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Slit2/Robo1 signaling is involved in angiogenesis of glomerular endothelial cells exposed to a diabetic-like environment

Angiogenesis volume 21, pages 237–249 (2018)Cite this article

Abstract

Abnormal angiogenesis plays a pathological role in diabetic nephropathy (DN), contributing to glomerular hypertrophy and microalbuminuria. Slit2/Robo1 signaling participates in angiogenesis in some pathological contexts, but whether it is involved in glomerular abnormal angiogenesis of early DN is unclear. The present study evaluated the effects of Slit2/Robo1 signaling pathway on angiogenesis of human renal glomerular endothelial cells (HRGECs) exposed to a diabetic-like environment or recombinant Slit2-N. To remove the effect of Slit2 derived from mesangial cells, human renal mesangial cells (HRMCs) grown in high glucose (HG) medium (33 mM) were transfected with Slit2 siRNA and then the HG-HRMCs-CM with Slit2 depletion was collected after 48 h. HRGECs were cultured in the HG-HRMCs-CM or recombinant Slit2-N for 0, 6, 12, 24, or 48 h. The mRNA and protein expressions of Slit2/Robo1, PI3K/Akt and HIF-1α/VEGF signaling pathways were detected by quantitative real-time PCR, western blotting, and ELISA, respectively. The CCK-8 cell proliferation assay, flow cytometry and the scratch wound-healing assay were used to assess cell proliferation, cycles, and migration, respectively. Matrigel was used to perform a tubule formation assay. Our results showed that the HG-HRMCs-CM with Slit2 depletion enhanced the activation of Slit2/Robo1, PI3K/Akt, and HIF-1α/VEGF signaling in HRGECs in time-dependent manner (0–24 h post-treatment). In addition, the HG-HRMCs-CM with Slit2 depletion significantly promoted HRGECs proliferation, migration, and tube formation. Pretreatment of HRGECs with Robo1 siRNA suppressed the activation of PI3K/Akt and HIF-1α/VEGF signaling and inhibited angiogenesis, whereas PI3K inhibitor suppressed HIF-1α/VEGF signaling, without influencing Robo1 expression. In the HRGECs treated with Slit2-N, Slit2-N time-dependently enhanced the activation of Robo1/PI3K/Akt/VEGF pathway but not HIF-1α activity, and promoted HRGECs proliferation, migration, and tube formation. The effects induced by Slit2 were also abolished by Robo1 siRNA and PI3K inhibitor. Taken together, our findings indicate that in a diabetic-like environment, in addition to mesangial cells, autocrine activation of Slit2/Robo1 signaling of HRGECs may contribute to angiogenesis of HRGECs through PI3K/Akt/VEGF pathway; therefore, Slit2/Robo1 signaling may be a potent therapeutic target for the treatment of abnormal angiogenesis in early DN and may have broad implications for the treatment of other diseases dependent on pathologic angiogenesis.

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References

  1. Costa PZ, Soares R (2013) Neovascularization in diabetes and its complications. Unraveling the angiogenic paradox. Life Sci 92(22):1037–1045. https://doi.org/10.1016/j.lfs.2013.04.001

    CAS  Article  PubMed  Google Scholar 

  2. Nakagawa T, Kosugi T, Haneda M, Rivard CJ, Long DA (2009) Abnormal angiogenesis in diabetic nephropathy. Diabetes 58(7):1471–1478. https://doi.org/10.2337/db09-0119

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  3. Hoeben A, Landuyt B, Highley MS, Wildiers H, Van Oosterom AT, De Bruijn EA (2004) Vascular endothelial growth factor and angiogenesis. Pharmacol Rev 56(4):549–580. https://doi.org/10.1124/pr.56.4.3

    CAS  Article  PubMed  Google Scholar 

  4. de Vriese AS, Tilton RG, Elger M, Stephan CC, Kriz W, Lameire NH (2001) Antibodies against vascular endothelial growth factor improve early renal dysfunction in experimental diabetes. J Am Soc Nephrol 12(5):993–1000

    PubMed  Google Scholar 

  5. Flyvbjerg A, Dagnaes-Hansen F, De Vriese AS, Schrijvers BF, Tilton RG, Rasch R (2002) Amelioration of long-term renal changes in obese type 2 diabetic mice by a neutralizing vascular endothelial growth factor antibody. Diabetes 51(10):3090–3094

    CAS  Article  PubMed  Google Scholar 

  6. Eremina V, Jefferson JA, Kowalewska J, Hochster H, Haas M, Weisstuch J, Richardson C, Kopp JB, Kabir MG, Backx PH, Gerber HP, Ferrara N, Barisoni L, Alpers CE, Quaggin SE (2008) VEGF inhibition and renal thrombotic microangiopathy. N Engl J Med 358(11):1129–1136. https://doi.org/10.1056/NEJMoa0707330

