/j.transproceed.2011.10.050

Transplantation Proceedings, 43, 3933–3938 (2011)

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endothelial cell surface can induce a signaling cascade, which leads to the differentiation and angiogenesis of endothelial cells.10 Galectin-3, a carbohydrate-binding protein, has been shown to be involved in the biological processes of tumor cell adhesion, proliferation, differentiation, angiogenesis, cancer progression, and metatasis.5-9 The effects of galectin-3 on proliferation and angiogenesis of endothelial cells differentiated from bone marrow mesenchymal stem cells have not been reported. The aim of our study was to investigate whether galectin-3 increases cell proliferation and in vitro tube formation in differentiated endothelial cells. MATERIALS AND METHODS Antibodies and Reagents LG-DMEM (Hyclone, San Diego, Calif), trypsin-EDTA solution (Beyotime, Jiangsa, China), fetal bovine serum (FBS; Sijiqing, China), percoll (Pharmacia, Kalamazoo, Mich), galectin-3 (Peprotechm Rocky HIll, NJ) vascular enthelial growth actor (VEGF; Peprotech), basic fibroblast growth factor (bFGF; Peprotech), polyclonal ntibody (Promega, Madiso, Wisc), rabbit–anti-mouse polyclonal antibodies against Von Willebrand factor (vWF; Promega), MTT (Amresco, Solon, Oh), propidine iodide (PI, Sigma, Louis, MO), Rnase (Sigma), Triton-X-100 (Biosharp, Mahwah, NJ), inverted phase contrast microscope (Olympus, Japan), microplate reader (ELX-800, BioTerk, WInooski, Vt), Flow Cytometry (Epics XL-MCL, Coulter, Fullerton, Calif), male SPF Sprague– Dawley rats, 100 –120 g (Animal Center of Anhui Province, China), and transmission electron microscopy (JEOL-2100F, Japan).

Isolation, Culture, and Purification of Bone Marrow–Derived Rat MSCs Bone marrow was collected from the femurs of 4- to 5-week-old male SPF Sprague-Dawley rats. Rats were humancly killed by cervical dislocation and then placed in 75% alcohol 10 minutes. All rat femurs were taken and stripped of adherent muscles of the knee and under sterile conditions. A needle was inserted into the marrow cavity and cells were aspirated followed by several flushes through the bone with a 5-mL injection set filled with phosphatebuffered saline (PBS) containing penicillin (100 ␮g/mL) and streptomycin (100 ␮g/mL); the bone marrow was flushed repeatedly 5 times. A similar procedure was performed again in the opposite of the bone as close to the tip as possible. Then, bone marrow aspirate was collected, and well mixed gently, and centrifuged at 1000 rpm for 10 minutes at room temperature. After centrifugation, the supermatant solution is decanted, and the marrow thus obtained was suspended in 4 mL of PBS containing penicillin (100 ␮g/mL) and streptomycin (100 ␮g/mL). The mononuclear fraction of the bone marrow was isolated with percoll solution (1.073 g/L), centrifuged at 2500 rpm for 20 minutes at room temperature. Mononculear cells were removed from the gradient interface and washed twice with PBS. The mononuclear cells were resuspended in DMEM-LG supplemented with penicillin 100 U/mL, streptomycin 100 U/mL, and 10% FBS, and seeded into 252 cm2 tissue-culture plastic flasks at a density of 2 ⫻ 106 cells/mL. MSC cultures grew at 37°C in 5% CO2. Nonadherent cells were removed after 48 hours by washing with PBS. The medium was changed subsequently every 2–3 days. The culture reached 80%–90% confluency 10 –14 days Later. MSCs were recovered using 0.25% Trypsin-EDTA and

WAN, ZHANG AND DING replaced at a density of 5000 – 6000 cells per square centimeter of surface area as passage 1 (P1) cells.

