Glucocorticoids Inducing Vascular Repair Disorders under Hypoxia via Inhibiting Cell Migration and Autocrine/paracrine: Bioinformatical Analysis Combined with Cytological Experiment
MA Jun, YANG Pei, WANG Kun-zheng
Department of Orthopedics, the Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an Shaanxi 710004, China
Glucocorticoids Inducing Vascular Repair Disorders under Hypoxia via Inhibiting Cell Migration and Autocrine/paracrine: Bioinformatical Analysis Combined with Cytological Experiment
MA Jun, YANG Pei, WANG Kun-zheng
Department of Orthopedics, the Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an Shaanxi 710004, China
摘要The exact molecular and cytological mechanism of how glucocorticoids induce vascular repair disorders in glucocorticoid-induced avascular necrosis of the femoral head is still unclear. We used bioinformatical tools for data mining and detected the biological behavior of endothelial cells (ECs) under hypoxia conditions and high dose dexamethasone to reveal the mechanisms above. Six differential expression miRNAs (DE-miRNAs) were filtered from Gene Expression Omnibus (GEO) database GSE60093 which contained ECs treated with high dose glucocorticoid and control samples. Enrichment and PPI network analyses of the DE-miRNAs target genes showed the most remarkable pathway was HIF-1 signaling pathway and high dose glucocorticoid as a negative regulator of cell differentiation, energy metabolism, migration and cytokines secretion. Glucocorticoids also reduced the activity of autocrine/paracrine via limiting ion channels and transmembrane transporter process. In cytological experiment, HUVECs were divided into four groups: hypoxia group (H), hypoxia + dexamethasone group (HD), dexamethasone group (D), the normal group (N). Cell activity detection and Live/Dead dyeing showed cell activity and the number of live cells in Group H was higher than the other three groups at 24 h after intervention, while cell activity, number and proportion of live cells in HD group were worst. Cytoskeleton staining showed HD group met cytoskeleton form disorders. The scratch assay showed cell migration ability of Group H was strongest while cell migration ability of the HD group was worst. MIF expression in HD group showed a trend of bimodal, the peak of VEGF-A secretion lagged behind the MIF's. Expression of MIF and VEGF-A in the HD group were low. High dose dexamethasone suppressed the active response of ECs to hypoxia stimulation via directly inhibiting the expression of MIF and interdicting autocrine/paracrine mechanism. We infered that the treatment with high dose glucocorticoid would inhibit neo-angiogenesis under hypoxia followed by aggravating hypoxia/ischemia and osteonecrosis.
Abstract：The exact molecular and cytological mechanism of how glucocorticoids induce vascular repair disorders in glucocorticoid-induced avascular necrosis of the femoral head is still unclear. We used bioinformatical tools for data mining and detected the biological behavior of endothelial cells (ECs) under hypoxia conditions and high dose dexamethasone to reveal the mechanisms above. Six differential expression miRNAs (DE-miRNAs) were filtered from Gene Expression Omnibus (GEO) database GSE60093 which contained ECs treated with high dose glucocorticoid and control samples. Enrichment and PPI network analyses of the DE-miRNAs target genes showed the most remarkable pathway was HIF-1 signaling pathway and high dose glucocorticoid as a negative regulator of cell differentiation, energy metabolism, migration and cytokines secretion. Glucocorticoids also reduced the activity of autocrine/paracrine via limiting ion channels and transmembrane transporter process. In cytological experiment, HUVECs were divided into four groups: hypoxia group (H), hypoxia + dexamethasone group (HD), dexamethasone group (D), the normal group (N). Cell activity detection and Live/Dead dyeing showed cell activity and the number of live cells in Group H was higher than the other three groups at 24 h after intervention, while cell activity, number and proportion of live cells in HD group were worst. Cytoskeleton staining showed HD group met cytoskeleton form disorders. The scratch assay showed cell migration ability of Group H was strongest while cell migration ability of the HD group was worst. MIF expression in HD group showed a trend of bimodal, the peak of VEGF-A secretion lagged behind the MIF's. Expression of MIF and VEGF-A in the HD group were low. High dose dexamethasone suppressed the active response of ECs to hypoxia stimulation via directly inhibiting the expression of MIF and interdicting autocrine/paracrine mechanism. We infered that the treatment with high dose glucocorticoid would inhibit neo-angiogenesis under hypoxia followed by aggravating hypoxia/ischemia and osteonecrosis.