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  7. Levy A, Albiges-Sauvin L, Massard C, Izzedine H, Ederhy S, Bahleda R, Gomez-Roca C, Chargari C, Brocheriou-Spelle I, Soria JC (2012) Reappraisal of treatment-induced renal dysfunction in patients receiving antiangiogenic agents in phase I trials. Investig New Drugs 30(3):1116–1120. https://doi.org/10.1007/s10637-011-9671-z

    CAS  Article  Google Scholar 

  8. Hayman SR, Leung N, Grande JP, Garovic VD (2012) VEGF inhibition, hypertension, and renal toxicity. Curr Oncol Rep 14(4):285–294. https://doi.org/10.1007/s11912-012-0242-z

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  9. Dickson BJ, Gilestro GF (2006) Regulation of commissural axon pathfinding by slit and its Robo receptors. Annu Rev Cell Dev Biol 22:651–675. https://doi.org/10.1146/annurev.cellbio.21.090704.151234

    CAS  Article  PubMed  Google Scholar 

  10. Wong K, Ren XR, Huang YZ, Xie Y, Liu G, Saito H, Tang H, Wen L, Brady-Kalnay SM, Mei L, Wu JY, Xiong WC, Rao Y (2001) Signal transduction in neuronal migration: roles of GTPase activating proteins and the small GTPase Cdc42 in the Slit–Robo pathway. Cell 107(2):209–221

    CAS  Article  PubMed  Google Scholar 

  11. Ypsilanti AR, Zagar Y, Chedotal A (2010) Moving away from the midline: new developments for Slit and Robo. Development 137(12):1939–1952. https://doi.org/10.1242/dev.044511

    CAS  Article  PubMed  Google Scholar 

  12. Hohenester E (2008) Structural insight into Slit–Robo signalling. Biochem Soc Trans 36(Pt 2):251–256. https://doi.org/10.1042/BST0360251

    CAS  Article  PubMed  Google Scholar 

  13. Yuen DA, Robinson LA (2013) Slit2–Robo signaling: a novel regulator of vascular injury. Curr Opin Nephrol Hypertens 22(4):445–451. https://doi.org/10.1097/MNH.0b013e32836235f4

    CAS  Article  PubMed  Google Scholar 

  14. Wang B, Xiao Y, Ding BB, Zhang N, Yuan X, Gui L, Qian KX, Duan S, Chen Z, Rao Y, Geng JG (2003) Induction of tumor angiogenesis by Slit–Robo signaling and inhibition of cancer growth by blocking Robo activity. Cancer Cell 4(1):19–29

    Article  PubMed  Google Scholar 

  15. Zhou W, Yu W, Xie W, Huang L, Xu Y, Li X (2011) The role of Slit–Robo signaling in proliferative diabetic retinopathy and retinal pigment epithelial cells. Mol Vis 17:1526–1536

    CAS  PubMed Central  PubMed  Google Scholar 

  16. Kang MK, Lim SS, Lee JY, Yeo KM, Kang YH (2013) Anthocyanin-rich purple corn extract inhibit diabetes-associated glomerular angiogenesis. PLoS ONE 8(11):e79823. https://doi.org/10.1371/journal.pone.0079823

    Article  PubMed Central  PubMed  Google Scholar 

  17. Xing Y, Lai J, Liu X, Zhang N, Ming J, Liu H, Zhang X (2017) Netrin-1 restores cell injury and impaired angiogenesis in vascular endothelial cells upon high glucose by PI3K/AKT-eNOS. J Mol Endocrinol 58(4):167–177. https://doi.org/10.1530/JME-16-0239

    CAS  Article  PubMed  Google Scholar 

  18. Patella F, Leucci E, Evangelista M, Parker B, Wen J, Mercatanti A, Rizzo M, Chiavacci E, Lund AH, Rainaldi G (2013) MiR-492 impairs the angiogenic potential of endothelial cells. J Cell Mol Med 17(8):1006–1015. https://doi.org/10.1111/jcmm.12085

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  19. Kitahara T, Hiromura K, Ikeuchi H, Yamashita S, Kobayashi S, Kuroiwa T, Kaneko Y, Ueki K, Nojima Y (2005) Mesangial cells stimulate differentiation of endothelial cells to form capillary-like networks in a three-dimensional culture system. Nephrol Dial Transplant 20(1):42–49. https://doi.org/10.1093/ndt/gfh572

    CAS  Article  PubMed  Google Scholar 

  20. Kolluru GK, Bir SC, Kevil CG (2012) Endothelial dysfunction and diabetes: effects on angiogenesis, vascular remodeling, and wound healing. Int J Vasc Med 2012:918267. https://doi.org/10.1155/2012/918267

