Vascular endothelial growth factor promotes the proliferation and osteogenic differentiation of mouse bone marrow mesenchymal stem cells

doi:10.3969/j.issn.2095-4344.2014.41.006

Abstract

Abstract:

BACKGROUND: Vascular endothelial growth factor (VEGF) is a crucial factor for regulating bone marrow mesenchymal stem cells in microenvironment, which can promote endothelial differentiation of bone marrow mesenchymal stem cells. But there is no report about VEGF effect on regulating the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells.

OBJECTIVE: To investigate the regulatory role of VEGF in the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells.

METHODS: The bone marrow mesenchymal stem cells were isolated and cultured from mouse long bones. Cell Counting Kit-8 was used to test the proliferation of bone marrow mesenchymal stem cells cultured with different concentrations of recombinant VEGF proteins. The appropriate concentration of VEGF recombinant protein was selected to test their effects on the osteogenic differentiation of bone marrow mesenchymal stem cells. The mRNA and protein levels of Osterix, Runx2, alkaline phosphatase, osteocalcin and heme oxygenase-1 in bone marrow mesenchymal stem cells under induction of VEGF were detected by molecular biology methods.

RESULTS AND CONCLUSION: (1) VEGF promoted the proliferation of bone marrow mesenchymal stem cells dose-dependently, and 100 μg/L was the optimum concentration. (2) VEGF promoted the expression of Osterix, Runx2, alkaline phosphatase and osteocalcin in bone marrow mesenchymal stem cells under osteogenic induction. Similar results were obtained by alizarin red staining and quantification of numbers of calcium nodules. (3) VEGF induced the expression of heme oxygenase-1 in bone marrow mesenchymal stem cells at mRNA and protein levels. These findings indicate that VEGF can induce the expression of heme oxygenase-1 i in bone marrow mesenchymal stem cells, and promote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

全文链接：

Key words: bone marrow, mesenchymal stem cells, vascular endothelial growth factors, heme oxygenase-1

CLC Number:

R394.2

Zhang Lian-fang, Kang Hui, Deng Lian-fu, Yang Hui-lin. Vascular endothelial growth factor promotes the proliferation and osteogenic differentiation of mouse bone marrow mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(41): 6591-6596.

Figures/Tables 3

2.1 血管内皮生长因子促进骨髓间充质干细胞的增殖 CCK8试剂盒检测在含不同质量浓度重组血管内皮生长因子蛋白的培养液下骨髓间充质干细胞的增殖情况，发现20，50，100，200 μg/L重组血管内皮生长因子蛋白培养下骨髓间充质干细胞的增殖率分别是对照组的1.44倍、1.90倍、2.81倍和2.92倍，可见骨髓间充质干细胞的增殖随培养液中血管内皮生长因子蛋白质量浓度的增加而增加，100 μg/L促增殖作用最显著，而200 μg/L较100 μg/L对骨髓间充质干细胞的促增殖作用无差异(图1)。这说明血管内皮生长因子对骨髓间充质干细胞的增殖有促进作用，且有浓度依赖性；200 μg/L组较100 μg/L组无差异，可能是因为在100 μg/L这一质量浓度时受体已经饱和的缘故，因而后面的实验选用100 μg/L这一质量浓度。 2.2 血管内皮生长因子促进骨髓间充质干细胞向成骨细胞分化骨髓间充质干细胞在有成骨细胞分化诱导剂的培养液中培养第3天，Real-time PCR方法检测其成骨细胞分化早期基因Osterix和Runx2的表达；培养第7天检测碱性磷酸酶和骨钙素的表达。发现培养3 d后100 μg/L血管内皮生长因子组骨髓间充质干细胞的Osterix和Runx2表达分别是对照组的4.61倍(P < 0.001)和3.52倍(P < 0.001) (图2A，B)；培养第7天，100 μg/L血管内皮生长因子组骨髓间充质干细胞中碱性磷酸酶和骨钙素的表达分别是对照组的9.79倍(P < 0.001)和5.25倍(P < 0.001)(图2C，D)。成骨诱导培养下第21天，对骨髓间充质干细胞行茜素红染色(图2E)，镜下计数钙结节，100 μg/L血管内皮生长因子组钙结节数量(31.78±3.14)是对照组(9.22±1.41)的3.45倍(P < 0.001)(图2 F)。结果证实血管内皮生长因子可显著促进骨髓间充质干细胞向成骨细胞方向分化。 2.3 血管内皮生长因子促进骨髓间充质干细胞内血红素氧化酶1的表达为研究血管内皮生长因子对血红素氧化酶1表达的影响，实验检测骨髓间充质干细胞中血红素氧化酶1的mRNA和蛋白表达。研究发现100 μg/L血管内皮生长因子组骨髓间充质干细胞中血红素氧化酶1的mRNA是对照组的4.45倍(P < 0.001)(图3A)。 Western blot检测结果同Real time PCR结果一致：血管内皮生长因子明显促进骨髓间充质干细胞中血红素氧化酶1的蛋白表达(图3B)。 "