Endothelial Cell Differentiation and Characterization We collected the third passage MSCs with no less than 50% confluence, decanted the medium, washed cells with PBS, then added the induced medium which contained L-DMEM, 5% FBS, VEGF (10 ng/mL), and bFGF (2 ng/mL). The culture were maintained for 14 –21 days by induced medium and exchanged every 3 days.

Immunocytochemistry The differentiated endothelial cells were seeded into 6-well plates, and then filled with slide and cultured to confluency. Paraformaldehyde fixed tissues samples were embedded in paraffin. Remove paraffin in toluene (2 ⫻ 20 min). Put through graded alcholos [25% (w/v), 5 minutes each], then rinsed in water. Block with methanolperoxide (0.3% H2O2 in MeOH) for 30 minutes. Sections were washed in PBS (10 mmol/L NaHPO4, pH 7.4, 0.15 mol NaCl) for 20 minutes; layered on PBS containing a 1/100 dilution of normal rabbit serum for 20 minutes; rinsed and layered on the primary antibody- rabbit–anti-mouse serum antibodies (1:40); diluted in PBS containing 1% (w/v) bovine serum albumin; and place in a humidity chamber overnight at 4°C, Cells were washed in PBS for 10 minutes then layered on secondary antibody (biotinylated Rabbit anti-Goat immunoglobulin G) diluted approximately 1/100 in PBS for 30 mimutes at ambient temperature. Then, they were washed in PBS for 10 minutes, VectaStain ABC reagent mixures was applied per the manufacturer’s instructions. Cell were developed in 0.05% (w/v) DAB in 0.05 mol/L Tris-HCl, pH 7.6, containing 0.075% H2O2 for 15 minutes at ambient temperature. After rinsing in cold water, cells counterstained in hematoxylin for 3–5 minutes, and rinsed in water, then graded alcohols (25% (w/v), 5 minutes each). After rinsing in 3 changes of toluene, cells were coversliped with permount and photographed by fluorescence microscopy.

Transmission Electron Microscopy Rinsed with PBS, monolayers of differentiated endothelial cells were strained into a microcentrifuge tube, fixed with 4% paraformaldehyde and 2.5% glutaraldehyde for 2 hours at 4°C, followed by 1% osmium tetroxide anchor cells. After dehydration by gradient ethanol, samples were embedded in araldite tesin. Then, ultrathin sections were cut using a diamond knife on an ultra microtome. Finally, the sections were stained with uranyl acetate and lead citrate. Cells were observed by transmission electron microscopy (TEM) at 100 kv.

In vitro Cell Proliferation Assay Subconfluent (70%– 80%) induced cells, which were in the exponential phase of growth, were trypsinized, seeded into a 96-well plate at 104 cells/well, and incubed in common culture medium that contained 10% FBS for 24 hours. The culture medium was then changed to serum-free medium for 12 hours and thereafter to LG-DMEM with 5% FBS and recombination galectin-3 protein solution (galectin-3 final concentration:0, 0.1, 1, and 5 ␮g/mL), and incubation was allowed to proceed for 24 hours. This process was repeated in 5 wells. At the end of the drug treatments, 20 ␮L of 3-(4,5-methylth-iazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) solution (5 mg/mL) was added into each well in the dark, and incubation was allowed to proceed for 4 hours at 37°C. Finally, 150 ␮L of DMSO was added into each well, and it was Shaken for 10 minutes at room temperature. The absorbance of he culture medium at 490 nm was measured using a microplate reader. The measurement was repeated 3 times.

GALECTIN-3 For flow cytometry, cultured cells in 6-well plate were washed in PBS and incubed with serum-free medium for 24 hours. They were exposed to LG-DMEM supplemented with 5% FBS and recombinant galectin-3 protein (galectin-3 final concentration: 0, 0.1, 1, and 5 ␮g/mL) for 24 hours. They were harvested and resuspended in fixation fluid at a density of 2 ⫻ 106/mL, and fixed with 70% ethanol for 12 hours at ⫺20°C, rinsed with PBS once, and added to 1 mL DNA staining solution (trisodium citrate 0.25 g, Triton-X-100 0.75 mL, PI0.025 g, Rnase 0.005 g, distilled water 250 mL). The cells were filtered 15 minutes later, and the cell cycle as detected by flow cytometry. Cell proliferation index (PI) ⫽ (S ⫹ G2M)/(G0/G1 ⫹ S ⫹ G2M). This measurement was repeated 3 times.