    PubMed Central  PubMed  Google Scholar 

  21. Rama N, Dubrac A, Mathivet T, Ni Charthaigh RA, Genet G, Cristofaro B, Pibouin-Fragner L, Ma L, Eichmann A, Chedotal A (2015) Slit2 signaling through Robo1 and Robo2 is required for retinal neovascularization. Nat Med 21(5):483–491. https://doi.org/10.1038/nm.3849

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  22. Fish JE, Wythe JD, Xiao T, Bruneau BG, Stainier DY, Srivastava D, Woo S (2011) A Slit/miR-218/Robo regulatory loop is required during heart tube formation in zebrafish. Development 138(7):1409–1419. https://doi.org/10.1242/dev.060046

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  23. Piper M, Georgas K, Yamada T, Little M (2000) Expression of the vertebrate Slit gene family and their putative receptors, the Robo genes, in the developing murine kidney. Mech Dev 94(1–2):213–217

    CAS  Article  PubMed  Google Scholar 

  24. Liu A, Dardik A, Ballermann BJ (1999) Neutralizing TGF-β1 antibody infusion in neonatal rat delays in vivo glomerular capillary formation 1. Kidney Int 56(4):1334–1348. https://doi.org/10.1046/j.1523-1755.1999.00661.x

    CAS  Article  PubMed  Google Scholar 

  25. Small EM, Sutherland LB, Rajagopalan KN, Wang S, Olson EN (2010) MicroRNA-218 regulates vascular patterning by modulation of Slit–Robo signaling. Circ Res 107(11):1336–1344. https://doi.org/10.1161/CIRCRESAHA.110.227926

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  26. Chen X, Li J, Li M, Zeng M, Li T, Xiao W, Li J, Wu Q, Ke X, Luo D, Tang S, Luo Y (2013) KH902 suppresses high glucose-induced migration and sprouting of human retinal endothelial cells by blocking VEGF and PIGF. Diabetes Obes Metab 15(3):224–233. https://doi.org/10.1111/dom.12008

    CAS  Article  PubMed  Google Scholar 

  27. Chaudagar KK, Mehta AA (2014) Effect of telmisartan on VEGF-induced and VEGF-independent angiogenic responsiveness of coronary endothelial cells in normal and streptozotocin (STZ)-induced diabetic rats. Clin Exp Hypertens 36(8):557–566. https://doi.org/10.3109/10641963.2014.881842

    CAS  Article  PubMed  Google Scholar 

  28. Zeng Z, Huang WD, Gao Q, Su ML, Yang YF, Liu ZC, Zhu BH (2015) Arnebin-1 promotes angiogenesis by inducing eNOS, VEGF and HIF-1α expression through the PI3K-dependent pathway. Int J Mol Med 36(3):685–697. https://doi.org/10.3892/ijmm.2015.2292

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  29. Massacesi C, Di Tomaso E, Urban P, Germa C, Quadt C, Trandafir L, Aimone P, Fretault N, Dharan B, Tavorath R, Hirawat S (2016) PI3K inhibitors as new cancer therapeutics: implications for clinical trial design. OncoTargets Ther 9:203–210. https://doi.org/10.2147/OTT.S89967

    CAS  Article  Google Scholar 

  30. Sasore T, Kennedy B (2014) Deciphering combinations of PI3K/AKT/mTOR pathway drugs augmenting anti-angiogenic efficacy in vivo. PLoS ONE 9(8):e105280. https://doi.org/10.1371/journal.pone.0105280

    Article  PubMed Central  PubMed  Google Scholar 

  31. Jacot JL, Sherris D (2011) Potential therapeutic roles for inhibition of the PI3K/Akt/mTOR pathway in the pathophysiology of diabetic retinopathy. J Ophthalmol 2011:589813. https://doi.org/10.1155/2011/589813

    PubMed Central  PubMed  Google Scholar 

  32. Graupera M, Potente M (2013) Regulation of angiogenesis by PI3K signaling networks. Exp Cell Res 319(9):1348–1355. https://doi.org/10.1016/j.yexcr.2013.02.021

    CAS  Article  PubMed  Google Scholar 

  33. Pugh CW, Ratcliffe PJ (2003) Regulation of angiogenesis by hypoxia: role of the HIF system. Nat Med 9(6):677–684. https://doi.org/10.1038/nm0603-677

    CAS  Article  PubMed  Google Scholar 

  34. Yu Z, Zhang T, Gong C, Sheng Y, Lu B, Zhou L, Ji L, Wang Z (2016) Erianin inhibits high glucose-induced retinal angiogenesis via blocking ERK1/2-regulated HIF-1α–VEGF/VEGFR2 signaling pathway. Sci Rep 6:34306. https://doi.org/10.1038/srep34306

