, Volume 20, Issue 1, pp 29–37 | Cite as

BIGH3 protein and macrophages in retinal endothelial cell apoptosis

  • Albert A. Mondragon
  • Brandi S. Betts-Obregon
  • Robert J. Moritz
  • Kalpana Parvathaneni
  • Mary M. Navarro
  • Hong Seok Kim
  • Chi Fung Lee
  • Richard G. LeBaron
  • Reto Asmis
  • Andrew T. TsinEmail author
Original Paper


Diabetes is a pandemic disease with a higher occurrence in minority populations. The molecular mechanism to initiate diabetes-associated retinal angiogenesis remains largely unknown. We propose an inflammatory pathway of diabetic retinopathy in which macrophages in the diabetic eye provide TGFβ to retinal endothelial cells (REC) in the retinal microvasculature. In response to TGFβ, REC synthesize and secrete a pro-apoptotic BIGH3 (TGFβ-Induced Gene Human Clone 3) protein, which acts in an autocrine loop to induce REC apoptosis. Rhesus monkey retinal endothelial cells (RhREC) were treated with dMCM (cell media of macrophages treated with high glucose and LDL) and assayed for apoptosis (TUNEL), BIGH3 mRNA (qPCR), and protein (Western blots) expressions. Cells were also treated with ΤGFβ1 and 2 for BIGH3 mRNA and protein expression. Inhibition assays were carried out using antibodies for TGFβ1 and for BIGH3 to block apoptosis and mRNA expression. BIGH3 in cultured RhREC cells were identified by immunohistochemistry (IHC). Distribution of BIGH3 and macrophages in the diabetic mouse retina was examined with IHC. RhRECs treated with dMCM or TGFβ showed a significant increase in apoptosis and BIGH3 protein expression. Recombinant BIGH3 added to RhREC culture medium led to a dose-dependent increase in apoptosis. Antibodies (Ab) directed against BIGH3 and TGFβ, as well as TGFβ receptor blocker resulted in a significant reduction in apoptosis induced by either dMCM, TGFβ or BIGH3. IHC showed that cultured RhREC constitutively expressed BIGH3. Macrophage and BIGH3 protein were co-localized to the inner retina of the diabetic mouse eye. Our results support a novel inflammatory pathway for diabetic retinopathy. This pathway is initiated by TGFβ released from macrophages, which promotes synthesis and release of BIGH3 protein by REC and REC apoptosis.


BIGH3 Macrophage Retinal endothelial cells Apoptosis Diabetic retinopathy TGFβ 



The authors thank the National Institute on Minority Health and Health Disparities (G12MD007591), the National Heart Lung and Blood Institute (R01HL70963) of the National Institutes of Health, and The San Antonio Life Sciences Institute Grant (SALSI) from Texas Higher Education Coordinating Board for their support. Authors thank Andrew S. Mendiola for technical and editorial assistance and Dr. Jeff Grigsby for insightful comments/suggestions.

Conflict of interest

The authors declare that they have no conflict of interest.



Supplementary material

10495_2014_1052_MOESM1_ESM.docx (723 kb)
Supplementary material 1 (DOCX 722 kb)


