Calcium dobesilate reduces VEGF signaling by interfering with heparan sulfate binding site and protects from vascular complications in diabetic mice

Autoři: Florence Njau aff001;  Nelli Shushakova aff001;  Heiko Schenk aff001;  Vera Christine Wulfmeyer aff001;  Robin Bollin aff001;  Jan Menne aff001;  Hermann Haller aff001
Působiště autorů: Division of Nephrology, Hannover Medical School, Hannover, Germany aff001
Vyšlo v časopise: PLoS ONE 15(1)
Kategorie: Research Article


Inhibiting vascular endothelial growth factor (VEGF) is a therapeutic option in diabetic microangiopathy. However, VEGF is needed at physiological concentrations to maintain glomerular integrity; complete VEGF blockade has deleterious effects on glomerular structure and function. Anti-VEGF therapy in diabetes raises the challenge of reducing VEGF-induced pathology without accelerating endothelial cell injury. Heparan sulfate (HS) act as a co-receptor for VEGF. Calcium dobesilate (CaD) is a small molecule with vasoprotective properties that has been used for the treatment of diabetic microangiopathy. Preliminary evidence suggests that CaD interferes with HS binding sites of fibroblast growth factor. We therefore tested the hypotheses that (1) CaD inhibits VEGF signaling in endothelial cells, (2) that this effect is mediated via interference between CaD and HS, and (3) that CaD ameliorates diabetic nephropathy in a streptozotocin-induced diabetic mouse model by VEGF inhibition. We found that CaD significantly inhibited VEGF165-induced endothelial cell migration, proliferation, and permeability. CaD significantly inhibited VEGF165-induced phosphorylation of VEGFR-2 and suppressed the activity of VEGFR-2 mediated signaling cascades. The effects of CaD in vitro were abrogated by heparin, suggesting the involvement of heparin-like domain in the interaction with CaD. In addition, VEGF121, an isoform which does not bind to heparin, was not inhibited by CaD. Using the proximity ligation approach, we detected inhibition of interaction in situ between HS and VEGF and between VEGF and VEGFR-2. Moreover, CaD reduced VEGF signaling in mice diabetic kidneys and ameliorated diabetic nephropathy and neuropathy, suggesting CaD as a VEGF inhibitor without the negative effects of complete VEGF blockade and therefore could be useful as a strategy in treating diabetic nephropathy.

Klíčová slova:

diabetes mellitus – Endothelial cells – Enzyme-linked immunoassays – Heparin – Kidneys – Mouse models – Phosphorylation – VEGF signaling


1. Flyvbjerg A, Dagnaes-Hansen F, De Vriese AS, Schrijvers BF, Tilton RG, Rasch R. Amelioration of long-term renal changes in obese type 2 diabetic mice by a neutralizing vascular endothelial growth factor antibody. Diabetes. 2002;51: 3090–3094. doi: 10.2337/diabetes.51.10.3090 12351452

2. Group TDC and CT of DI and CR. Retinopathy and Nephropathy in Patients with Type 1 Diabetes Four Years after a Trial of Intensive Therapy. N Engl J Med. 2000;342: 381–389. doi: 10.1056/NEJM200002103420603 10666428

3. Parving HH, Andersen AR, Smidt UM, Hommel E, Mathiesen ER, Svendsen PA. Effect of antihypertensive treatment on kidney function in diabetic nephropathy. Br Med J (Clin Res Ed). 1987;294: 1443–7. doi: 10.1136/bmj.294.6585.1443 3111583

4. Bakris GL, Ritz E. The message for World Kidney Day 2009: Hypertension and kidney disease—A marriage that should be prevented. Journal of Hypertension. 2009. pp. 666–669. doi: 10.1097/HJH.0b013e328327706a 19262237

5. Wu HY, Huang JW, Lin HJ, Liao W-CC, Peng Y-SS, Hung KY, et al. Comparative effectiveness of renin-angiotensin system blockers and other antihypertensive drugs in patients with diabetes: systematic review and bayesian network meta-analysis. BMJ. 2013;347: f6008. doi: 10.1136/bmj.f6008 24157497

6. Collins AJ, Foley RN, Chavers B, Gilbertson D, Herzog C, Johansen K, et al. US renal data system 2011 Annual data report. Am J Kidney Dis. 2012;59: 6386. doi: 10.1053/j.ajkd.2011.11.015