基金资助:National Natural Science Foundation of China; grant number:81301562 and 81572147
通讯作者:
WANG Kun-zheng. E-mail: wkunzheng1955@126.com
引用本文:
MA Jun, YANG Pei, WANG Kun-zheng. Glucocorticoids Inducing Vascular Repair Disorders under Hypoxia via Inhibiting Cell Migration and Autocrine/paracrine: Bioinformatical Analysis Combined with Cytological Experiment[J]. 中国生物医学工程学报(英文版）, 2021, 30(2): 75-92.
MA Jun, YANG Pei, WANG Kun-zheng. Glucocorticoids Inducing Vascular Repair Disorders under Hypoxia via Inhibiting Cell Migration and Autocrine/paracrine: Bioinformatical Analysis Combined with Cytological Experiment. Chinese Journal of Biomedical Engineering, 2021, 30(2): 75-92.
[1] Xie XH, Wang XL, Yang HL, et al.Steroid-associated osteonecrosis: Epidemiology, pathophysiology, animal model, prevention, and potential treatments (an overview)[J]. J Orthop Translat, 2015, 3(2): 58-70. [2] Hernigou P, Trousselier M, Roubineau F, et al.Stem cell therapy for the treatment of hip osteonecrosis: A 30-Year review of progress[J]. Clin Orthop Surg, 2016, 8(1): 1-8. [3] Weinstein RS.Glucocorticoid-induced osteoporosis and osteonecrosis[J]. Endocrinol Metab Clin North Am, 2012, 41(3): 595-611. [4] Seamon J, Keller T, Saleh J, et al.The pathogenesis of nontraumatic osteonecrosis. Arthritis[J]. 2012, 2012: 601763. [5] Zhao FC, Li ZR, Guo KJ.Clinical analysis of osteonecrosis of the femoral head induced by steroids[J]. Orthop Surg, 2012, 4(1): 28-34. [6] van der Jagt D. Osteonecrosis of the femoral head: evaluation and treatment[J]. J Am Acad Orthop Surg, 2015, 23(2): 69-70. [7] Fan Lihong, Li Jia, Yu Zefeng, et al.Hypoxia-inducible factor prolyl hydroxylase inhibitor prevents steroid-associated osteonecrosis of the femoral head in rabbits by promoting angiogenesis and inhibiting apoptosis[J]. PLoS One, 2014, 9(9): e107774. [8] Zhang Chen, Li Xinghua, Li Miao, et al.Repair effect of coexpression of the hVEGF and hBMP genes via an adeno-associated virus vector in a rabbit model of early steroid-induced avascular necrosis of the femoral head[J]. Transl Res, 2015, 166(3): 269-280. [9] Kusumbe AP, Ramasamy SK, Adams RH.Coupling of angiogenesis and osteogenesis by a specific vessel subtype in bone[J]. Nature, 2014, 507(7492): 323-328. [10] Schipani E, Maes C, Garmeliet G, et al.Regulation of osteogenesis-angiogenesis coupling by HIFs and VEGF[J]. J Bone Miner Res, 2009, 24(8): 1347-1353. [11] Gotz W, Reichert C, Canullo L, et al.Coupling of osteogenesis and angiogenesis in bone substitute healing-a brief overview[J]. Ann Anat, 2012, 194(2): 171-173. [12] Asare Y, Schmitt M, Bernhagen J.The vascular biology of macrophage migration inhibitory factor (MIF). Expression and effects in inflammation, atherogenesis and angiogenesis[J]. Thromb Haemost, 2013, 109(3): 391-398. [13] Harvey TW, Engel JE, Chade AR.Vascular endothelial growth factor and podocyte protection in chronic hypoxia: Effects of endothelin-A receptor antagonism[J]. Am J Nephrol, 2016, 43(2): 74-84. [14] Elsby LM, Donn R, Alourfi Z, et al.Hypoxia and glucocorticoid signaling converge to regulate macrophage migration inhibitory factor gene expression[J]. Arthritis Rheum, 2009, 60(8): 2220-2231. [15] David JR.Delayed hypersensitivity in vitro: its mediation by cell-free substances formed by lymphoid cell-antigen interaction[J]. Proc Natl Acad Sci U S A, 1966, 56(1): 72-77. [16] Agarwal V, Bell GW, Nam JW, et al.Predicting effective microRNA target sites in mammalian mRNAs[J]. Elife, 2015, 4: e05005. [17] Su G, Morris JH, Demchak B, et al. Biological network exploration with Cytoscape 3[J]. Curr Protoc Bioinformatics, 2014, 