References

[1] Friedenstein AJ, Chailakhjan RK, Lalykina KS.The development of fibroblast colonies in monolayer cultures of guinea-pig bone marrow and spleen cells.Cell Tissue Kinet. 1970;3(4):393-403.
[2] Castro-Malaspina H, Gay RE, Resnick G,et al. Characterization of human bone marrow fibroblast colony-forming cells (CFU-F) and their progeny.Blood. 1980;56(2):289-301.
[3] Caplan AI.Mesenchymal stem cells.J Orthop Res. 1991;9(5): 641-650.
[4] Caplan AI.Adult mesenchymal stem cells for tissue engineering versus regenerative medicine.J Cell Physiol. 2007; 213(2):341-347.
[5] Bianco P, Robey PG, Simmons PJ.Mesenchymal stem cells: revisiting history, concepts, and assays.Cell Stem Cell. 2008; 2(4):313-319.
[6] Liu Y, Teoh SH, Chong MS,et al.Vasculogenic and osteogenesis-enhancing potential of human umbilical cord blood endothelial colony-forming cells.Stem Cells. 2012; 30(9):1911-1924.
[7] Liu CH, Hwang SM.Cytokine interactions in mesenchymal stem cells from cord blood.Cytokine. 2005;32(6):270-279.
[8] Busletta C, Novo E, Valfrè Di Bonzo L,et al. Dissection of the biphasic nature of hypoxia-induced motogenic action in bone marrow-derived human mesenchymal stem cells.Stem Cells. 2011 Jun;29(6):952-963.
[9] 王培蕊,马学玲,王心蕊,等.人骨髓间充质干细胞分离培养及血管内皮生长因子对其产生的诱导分化作用[J].中国组织工程研究与临床康复,2007,11(46):9250-9254.
[10] 刘傥,张湘生,张庆,等.骨髓间充质干细胞成骨分化中血管内皮生长因子和骨形态发生蛋白2的表达[J].中国组织工程研究,2012, 16(36):6651-6657.
[11] Wang Y, Wan C, Deng L,et al.The hypoxia-inducible factor alpha pathway couples angiogenesis to osteogenesis during skeletal development.J Clin Invest. 2007;117(6): 1616-1626.
[12] Bakken AF, Thaler MM, Schmid R.Metabolic regulation of heme catabolism and bilirubin production. I. Hormonal control of hepatic heme oxygenase activity.J Clin Invest. 1972;51(3): 530-536.
[13] Pimstone NR, Engel P, Tenhunen R,et al.Inducible heme oxygenase in the kidney: a model for the homeostatic control of hemoglobin catabolism.J Clin Invest. 1971;50(10): 2042-2050.
[14] Tenhunen R, Marver HS, Schmid R.Microsomal heme oxygenase. Characterization of the enzyme.J Biol Chem. 1969;244(23):6388-6394.
[15] Tenhunen R, Marver HS, Schmid R.The enzymatic conversion of heme to bilirubin by microsomal heme oxygenase.Proc Natl Acad Sci U S A. 1968;61(2):748-755.
[16] Vanella L, Kim DH, Asprinio D,et al.HO-1 expression increases mesenchymal stem cell-derived osteoblasts but decreases adipocyte lineage.Bone. 2010;46(1):236-243.
[17] Barbagallo I, Vanella A, Peterson SJ,et al.Overexpression of heme oxygenase-1 increases human osteoblast stem cell differentiation.J Bone Miner Metab. 2010;28(3):276-288.
[18] Vanella L, Sanford C Jr, Kim DH,et al. Oxidative stress and heme oxygenase-1 regulated human mesenchymal stem cells differentiation.Int J Hypertens. 2012;2012:890671.
[19] Soleimani M, Nadri S.A protocol for isolation and culture of mesenchymal stem cells from mouse bone marrow.Nat Protoc. 2009;4(1):102-106.
[20] Hsiao FS, Cheng CC, Peng SY,et al.Isolation of therapeutically functional mouse bone marrow mesenchymal stem cells within 3 h by an effective single-step plastic-adherent method.Cell Prolif. 2010;43(3):235-248.