7. Nelson RG, Newman JM, Knowler WC, Sievers ML, Kunzelman CL, Pettitt DJ, et al. Incidence of end-stage renal disease in Type 2 (non-insulin-dependent) diabetes mellitus in pima indians. Diabetologia. 1988;31: 730–736. doi: 10.1007/bf00274774 3240833

8. Calles-Escandon J, Cipolla M. Diabetes and endothelial dysfunction: A clinical perspective. Endocrine Reviews. 2001. pp. 36–52. doi: 10.1210/edrv.22.1.0417 11159815

9. Cooper ME, Vranes D, Youssef S, Stacker SA, Cox AJ, Rizkalla B, et al. Increased renal expression of vascular endothelial growth factor (VEGF) and its receptor VEGFR-2 in experimental diabetes. Diabetes. 1999;48: 2229–2239. doi: 10.2337/diabetes.48.11.2229 10535459

10. Braun L, Kardon T, Reisz-Porszasz ZS, Banhegyi G, Mandl J. The regulation of the induction of vascular endothelial growth factor at the onset of diabetes in spontaneously diabetic rats. Life Sci. 2001;69: 2533–42. doi: 10.1016/s0024-3205(01)01327-3 11693260

11. Hohenstein B, Hausknecht B, Boehmer K, Riess R, Brekken RA, Hugo CPM. Local VEGF activity but not VEGF expression is tightly regulated during diabetic nephropathy in man. Kidney Int. 2006;69: 1654–1661. 16541023

12. Schrijvers BF, Flyvbjerg A, De Vriese AS. The role of vascular endothelial growth factor (VEGF) in renal pathophysiology. Kidney International. 2004. pp. 2003–2017. 15149314

13. Wang W, Merrill MJ, Borchardt RT. Vascular endothelial growth factor affects permeability of brain microvessel endothelial cells in vitro. Am J Physiol. 1996;271: C1973–80. doi: 10.1152/ajpcell.1996.271.6.C1973 8997200

14. De Vriese AS, Tilton RG, Elger M, Stephan CC, Kriz W, Lameire NH. Antibodies against vascular endothelial growth factor improve early renal dysfunction in experimental diabetes. JAmSocNephrol. 2001;12: 993–1000.

15. Ku DD, Zaleski JK, Liu S, Brock TA. Vascular endothelial growth factor induces EDRF-dependent relaxation in coronary arteries. Am J Physiol. 1993;265: H586–92. doi: 10.1152/ajpheart.1993.265.2.H586 8368362

16. Nakagawa T, Kosugi T, Haneda M, Rivard CJ, Long DA. Abnormal angiogenesis in diabetic nephropathy. Diabetes. 2009;58: 1471–1478. doi: 10.2337/db09-0119 19564458

17. Barleon B, Sozzani S, Zhou D, Weich HA, Mantovani A, Marmé D. Migration of human monocytes in response to vascular endothelial growth factor (VEGF) is mediated via the VEGF receptor flt-1. Blood. 1996;87: 3336–3343. 8605350

18. Schrijvers BF, Flyvbjerg A, Tilton RG, Lameire NH, De Vriese AS. A neutralizing VEGF antibody prevents glomerular hypertrophy in a model of obese type 2 diabetes, the Zucker diabetic fatty rat. Nephrol Dial Transplant. 2006;21: 324–329. doi: 10.1093/ndt/gfi217 16249198

19. Lai X-X, Xu R-A, Yu-Ping L, Yang H. Risk of adverse events with bevacizumab addition to therapy in advanced non-small-cell lung cancer: a meta-analysis of randomized controlled trials. Onco Targets Ther. 2016;9: 2421–8. doi: 10.2147/OTT.S96156 27143937

20. Estrada CC, Maldonado A, Mallipattu SK. Therapeutic inhibition of VEGF signaling and associated nephrotoxicities. Journal of the American Society of Nephrology. 2019. doi: 10.1681/ASN.2018080853 30642877

21. Eremina V, Quaggin SE. Biology of anti-angiogenic therapy-induced thrombotic microangiopathy. Semin Nephrol. 2010;30: 582–590. doi: 10.1016/j.semnephrol.2010.09.006 21146123