3935 randomly for each well. All groups were studied triplicate in 3 independent experiments. Data are expressed as relative tubular length (%) compared with the control.

Statistical Analysis All the data are expressed as mean values ⫾ standard error of the mean. Statistical analysis was performed with the Statview SE software package (SPSS Inc., Chicago, Il). The data were normally distributed, and values obtained in the different groups were compared using one-way ANOVA. Statistical difference was accept at P ⬍ .05. This study was approved by the local institutional review board.

Tube Formation Assay

RESULTS Cell Culture

We used 50 ␮L of growth factor–reduced matrigel to coat a 96-well plate at 4°C and allowed it to polymerize for 45 minutes at 37°C. Differentiated endothelial cells (5000 cells) were seeded on the surface of the matrigel well, and were serum starved in LG-DMEM medium for 2 hours. Thereafter, they were exposed to LG-DMEM supplemented with 5% FBS and recombinant galectin-3 protein (galectin-3 final concentration: 0, 0.1, 1, and 5 ␮g/mL) for 24 hours. Tube formation was visualized using a microscope. Quantification of angiogenic activity was calculated by measuring the length of tube walls formed between endothelial cells in each well. Total tubular length per well was determined by computer-assisted image analysis using Image Pro Plus software. Three culture wells were used for each sample, and 3 microscopic fields were examined

Cells were adhered in plastic flasks, P0 (passage zero) cells appeared in flasks after 2 days of plating with a fibroblast-like, spindle-shaped or polygonal morphology. These cells began to proliferate at about day 4 and gradually grew to form small colonies (Fig 1A). By day 7– 8, the number of cellular colonies of different sizes had obviously increased. After approximately 12 days, cells became confluent (Fig 1B). The behavior of passaged MSC was similar to that in primary culture, 0 in the later passage 3, the spindle-shaped cells were converted to broadened and flat morphology and replicated faster (Fig 1C). The MSCs in subcultures, which were differentiated

Fig 1. Morphologic characteristics of mesenchymal stem cells from rat bone morrow before and after induction. (A) MSCs primary culture day 4 (original magnification, ⫻100). (B) Primary culture day 12 (original magnification, ⫻100). (C) MSCs in passage 3 and day 3 (original magnification, ⫻100). (D) The induced MSCs day 14 (original magnification, ⫻100). (E) Immunohistochemistry staining with rabbit-anti-mouse serum antibodies against v-WF. The positive cells showed greenyellow fluorescence in cytoplasm and clear cell shape (original magnification, ⫻200). (F) A typical endothelial cell with mitochondria and specific endothelial organelles called Weibel-Palade bodies (white arrow) was observed (original magnification, ⫻22,000).

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with control group (P ⬍ .05). Galectin-3 stimulated proliferation of endothelial cells in a dose–response relationship, suggesting that galectin-3 protein was involved in the proliferation process of endothelial cells. Endothelial cells proliferation detected by flow cytometry is shown in Table 1 and Fig 3. The S-phase and PI fractions may properly reflect their proliferation, which increased remarkably 1 compared with the control group. There was a significant dosedependent correlation among the effects of galectine-3. The G2M-phase fracton increased in the concentrations of 1 and 5 ␮g/mL (P ⬍ .05) compared with the control group. In Vitro Tube Formation

FRQWURO







*ODFWLQʹJPO Fig 2. Proliferation of differentiated endothelial cells detected by MTT assay. Absorbance at 490 nm after exposure to different concentrations of recombinant galectin-3 protein for 24 hours (n ⫽ 3). *P ⬍ .05 versus control group. **P ⬍ .01 versus control group.