47: 8.13.1-8.13.24. [18] Zhou Yingyao, Zhou Bin, Pache L, et al.Metascape provides a biologist-oriented resource for the analysis of systems-level datasets[J]. Nat Commun, 2019, 10(1): 1523. [19] Szklarczyk D, Gable AL, Lyon D, et al.STRING v11: protein-protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets[J]. Nucleic Acids Res, 2019, 47(D1): D607-D613. [20] Yu D, Lim J, Wang X, et al.Enhanced construction of gene regulatory networks using hub gene information[J]. BMC Bioinformatics, 2017, 18(1): 186. [21] Liu Xiaojun, Xiao Junjie, Zhu Han, et al.miR-222 is necessary for exercise-induced cardiac growth and protects against pathological cardiac remodeling[J]. Cell Metab, 2015, 21(4): 584-595. [22] Wang Tao, Zhong Donghuo, Qin Zhongjun, et al.miR-100-3p inhibits the adipogenic differentiation of hMSCs by targeting PIK3R1 via the PI3K/AKT signaling pathway[J]. Aging (Albany NY), 2020, 12(24): 25090-25100. [23] Yang Jie, Li Ying, Yu Zuochun, et al.Circular RNA Circ100084 functions as sponge of miR23a5p to regulate IGF2 expression in hepatocellular carcinoma[J]. Mol Med Rep, 2020, 21(6): 2395-2404. [24] Jansson MD, Damas ND, Lees M, et al.miR-339-5p regulates the p53 tumor-suppressor pathway by targeting MDM2[J]. Oncogene, 2015, 34(15): 1908-1918. [25] Qian Zhengjiang, Li Yanjiao, Yang Haiyang.PDGFBB promotes proliferation and migration via regulating miR-1181/STAT3 axis in human pulmonary arterial smooth muscle cells[J]. Am J Physiol Lung Cell Mol Physiol, 2018, 315(6): L965-L976. [26] Baugh JA, Gantier M, Li L, et al.Dual regulation of macrophage migration inhibitory factor (MIF) expression in hypoxia by CREB and HIF-1[J]. Biochem Biophys Res Commun, 2006, 347(4): 895-903. [27] Fu Hua, Luo Fengming, Yang Li, et al.Hypoxia stimulates the expression of macrophage migration inhibitory factor in human vascular smooth muscle cells via HIF-1alpha dependent pathway[J]. BMC Cell Biol, 2010, 11: 66. [28] Geenen ILA, Molin DGM, van den Akker NMS, et al. Endothelial cells (ECs) for vascular tissue engineering: venous ECs are less thrombogenic than arterial ECs[J]. J Tissue Eng Regen Med, 2015, 9(5): 564-576. [29] Dehghanian F, Hojati Z, Kay M.New insights into VEGF-A alternative splicing: Key regulatory switching in the pathological process[J]. Avicenna J Med Biotechnol, 2014, 6(4): 192-199. [30] Malinda KM.In vivo matrigel migration and angiogenesis assays[J]. Methods Mol Med, 2003, 78: 329-335. [31] Cluitmans FHM, Esendam BHJ, Veenhof WFJ, et al.The role of cytokines and hematopoietic growth factors in the autocrine/paracrine regulation of inducible hematopoiesis[J]. Ann Hematol, 1997, 75(1-2): 27-31. [32] Janowska-Wieczorek A, Majka M, Ratajczak J, et al.Autocrine/paracrine mechanisms in human hematopoiesis[J]. Stem Cells, 2001, 19(2): 99-107. [33] Simons D, Grieb G, Hristov M, et al.Hypoxia-induced endothelial secretion of macrophage migration inhibitory factor and role in endothelial progenitor cell recruitment[J]. J Cell Mol Med, 2011, 15(3): 668-678. [34] Alcami A.Viral mimicry of cytokines, chemokines and their receptors[J]. Nat Rev Immunol, 2003, 3(1): 36-50. [35] Porter RM, Huckle WR, Goldstein AS.Effect of dexamethasone withdrawal on osteoblastic differentiation of bone marrow stromal cells[J]. J Cell Biochem, 2003, 90(1): 13-22. [36] Annane D.Glucocorticoids in the treatment of severe sepsis and septic shock[J]. Curr Opin Crit Care, 2005, 11(5): 449-453. [37] Edgar AR, Judith PY, Elisa DM, et al.Glucocorticoids and estrogens modulate the NF-kappaB pathway differently in the micro- and macrovasculature[J]. Med Hypotheses, 2013, 81(6): 1078-1082.