22. Eremina V, Jefferson JA, Kowalewska J, Hochster H, Haas M, Weisstuch J, et al. VEGF Inhibition and Renal Thrombotic Microangiopathy. N Engl J Med. 2008;358: 1129–1136. doi: 10.1056/NEJMoa0707330 18337603

23. Zhang XY, Liu W, Wu SS, Jin JL, Li WH, Wang NL. Calcium dobesilate for diabetic retinopathy: a systematic review and meta-analysis. Sci China Life Sci. 2014;58: 101–107. doi: 10.1007/s11427-014-4792-1 25528255

24. Javadzadeh A, Ghorbanihaghjo A, Adl FH, Andalib D, Khojasteh-Jafari H, Ghabili K. Calcium dobesilate reduces endothelin-1 and high-sensitivity C-reactive protein serum levels in patients with diabetic retinopathy. Mol Vis. 2013;19: 62–68. 23335852

25. Zhou Y, Yuan J, Qi C, Shao X, Mou S, Ni Z. Calcium dobesilate may alleviate diabetes-induced endothelial dysfunction and inflammation. Mol Med Rep. 2017;16: 8635–8642. doi: 10.3892/mmr.2017.7740 29039485

26. Zhang X. Therapeutic effects of calcium dobesilate on diabetic nephropathy mediated through reduction of expression of PAI-1. Exp Ther Med. 2013;5: 295–299. doi: 10.3892/etm.2012.755 23251286

27. Fernández IS, Cuevas P, Angulo J, López-Navajas P, Canales-Mayordomo Á, González-Corrochano R, et al. Gentisic acid, a compound associated with plant defense and a metabolite of aspirin, heads a new class of in vivo fibroblast growth factor inhibitors. J Biol Chem. 2010;285: 11714–11729. doi: 10.1074/jbc.M109.064618 20145243

28. Angulo J, Cuevas P, Cuevas B, El Youssef M, Fernández A, Martínez-Salamanca E, et al. Diacetyloxyl derivatization of the fibroblast growth factor inhibitor dobesilate enhances its anti-inflammatory, anti-angiogenic and anti-tumoral activities. J Transl Med. 2015. doi: 10.1186/s12967-015-0413-4 25638171

29. Thamm K, Njau F, Van Slyke P, Dumont DJ, Park J-K, Haller H, et al. Pharmacological Tie2 activation in kidney transplantation. World J Transplant. 2016;6: 573. doi: 10.5500/wjt.v6.i3.573 27683636

30. Martins-Green M, Petreaca M, Yao M. Chapter 8 An Assay System for In Vitro Detection of Permeability in Human “Endothelium.” Methods in Enzymology. 2008. pp. 137–153. doi: 10.1016/S0076-6879(08)02008-9

31. Goncalves V, Gautier B, Garbay C, Vidal M, Inguimbert N. Development of a chemiluminescent screening assay for detection of vascular endothelial growth factor receptor 1 ligands. Anal Biochem. 2007;366: 108–110. doi: 10.1016/j.ab.2007.03.027 17482136

32. Angulo J, Peiró C, Romacho T, Fernández A, Cuevas B, González-Corrochano R, et al. Inhibition of vascular endothelial growth factor (VEGF)-induced endothelial proliferation, arterial relaxation, vascular permeability and angiogenesis by dobesilate. Eur J Pharmacol. 2011;667: 153–159. doi: 10.1016/j.ejphar.2011.06.015 21703259

33. Tornavaca O, Chia M, Dufton N, Almagro LO, Conway DE, Randi AM, et al. ZO-1 controls endothelial adherens junctions, cell-cell tension, angiogenesis, and barrier formation. J Cell Biol. 2015. doi: 10.1083/jcb.201404140 25753039

34. González-Mariscal L, Betanzos A, Ávila-Flores A. MAGUK proteins: Structure and role in the tight junction. Semin Cell Dev Biol. 2000. doi: 10.1006/scdb.2000.0178 10966866

35. Edward Conrad H, Edward Conrad H. Chapter 2 –Structures of Heparinoids. Heparin-Binding Proteins. 1998. pp. 7–60. doi: 10.1016/B978-012186060-8/50003-6