[21] Dai J, Rabie AB.VEGF: an essential mediator of both angiogenesis and endochondral ossification.J Dent Res. 2007;86(10):937-950.
[22] Drake CJ, LaRue A, Ferrara N,et al.VEGF regulates cell behavior during vasculogenesis.Dev Biol. 2000;224(2): 178-188.
[23] Engsig MT, Chen QJ, Vu TH,et al.Matrix metalloproteinase 9 and vascular endothelial growth factor are essential for osteoclast recruitment into developing long bones.J Cell Biol. 2000;151(4):879-889.
[24] Hiltunen MO, Ruuskanen M, Huuskonen J,et al. Adenovirus- mediated VEGF-A gene transfer induces bone formation in vivo. FASEB J. 2003;17(9):1147-1149.
[25] Claffey KP, Abrams K, Shih SC, et al.Fibroblast growth factor 2 activation of stromal cell vascular endothelial growth factor expression and angiogenesis.Lab Invest. 2001;81(1):61-75.
[26] Deckers MM, van Bezooijen RL, van der Horst G,et al.Bone morphogenetic proteins stimulate angiogenesis through osteoblast-derived vascular endothelial growth factor A. Endocrinology. 2002;143(4):1545-1553.
[27] Peng H, Wright V, Usas A,et al.Synergistic enhancement of bone formation and healing by stem cell-expressed VEGF and bone morphogenetic protein-4.J Clin Invest. 2002;110(6): 751-759.
[28] Yeh LC, Lee JC.Osteogenic protein-1 increases gene expression of vascular endothelial growth factor in primary cultures of fetal rat calvaria cells.Mol Cell Endocrinol. 1999; 153(1-2):113-124.
[29] Ball SG, Shuttleworth CA, Kielty CM.Vascular endothelial growth factor can signal through platelet-derived growth factor receptors.J Cell Biol. 2007;177(3):489-500.
[30] Abraham NG, Kappas A.Pharmacological and clinical aspects of heme oxygenase.Pharmacol Rev. 2008;60(1):79-127.
[31] Komatsu DE, Hadjiargyrou M.Activation of the transcription factor HIF-1 and its target genes, VEGF, HO-1, iNOS, during fracture repair.Bone. 2004;34(4):680-688.
[32] Bussolati B, Ahmed A, Pemberton H,et al.Bifunctional role for VEGF-induced heme oxygenase-1 in vivo: induction of angiogenesis and inhibition of leukocytic infiltration.Blood. 2004;103(3):761-766.
[33] Lin HH, Lai SC, Chau LY.Heme oxygenase-1/carbon monoxide induces vascular endothelial growth factor expression via p38 kinase-dependent activation of Sp1.J Biol Chem. 2011;286(5):3829-3838.
[34] Vanella L, Kim DH, Asprinio D,et al.HO-1 expression increases mesenchymal stem cell-derived osteoblasts but decreases adipocyte lineage.Bone. 2010;46(1):236-243.
[35] Barbagallo I, Vanella A, Peterson SJ,et al.Overexpression of heme oxygenase-1 increases human osteoblast stem cell differentiation.J Bone Miner Metab. 2010;28(3):276-288.
[36] Mundy G, Garrett R, Harris S,et al.Stimulation of bone formation in vitro and in rodents by statins.Science. 1999; 286(5446):1946-1949.
[37] Armour KE, Armour KJ, Gallagher ME,et al.Defective bone formation and anabolic response to exogenous estrogen in mice with targeted disruption of endothelial nitric oxide synthase.Endocrinology. 2001;142(2):760-766.
[38] Garrett IR, Gutierrez G, Mundy GR.Statins and bone formation.Curr Pharm Des. 2001;7(8):715-736.
[39] Penna C, Perrelli MG, Karam JP,et al.Pharmacologically active microcarriers influence VEGF-A effects on mesenchymal stem cell survival.J Cell Mol Med. 2013;17(1): 192-204.