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  35. Shao Y, Lv C, Wu C, Zhou Y, Wang Q (2016) Mir-217 promotes inflammation and fibrosis in high glucose cultured rat glomerular mesangial cells via Sirt1/HIF-1α signaling pathway. Diabetes Metab Res Rev 32(6):534–543. https://doi.org/10.1002/dmrr.2788

    CAS  Article  PubMed  Google Scholar 

  36. Isoe T, Makino Y, Mizumoto K, Sakagami H, Fujita Y, Honjo J, Takiyama Y, Itoh H, Haneda M (2010) High glucose activates HIF-1-mediated signal transduction in glomerular mesangial cells through a carbohydrate response element binding protein. Kidney Int 78(1):48–59. https://doi.org/10.1038/ki.2010.99

    CAS  Article  PubMed  Google Scholar 

  37. Makino H, Miyamoto Y, Sawai K, Mori K, Mukoyama M, Nakao K, Yoshimasa Y, Suga S (2006) Altered gene expression related to glomerulogenesis and podocyte structure in early diabetic nephropathy of db/db mice and its restoration by pioglitazone. Diabetes 55(10):2747–2756. https://doi.org/10.2337/db05-1683

    CAS  Article  PubMed  Google Scholar 

  38. Liu Y, Cox SR, Morita T, Kourembanas S (1995) Hypoxia regulates vascular endothelial growth factor gene expression in endothelial cells. Identification of a 5′ enhancer. Circ Res 77(3):638–643

    CAS  Article  PubMed  Google Scholar 

  39. Gerber HP, Condorelli F, Park J, Ferrara N (1997) Differential transcriptional regulation of the two vascular endothelial growth factor receptor genes. Flt-1, but not Flk-1/KDR, is up-regulated by hypoxia. J Biol Chem 272(38):23659–23667

    CAS  Article  PubMed  Google Scholar 

  40. Ben-Yosef Y, Miller A, Shapiro S, Lahat N (2005) Hypoxia of endothelial cells leads to MMP-2-dependent survival and death. Am J Physiol Cell Physiol 289(5):C1321–C1331. https://doi.org/10.1152/ajpcell.00079.2005

    CAS  Article  PubMed  Google Scholar 

  41. Keely S, Glover LE, MacManus CF, Campbell EL, Scully MM, Furuta GT, Colgan SP (2009) Selective induction of integrin β1 by hypoxia-inducible factor: implications for wound healing. FASEB J 23(5):1338–1346. https://doi.org/10.1096/fj.08-125344

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  42. Nomura M, Yamagishi S, Harada S, Hayashi Y, Yamashima T, Yamashita J, Yamamoto H (1995) Possible participation of autocrine and paracrine vascular endothelial growth factors in hypoxia-induced proliferation of endothelial cells and pericytes. J Biol Chem 270(47):28316–28324

    CAS  Article  PubMed  Google Scholar 

  43. Skuli N, Liu L, Runge A, Wang T, Yuan L, Patel S, Iruela-Arispe L, Simon MC, Keith B (2009) Endothelial deletion of hypoxia-inducible factor-2α (HIF-2α) alters vascular function and tumor angiogenesis. Blood 114(2):469–477. https://doi.org/10.1182/blood-2008-12-193581

    CAS  Article  PubMed Central  PubMed  Google Scholar 

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Acknowledgements

We are grateful to Dr. Xiaohua Guo (University of Utah) for his invaluable advice and to Yunan Li for assistance in manuscript preparation.

Funding

This study was supported by the National Natural Science Foundation of China (No. 81370821), The National Key technology Research and Development (R&D) Program (No. 2015BAI12B05), and the Natural Science Foundation of Chongqing (No. cstc2013jjB10023).

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  1. Junhui Liu and Weiping Hou contributed equally to this work.

Affiliations

  1. Department of Nephrology, Xinqiao Hospital, Third Military Medical University, Chongqing, 400037, China

    Junhui Liu, Weiping Hou, Tao Guan, Luyao Tang, Xufei Zhu, Yi Li, Shihui Hou, Hua Chen & Yunjian Huang

  2. Department of Pathology, Xinqiao Hospital, Third Military Medical University, Chongqing, 400037, China

    Jun Zhang

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  1. Junhui Liu
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Liu, J., Hou, W., Guan, T. et al. Slit2/Robo1 signaling is involved in angiogenesis of glomerular endothelial cells exposed to a diabetic-like environment. Angiogenesis 21, 237–249 (2018). https://doi.org/10.1007/s10456-017-9592-3

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Keywords

  • Slit2/Robo1 signaling
  • Angiogenesis
  • Glomerular endothelial cells
  • PI3K/Akt signaling
  • HIF-1α/VEGF signaling
  • Diabetic nephropathy