for 1 week, showed polygon morphology and, thereafter, the induced cells presented cobblestone-like similarly with endothelial-like cells for 2 weeks (Fig 1D). Immunocytochemistry

Immunohistochemistry staining revealed that most of the endothelial-cell like MSCs were positive for vWF with green-yellow fluorescence in cytoplasm and clear cell shape (Fig 1E). Undifferentiated MSCs showed that no specific fluorescence was observed under fluorescent microscope. TEM

TEM analysis of the differentiated cells showed typical features of the endothelial cells with numerous caveolae and pinocytic vesicles. There was a dense nucleus and nucleolus compared with MSCs. We also observed a typical endothelial cell with mitochondria and specific endothelial organelles called Weibel–Palade (W-P) bodies (Fig 1F). Proliferation of Endothelial Cells

According to MTT assay (Fig 2), galectin-3 at concentrations of 1 and 5 ␮g/mL can increase proliferation compared

In this study, endothelial-like cells derived from MSCs were examined for their ability to form an in vitro capillary network. New capillary formation is required for the initial steps of angiogenesis, which involves processes such as endothelial cell activation, proliferation, and migration. Biological effects of galectin-3 were evaluated in an in vitro tube formation assay. When endothelial cell were cultured in matrigel in the presence of every concentrations of galcetin-3, apparent tubeformation was observed. With regard to galectin-3, a significant increase in the total length of the tubular network formation was observed at the treated groups (P ⬍ .01; Fig 4) compared with the control groups. Galectin-3 stimulated the formation of capillarylike structures by differentiated endothelial cells. DISCUSSION

MSCs have shown all criteria of true stem cells, including self-renewal, multilineage differential, and reconstitution of tissue.10 The use of autologous vascular endothelial progenitor cells seems attractive to the development of engineered vessels as well as to the vascularization of engineered tissues, and may be useful for augment vessel growth in ishcemic tissue.11,12 MSCs have potency of oriented differentiation into vessel endothelial cells under certain conditions and can be used in autotransplant for the therapy of ischemic disease of lower extremity as well as, which plays important roles in tissue engineering and regeneration. Ischemic injury caused by inflammatory 2 reaction and thrombosis are the primary causes of pathologic lesion in the ischemic disease of lower extremity.13,14 Meantime, some findings show that endothelial cells induced from bone marrow MSCs have antithrombotic effects; further-

Table 1. Differentiated Endothelial Cell Cycle Detected by Flow Cytometry (%) (n ⴝ 3, x៮ ⴞ S) Group

I (negative control) II III IV

Galectin-3 (␮g/mL)

G0/G1 (%)

S (%)

G2/M (%)

PI (%)

0

77.69 ⫾ 3.13

20.40 ⫾ 1.81

1.90 ⫾ 1.74

22.30 ⫾ 3.12

0.1 1.0 5.0

67.55 ⫾ 0.94 60.55 ⫾ 0.76 42.16 ⫾ 2.52

29.42 ⫾ 0.45** 34.56 ⫾ 0.82** 52.58 ⫾ 2.84**

3.02 ⫾ 0.49 4.88 ⫾ 1.12ⴱ 5.26 ⫾ 0.45**

32.45 ⫾ 0.94** 39.44 ⫾ 0.75** 57.84 ⫾ 2.52**

Differentiated endothelial cells exposured to different concentrations of recombinant galectin-3 protein for 24 hours (n ⫽ 3). **P ⬍ .01 versus negative control group.