36. Ashikari-Hada S, Habuchi H, Kariya Y, Kimata K. Heparin Regulates Vascular Endothelial Growth Factor 165 -dependent Mitogenic Activity, Tube Formation, and Its Receptor Phosphorylation of Human Endothelial Cells. J Biol Chem. 2005;280: 31508–31515. doi: 10.1074/jbc.M414581200 16027124

37. Gitay-Goren H, Soker S, Vlodavsky I, Neufeld G. The binding of vascular endothelial growth factor to its receptors is dependent on cell surface-associated heparin-like molecules. J Biol Chem. 1992;267: 6093–6098. 1556117

38. Wijelath E, Namekata M, Murray J, Furuyashiki M, Zhang S, Coan D, et al. Multiple mechanisms for exogenous heparin modulation of vascular endothelial growth factor activity. J Cell Biochem. 2010;111: 461–468. doi: 10.1002/jcb.22727 20524207

39. Park M, Lee ST. The fourth immunoglobulin-like loop in the extracellular domain of FLT-1, a VEGF receptor, includes a major heparin-binding site. Biochem Biophys Res Commun. 1999;264: 730–734. doi: 10.1006/bbrc.1999.1580 10544000

40. Dougher AM, Wasserstrom H, Torley L, Shridaran L, Westdock P, Hileman RE, et al. Identification of a heparin binding peptide on the extracellular domain of the KDR VEGF receptor. Growth Factors. 1997;14: 257–268. doi: 10.3109/08977199709021524 9386990

41. Nishiguchi KM, Kataoka K, Kachi S, Komeima K, Terasaki H. Regulation of pathologic retinal angiogenesis in mice and inhibition of VEGF-VEGFR2 binding by soluble heparan sulfate. PLoS One. 2010;5. doi: 10.1371/journal.pone.0013493 20975989

42. Tessler S, Rockwell P, Hicklin D, Cohen T, Levi BZ, Witte L, et al. Heparin modulates the interaction of VEGF165 with soluble and cell associated flk-1 receptors. J Biol Chem. 1994.

43. Cuevas P, Arrazola JM. Dobesilate in the treatment of plaque psoriasis. Eur J Med Res. 2005;10: 373–376. 16183548

44. Cuevas P, Arrazola JM. Therapeutic response of rosacea to dobesilate. Eur J Med Res. 2005;10: 454–456. 16287608

45. Yang W, Yu X, Zhang Q, Lu Q, Wang J, Cui W, et al. Attenuation of streptozotocin-induced diabetic retinopathy with low molecular weight fucoidan via inhibition of vascular endothelial growth factor. Exp Eye Res. 2013;115: 96–105. doi: 10.1016/j.exer.2013.06.011 23810809

46. Haller H, Ji L, Stahl K, Bertram A, Menne J. Molecular Mechanisms and Treatment Strategies in Diabetic Nephropathy: New Avenues for Calcium Dobesilate—Free Radical Scavenger and Growth Factor Inhibition. Biomed Res Int. 2017;2017: 1–11. doi: 10.1155/2017/1909258 29082239

47. Holmes K, Roberts OL, Thomas AM, Cross MJ. Vascular endothelial growth factor receptor-2: Structure, function, intracellular signalling and therapeutic inhibition. Cellular Signalling. 2007. pp. 2003–2012. doi: 10.1016/j.cellsig.2007.05.013 17658244

48. Shibuya M. Vascular Endothelial Growth Factor (VEGF) and Its Receptor (VEGFR) Signaling in Angiogenesis: A Crucial Target for Anti- and Pro-Angiogenic Therapies. Genes and Cancer. 2011;2: 1097–1105. doi: 10.1177/1947601911423031 22866201

49. Cerezo AB, Hornedo-Ortega R, Álvarez-Fernández MA, Troncoso AM, García-Parrilla MC. Inhibition of VEGF-induced VEGFR-2 activation and HUVEC migration by melatonin and other bioactive indolic compounds. Nutrients. 2017;9. doi: 10.3390/nu9030249 28282869

50. Fish JE, Gutierrez MC, Dang LT, Khyzha N, Chen Z, Veitch S, et al. Dynamic regulation of VEGF-inducible genes by an ERK/ERG/p300 transcriptional network. Dev. 2017. doi: 10.1242/dev.146050 28536097