  1. 1.
    Romeo G, Liu WH, Asnaghi V, Kern TS, Lorenzi M (2002) Activation of nuclear factor-kappaB induced by diabetes and high glucose regulates a proapoptotic program in retinal pericytes. Diabetes 51:2241–2248PubMedCrossRefGoogle Scholar
  2. 2.
    Geraldes P, Hiraoka-Yamamoto J, Matsumoto M et al (2009) Activation of PKC-delta and SHP-1 by hyperglycemia causes vascular cell apoptosis and diabetic retinopathy. Nat Med 15:1298–1306PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    Han JH, Ha SW, Lee IK, Kim BW, Kim JG (2010) High glucose-induced apoptosis in bovine retinal pericytes is associated with transforming growth factor beta and betaIG-H3: betaIG-H3 induces apoptosis in retinal pericytes by releasing Arg-Gly-Asp peptides. Clin Exp Ophthalmol 38:620–628CrossRefGoogle Scholar
  4. 4.
    Busik JV, Mohr S, Grant MB (2008) Hyperglycemia-induced reactive oxygen species toxicity to endothelial cells is dependent on paracrine mediators. Diabetes 57:1952–1965PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Fadok VA, Bratton D, Konowal A, Freed PW, Westcott JY, Henson PM (1998) Macrophages that have ingested apoptotic cells in vitro inhibit proinflammatory cytokine production through autocrine/paracrine mechanisms involving TGF-beta, PGE2, and PAF. J Clin Investig 101:890–898PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    McDonald P, Fadock V, Bratton D, Henson PM (1999) Transcriptional and translational regulation of inflammatory mediator production by endogenous TGF-B in macrophages that have ingested apoptotic cells. J Immunol 163:6164–6172PubMedGoogle Scholar
  7. 7.
    Asmis R, Qiao M, Rossi RR, Cholewa J, Xu L, Asmis LM (2006) Adriamycin promotes macrophage dysfunction in mice. Free Radic Biol Med 41:165–174PubMedCrossRefGoogle Scholar
  8. 8.
    Skonier J, Bennett K, Rothwell V, Kosowski S, Plowman G, Wallace P, Edelhoff S, Disteche C, Neubauer M, Marquardt H et al (1994) beta ig-h3: a transforming growth factor-beta-responsive gene encoding a secreted protein that inhibits cell attachment in vitro and suppresses the growth of CHO cells in nude mice. DNA Cell Biol 13:571–584PubMedCrossRefGoogle Scholar
  9. 9.
    Porreca E, DiFebbo C, Mincione G, Reale M, Baccante G, Guglielmi MD, Cuccurullo F, Colletta G (1997) Increased transforming growth factor-beta production and gene expression by peripheral blood monocytes of hypertensive patients. Hypertension 30:134–139PubMedCrossRefGoogle Scholar
  10. 10.
    Zamilpa R, Rupaimoole R, Phelix CF et al (2009) C-terminal fragment of transforming growth factor beta-induced protein (TGFBIp) is required for apoptosis in human osteosarcoma cells. Matrix Biol 28:347–353PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Thapa N, Kang KB, Kim IS (2005) Beta ig-h3 mediates osteoblast adhesion and inhibits differentiation. Bone 36:232–242PubMedCrossRefGoogle Scholar
  12. 12.
    Lee BHBJ, Park RW, Kim JE, Park JY, Kim IS (2006) Betaig-h3 triggers signaling pathways mediating adhesion and migration of vascular smooth muscle cells through alphavbeta5 integrin. Exp Mol Med 38:153–161PubMedCrossRefGoogle Scholar
  13. 13.
    Kim JE, Kim SJ, Lee BH, Park RW, Kim KS, Kim IS. (2000) Identification of motifs for cell adhesion within the repeated domains of transforming growth factor-beta-induced gene, betaig-h3. J Biol Chem 40.Google Scholar
  14. 14.
    Kim JE, Jeong HW, Nam JO, Lee BH, Choi JY, Park RW, Park JY, Kim IS (2002) Identification of motifs in the fasciclin domains of the transforming growth factor-beta-induced matrix protein betaig-h3 that interact with the alphavbeta5 integrin. J Biol Chem 277:46159–46165PubMedCrossRefGoogle Scholar
  15. 15.
    Liu W, Ahmad SA, Reinmuth N, Shaheen RM, Jung YD, Fan F, Ellis LM (2000) Endothelial cell survival and apoptosis in the tumor vasculature. Apoptosis 5:323–328PubMedCrossRefGoogle Scholar
  16. 16.
    Nam JO, Kim JE, Jeong HW, Lee SJ, Lee BH, Choi JY, Park RW, Kim IS (2003) Identification of the alphavbeta3 integrin-interacting motif of betaig-h3 and its anti-angiogenic effect. J Biol Chem 278:25902–25909PubMedCrossRefGoogle Scholar
  17. 17.