GALECTIN-3

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Fig 3. Differentiated endothelial cell cycle detected by flow cytometry. The induced endothelial cells were exposed to different concentrations of recombinant galcetin-3 protein for 24 hours: (A) Negative control; (B) 0.1 ␮g/mL; (C) 14 ␮g/mL; (D) 5 ␮g/mL.

more, the adherence capability of endothelial cells induced from bone marrow MSCs was superior to endothelial cells derived from venous.15,16 In our study, when recombinant galectin-3 protein was applied to differentiated endothelial cells for 24 hours the proliferation assays suggested that the absorbance values at the concentrations of 1 and 5 ␮g/mL increased significantly compared with control group, while 0.1 ␮g/mL group had no significant difference compared with control group. Flow cytometry detection showed that S-phase cells (%) increased remarkably compared with control group; G2M phase cells increased significantly in the concentrations of 1

and 5 ␮g/mL. Galectin-3 at 1 and 5 ␮g/mL had effects on differentiated endothelial cells in the S and G2M phases; however, galectin-3 at 0.1 ␮g/mL only had an effect in S phase. These results of MTT and were consistent with that of flow cytometry, suggesting that galectin-3 was effective in the promotion of endothelial cell proliferation. Meanwhile, our data demonstrated that the tube length increased significantly compared with control gropu, which indicated that galectin-3 was a potent stimulator for endothelial cell proliferation and angiogenesis. In recent years, numerous studies have shown that angiogenic factors can be divided into 3 groups of extracellular

A

B

Relative total tubular length ˄% of control˅



**

 

**

**

0.1

1.0

    control

5.0

Galectin-3˄μg/ml˅

C

D

E

Fig 4. Galectin-3 enhance differentiated endothelial cells tube formation on matrigel. Differentiated endothelial cells were plated in Matrigel-coated 96-well plates, and then exposed to different concentrations of recombinant galectin-3 protein for 24 hours. (A–D) Vasculogenesis images of endothelial cells differentiated from MSCs cultured on matrigel for 12 hors of incubation in vitro (original magnification, ⫻100). (A) Negative control; (B) 0.1 ␮g/mL; (C) 1 ␮g/mL; and (D) 5 ␮g/mL. (E) The total length of the tublar network formation was observed in each groups (mean ⫾ SEM, n ⫽ 3). **P ⬍ .01 versus negative control group.

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signals.17 The first group affects endothelial cell growth and differentiation like VEGF; the second group inhibits proliferation and enhances differentiation of endothelial cells as transforming growth factor (TGF)-␤, the third group 3 contributes to angiogenic regulation and includes angiostatin. Angiogenesis is the generation of new blood vessels from preexisting microvasculature and is regulated by a number of growth factors.18 Each of these procedures is tightly regulated by a net balance between positive and negative angiogenic factors under physiolog conditions. Once the net balance is disrupted, various diseases are caused, such as cancer, diabetic retinopathy, and limb ischemia. Galectin-3 a member of the ␤-galactoside– binding gene family with molecular masses ranging from 29 to 35 kd, is a multifunctional protein implicated in a variety of biological functions. It has a unique chimera type structure consisting of 3 different structural domains: A short NH2-terminal domain of 12 amino acids, a repeated collagen-like sequence rich in glycine, tyrosine, and proline amino acid residues, and a COOH-terminal carbohydrate-recognition domain.19,20 Many studies on physicolchemical characteristics of galectin-3 suggested their profound structural and functional differences among these 3 domains.21,22 One study demonstrated that galectin-3 treatment dose-dependently stimulated the proliferation of endothelial cells and neural progenitors in the in vitro setting, and may play a role in postischemic tissue remodeling by enhancing angiogenesis and neurogenesis.23 Studies on endothelial cells have shown that galectin-3 stimulates proliferation and angiogenesis,17 but no report on the effect of galectin-3 on differentiated cells from MSCs has been published. Our study demonstrated galectin-3 can enhance proliferation and angiogenesis of endothelial cell induced from bone marrow MSCs. Galectin-3 provides a new therapy method and potential choice for angiogenesis. It may be worthwhile to examine whether the angiogenic-promoting activity of galectin-3 can be used 4 as a therapeutic adjuvant strategy in some ischemia-related diseases and tissue engineered vascular grafts. However, the biosafety of galectin-3 has not been published in clinical therapy, particularly in the endothelial cell induced from bone marrow MSCs. REFERENCES 1. Carmeliet P, Jain K: Angiogenesis in cancer and other diseases. Nature 407:248, 2000 2. Kitlinska J, Abe K, Kuo L, et al: Differential effects of neuropeptide Y on the growth and vascularization of neural crest-derived tumors. Cancer Res 56:1719, 2005