51. Cuevas P, Díaz-Gonzalez D, García-Martín-Córdova C, Sánchez I, Lozano RM, Giménez-Gallego G, et al. Dobesilate diminishes activation of the mitogen—activated protein kinase ERK1/2 in glioma cells. J Cell Mol Med. 2006;10: 225–230. doi: 10.1111/j.1582-4934.2006.tb00303.x 16563234

52. Leal EC, Martins J, Voabil P, Liberal J, Chiavaroli C, Bauer J, et al. Calcium dobesilate inhibits the alterations in tight junction proteins and leukocyte adhesion to retinal endothelial cells induced by diabetes. Diabetes. 2010;59: 2637–2645. doi: 10.2337/db09-1421 20627932

53. Solà-Adell C, Bogdanov P, Hernández C, Sampedro J, Valeri M, Garcia-Ramirez M, et al. Calcium Dobesilate Prevents Neurodegeneration and Vascular Leakage in Experimental Diabetes. Curr Eye Res. 2017;42: 1273–1286. doi: 10.1080/02713683.2017.1302591 28574750

54. Liu E, Morimoto M, Kitajima S, Koike T, Yu Y, Shiiki H, et al. Increased Expression of Vascular Endothelial Growth Factor in Kidney Leads to Progressive Impairment of Glomerular Functions. J Am Soc Nephrol. 2007;18: 2094–2104. doi: 10.1681/ASN.2006010075 17554151

55. Teran M, Nugent MA. Synergistic binding of vascular endothelial growth factor-a and its receptors to heparin selectively modulates complex affinity. J Biol Chem. 2015. doi: 10.1074/jbc.M114.627372 25979342

56. Leung D, Cachianes G, Kuang W, Goeddel D, Ferrara N. Vascular endothelial growth factor is a secreted angiogenic mitogen. Science (80-). 1989;246: 1306–1309. doi: 10.1126/science.2479986 2479986

57. Kiyan Y, Tkachuk S, Kurselis K, Shushakova N, Stahl K, Dawodu D, et al. Heparanase-2 protects from LPS-mediated endothelial injury by inhibiting TLR4 signalling. Sci Rep. 2019. doi: 10.1038/s41598-019-50068-5 31537875

58. Loges S, Mazzone M, Hohensinner P, Carmeliet P. Silencing or Fueling Metastasis with VEGF Inhibitors: Antiangiogenesis Revisited. Cancer Cell. 2009. pp. 167–170. doi: 10.1016/j.ccr.2009.02.007 19249675

59. Bogdanov P, Solà-Adell C, Hernández C, García-Ramírez M, Sampedro J, Simó-Servat O, et al. Calcium dobesilate prevents the oxidative stress and inflammation induced by diabetes in the retina of db/db mice. J Diabetes Complications. 2017;31: 1481–1490. doi: 10.1016/j.jdiacomp.2017.07.009 28847447

60. Zhou Y, Qi C, Li S, Shao X, Mou S, Ni Z. Diabetic nephropathy can be treated with calcium dobesilate by alleviating the chronic inflammatory state and improving endothelial cell function. Cell Physiol Biochem. 2018. doi: 10.1159/000495491 30476916

61. Solà-Adell C, Bogdanov P, Hernández C, Sampedro J, Valeri M, Garcia-Ramirez M, et al. Calcium Dobesilate Prevents Neurodegeneration and Vascular Leakage in Experimental Diabetes. Curr Eye Res. 2017;42: 1273–1286. doi: 10.1080/02713683.2017.1302591 28574750

62. Han K, Liu C, Shi X, Rao X. Effects of alprostadil combined with calcium dobesilate in patients with diabetic peripheral neuropathy. Neuroendocrinol Lett. 2018.

63. Tejerina T, Ruiz E. Calcium dobesilate: Pharmacology and future approaches. General Pharmacology. 1998. pp. 357–360. doi: 10.1016/S0306-3623(98)00040-8

64. Garay RP, Hannaert P, Chiavaroli C. Calcium dobesilate in the treatment of diabetic retinopathy. Treatments in Endocrinology. 2005. pp. 221–232. doi: 10.2165/00024677-200504040-00003 16053339

65. Tanabe K, Maeshima Y, Sato Y, Wada J. Antiangiogenic Therapy for Diabetic Nephropathy. Biomed Res Int. 2017;2017: 1–12. doi: 10.1155/2017/5724069 28835895

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