    Thapa N, LeeBH Kim IS (2007) TGFBIp/betaig-h3 protein: a versatile matrix molecule induced by TGF-beta. Int J Biochem Cell Biol 39:2183–2194PubMedCrossRefGoogle Scholar
  18. 18.
    Han B, Qi S, Hu B, Luo H, Wu J (2011) TGF-beta i promotes islet beta-cell function and regeneration. J Immunol 186:5833–5844PubMedCrossRefGoogle Scholar
  19. 19.
    Han B, Luo H, Raelson J, Huang J, Li Y, Temblay J, Hu B, Qi S, Wu J (2014) TGFBI (betaIG-H3) is a diabetes risk gene based on mouse and human genetic studies. Hum Mol Genet 23:4597–4611PubMedCrossRefGoogle Scholar
  20. 20.
    Grigsby JG, Parvathaneni K, Almanza MA, Botello AM, Mondragon AA, Allen DM, Tsin AT (2011) Effects of tamoxifen versus raloxifene on retinal capillary endothelial cell proliferation. J Ocul Pharmacol Ther 27:225–233PubMedCentralPubMedCrossRefGoogle Scholar
  21. 21.
    Wintergerst ES, Jelk J, Asmis R (1998) Differential expression of CD14, CD36 and the LDL receptor on human monocyte-derived macrophages. A novel cell culture system to study macrophage differentiation and heterogeneity. Histochem Cell Biol 110:231–241PubMedCrossRefGoogle Scholar
  22. 22.
    Qiao M, Zhao Q, Lee CF, Tannock LR, Smart EJ, LeBaron RG, Phelix CF, Rangel Y, Asmis R (2009) Thiol oxidative stress induced by metabolic disorders amplifies macrophage chemotactic responses and accelerates atherogenesis and kidney injury in LDL receptor-deficient mice. Arterioscler Thromb Vasc Biol 29:1779–1786PubMedCentralPubMedCrossRefGoogle Scholar
  23. 23.
    Villanza-Espinoza E, Hatch A, Tsin ATC (2006) Effect of light exposure on accumulation depletion of retinyl ester in the chicken retina. Exp Eye Res 83:871–876CrossRefGoogle Scholar
  24. 24.
    Ferguson JW, Thoma BS, Mikesh MF, Kramer RH, Bennett KL, Purchio A, Bellard BJ, LeBaron RG (2003) The extracellular matrix protein betaIG-H3 is expressed at myotendinous junctions and supports muscle cell adhesion. Cell Tissue Res 313:93–105PubMedCrossRefGoogle Scholar
  25. 25.
    Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:402–408PubMedCrossRefGoogle Scholar
  26. 26.
    LeBaron RG, Bezverkov KI, Zimber MP, Pavelec R, Skonier J, Purchio AF (1995) BIG-H3, a novel secretory protein inducible by transforming growth factor-β, is present in normal skin and promotes the adhesion and spreading of dermal fibroblasts in vitro. J Invest Dermatol 104:844–849PubMedCrossRefGoogle Scholar
  27. 27.
    Omri S, Behar-Cohen F, de Kozak Y, Sennlaub F, Verissimo LM, Jonet L, Savoldelli M, Omri B, Crisanti P (2011) Microglia/macrophages migrate through retinal epithelium barrier by a transcellular route in diabetic retinopathy: role of PKCζ in the Goto Kakizaki rat model. Am J Pathol 179:942–953PubMedCentralPubMedCrossRefGoogle Scholar
  28. 28.
    Ferguson JW, Mikesh MF, Wheeler EF, LeBaron RG (2003) Developmental expression patterns of beta-ig (βIG-H3) and its function as a cell adhesion protein. Mech Dev 120:851–864PubMedCrossRefGoogle Scholar
  29. 29.
    Ochiai Y, Ochiai H (2002) Higher concentration of transforming growth factor-beta in aqueous humor of glaucomatous eyes and diabetic eyes. Jpn J Ophthalmol 46:249–253PubMedCrossRefGoogle Scholar
  30. 30.
    Hirase K, Ikeda T, Sotozono C, Nishida K, Sawa H, Kinoshita S (1998) Transforming growth factor beta2 in the vitreous in proliferative diabetic retinopathy. Arch Ophthalmol 116:738–741PubMedCrossRefGoogle Scholar
  31. 31.
    Constam DB, Philipp J, Malipiero UV, ten Dijke P, Schachner M, Fontana A (1992) Differential expression of transforming growth factor-beta 1, -beta 2, and -beta 3 by glioblastoma cells, astrocytes, and microglia. J Immunol 148:1404–1410PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Albert A. Mondragon
    • 1
  • Brandi S. Betts-Obregon
    • 1
  • Robert J. Moritz
    • 1
  • Kalpana Parvathaneni
    • 1
  • Mary M. Navarro
    • 1
  • Hong Seok Kim
    • 2
  • Chi Fung Lee
    • 2
  • Richard G. LeBaron
    • 1
  • Reto Asmis
    • 2
  • Andrew T. Tsin
    • 1
    • 2
    Email author
  1. 1.Department of BiologyThe University of Texas at San AntonioSan AntonioUSA
  2. 2.The University of Texas Health Science Center at San AntonioSan AntonioUSA

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