WAN, ZHANG AND DING 3. Risau W: Mechanisms of angiogenesis. Nature 386:671, 1971 4. Falanga V: Wound healing and its impairment in the diabetic foot. Lancet 366:1736, 2005 5. Lippert E, Falk W, Bataille F, et al: Soluble galectin-3 is a strong, colonic epithelial-cell– derived, lamina proprialtib roblast– stimulating factor. Gut 56:43, 2007 6. Henderson NC, Mackinnon AC, Farnworth SL, et al: Galectin-3 expression and secretion links macrophages to the promotion of renal fibrosis. Am J Pathol 172:288, 2008 7. Nishi Y, Sano H, Kawashima T, et al: Role of galectin-3 in human pulmonary fibrosis. Allergol Int 56:57, 2007 8. Nangia-Makker P, Nakahara S, Hogan V, et al: Galectin-3 in apoptosis, a novel therapeutic target. J Bioenerg Biomembr 39:79, 2007 9. Kiwaki K, Novak CM, Hsu DK, et al: Galectin-3 stimulates preadipocyte proliferation and is 5 up-regulated in growing adipose tissue. Obesity 15:32, 2007 10. Verfaillie CM: Adult stem cells: assessing the case for pluripotency. Trends Cell Biol 12:502, 2002 11. Barry F, Boynton R, Murphy M, et al: The SH-3 and SH-4 antibodies recognize distinct epitopes on CD73 from human mesenchymal stem cells. Biochem Biophys Res Commun 289:519, 2001 12. Verfaillie CM: Adult stem cells: assessing the case for pluripotency. Trends Cell Biol 12:502, 2002 13. Harder Y, Amon M, Emi D, et al: Evolution of isclpemic tissue injury in a random pattern flap: a new mouse model using intralvital microscopy. J Surg Res 121:197, 2004 14. Tatlidede, Murphy AD, McCormack MC, et al: Improved survival of murine islandskin flaps by prevention of reperfusion mjury. Plast Reconstr Surg 123:1431, 2009 15. Fang NT, Zhang YH, Sun HL, et al: Bone mesenchymal stem cell derived endothelial cells for constructing tissue engineered heart value and its anti-thrombotic effects. Chinese Journal of Clinical Rehabilitative Tissue Engineering Research 10:27, 2006 16. Wei XY, Liu WY, OuY H, et al: Using bone marrow and vein derived interstitial cells as seed cells of TEHV. Chinese journal of experimental surgery 22:777, 2005 17. Nangia-Makker P, Honjo Y, Sarvis R, et al: Galectin-3 induces endothelial cell morphogenesis and angiogenesis. Am J Pathol 156:899, 2000 18. Ferrara N. Vascular endothelial growth factor: basic science and clinical progress. Endocr Rev 25:581, 2004 19. Barondes SH, Cooper DN, Gitt MA, et al: Galectins structure and function of a large family of animal lectins. J Biol Chem 269:20807, 1994 20. Gong HC, Honjo Y, Nangia-Makker P, et al: The NH2 terminus of galectin-3 governs 6 cellular compartmentalizationand functions in cancer cells. Cancer Res 59:6239, 1999 21. Hsu DK, Zuberi RI, Liu FT: Biochemical and biophysical characterization of human recombinant IgE-binding protein, an S-type animal lectin. J Biol Chem 267:14167, 1992 22. Agrwal N, Sun Q, Wang SY, et al: Carbohydrate-binding protein 35. I. Properties of the recombinant polypeptide and the individuality of the domains.J Biol Chem 268:14932, 1993 23. Yan YP, Lang BT, Vemuganti R, et al: Galectin-3 mediates post-ischemic tissue remodeling. Brain Res 1288:116, 2009