Skip to main content
SpringerLink
Log in
Book cover

CCN Proteins pp 543–556Cite as

Analysis of CCN Protein Expression and Activities in Vasoproliferative Retinopathies

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1489))

Abstract

The retina is a complex neurovascular structure that conveys light/visual image through the optic nerve to the visual cortex of the brain. Neuronal and vascular activities in the retina are physically and functionally intertwined, and vascular alterations are consequential to the proper function of the entire visual system. In particular, alteration of the structure and barrier function of the retinal vasculature is commonly associated with the development of vasoproliferative ischemic retinopathy, a set of clinically well-defined chronic ocular microvascular complications causing blindness in all age groups. Experimentally, the retinal tissue provides researchers with a convenient, easily accessible, and directly observable model suitable to investigate whether and how newly identified genes regulate vascular development and regeneration. The six mammalian CCN gene-encoded proteins are part of an extracellular network of bioactive molecules that regulate various aspects of organ system development and diseases. Whether and how these molecules regulate the fundamental aspects of blood vessel development and pathology and subsequently the neurovascular link in the retina are open-ended questions. Sophisticated methods have been developed to gain insight into the pathogenesis of retinal vasculopathy. This chapter describes several useful methodologies and animal models to investigate the regulation and potential relevance of the CCN proteins in vasoproliferative diseases of the retina.

This is a preview of subscription content, log in via an institution.

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Parr JC (1980) The peculiar circulation. A review of retinal blood supply. Trans Ophthalmol Soc N Z 32:40–48

    CAS  PubMed  Google Scholar 

  2. Wechsler-Reya RJ, Barres BA (1997) Retinal development: communication helps you see the light. Curr Biol 7:R433–R436

    Article  CAS  PubMed  Google Scholar 

  3. Henkind P (1981) Retinal blood vessels. Neovascularisation, collaterals, and shunts. Trans Ophthalmol Soc N Z 33:46–50

    CAS  PubMed  Google Scholar 

  4. Kubota S, Takigawa M (2013) The CCN family acting throughout the body: recent research developments. Biomol Concepts 4:477–494

    Article  CAS  PubMed  Google Scholar 

  5. Brigstock DR (2003) The CCN family: a new stimulus package. J Endocrinol 178:169–175

    Article  CAS  PubMed  Google Scholar 

  6. Krupska I, Bruford EA, Chaqour B (2015) Eyeing the Cyr61/CTGF/NOV (CCN) group of genes in development and diseases: highlights of their structural likenesses and functional dissimilarities. Hum Genomics 9:24

    Article  PubMed  PubMed Central  Google Scholar 

  7. Lau LF (2011) CCN1/CYR61: the very model of a modern matricellular protein. Cell Mol Life Sci 68:3149–3163

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Yan L, Chaqour B (2013) Cysteine-rich protein 61 (CCN1) and connective tissue growth factor (CCN2) at the crosshairs of ocular neovascular and fibrovascular disease therapy. J Cell Commun Signal 7:253–263

    Article  PubMed  PubMed Central  Google Scholar 

  9. Klaassen I, van Geest RJ, Kuiper EJ, van Noorden CJ, Schlingemann RO (2015) The role of CTGF in diabetic retinopathy. Exp Eye Res 133:37–48

    Article  CAS  PubMed  Google Scholar 

  10. Chintala H, Krupska I, Yan LL, Lau L, Grant M, Chaqour B (2015) The matricellular protein CCN1 controls retinal angiogenesis by targeting VEGF, Src homology 2 domain phosphatase-1 and Notch signaling. Development 142:2364–2374

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Karaca M, Maechler P (2014) Development of mice with brain-specific deletion of floxed glud1 (glutamate dehydrogenase 1) using cre recombinase driven by the nestin promoter. Neurochem Res 39:456–459

    Article  CAS  PubMed  Google Scholar 

  12. Wang X (2009) Cre transgenic mouse lines. Methods Mol Biol 561:265–273

    Article  CAS  PubMed  Google Scholar 

  13. Campochiaro PA (2000) Retinal and choroidal neovascularization. J Cell Physiol 184:301–310

    Article  CAS  PubMed  Google Scholar 

  14. Gong S, Zheng C, Doughty ML, Losos K, Didkovsky N, Schambra UB, Nowak NJ, Joyner A, Leblanc G, Hatten ME et al (2003) A gene expression atlas of the central nervous system based on bacterial artificial chromosomes. Nature 425:917–925

    Article  CAS  PubMed  Google Scholar 

  15. Gibson DJ, Pi L, Sriram S, Mao C, Petersen BE, Scott EW, Leask A, Schultz GS (2014) Conditional knockout of CTGF affects corneal wound healing. Invest Ophthalmol Vis Sci 55:2062–2070

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Denton CP, Khan K, Hoyles RK, Shiwen X, Leoni P, Chen Y, Eastwood M, Abraham DJ (2009) Inducible lineage-specific deletion of TbetaRII in fibroblasts defines a pivotal regulatory role during adult skin wound healing. J Invest Dermatol 129:194–204

    Article  CAS  PubMed  Google Scholar 

  17. Kim KH, Chen CC, Monzon RI, Lau LF (2013) Matricellular protein CCN1 promotes regression of liver fibrosis through induction of cellular senescence in hepatic myofibroblasts. Mol Cell Biol 33:2078–2090

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Liu S, Leask A (2011) CCN2 is not required for skin development. J Cell Commun Signal 5:179–182

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Fontes MS, Kessler EL, van Stuijvenberg L, Brans MA, Falke LL, Kok B, Leask A, van Rijen HV, Vos MA, Goldschmeding R et al (2015) CTGF knockout does not affect cardiac hypertrophy and fibrosis formation upon chronic pressure overload. J Mol Cell Cardiol 88:82–90

    Article  CAS  PubMed  Google Scholar 

  20. Smith LE, Wesolowski E, McLellan A, Kostyk SK, D'Amato R, Sullivan R, D'Amore PA (1994) Oxygen-induced retinopathy in the mouse. Invest Ophthalmol Vis Sci 35:101–111

    CAS  PubMed  Google Scholar 

  21. Liang XL, Li J, Chen F, Ding XY, Yang XX, Long LX (2012) A comparing study of quantitative staining techniques for retinal neovascularization in a mouse model of oxygen-induced retinopathy. Int J Ophthalmol 5:1–6

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Stone J, Itin A, Alon T, Pe'er J, Gnessin H, Chan-Ling T, Keshet E (1995) Development of retinal vasculature is mediated by hypoxia-induced vascular endothelial growth factor (VEGF) expression by neuroglia. J Neurosci 15:4738–4747

    CAS  PubMed  Google Scholar 

  23. Scott A, Fruttiger M (2010) Oxygen-induced retinopathy: a model for vascular pathology in the retina. Eye (Lond) 24:416–421

    Article  CAS  Google Scholar 

  24. Chintala H, Liu H, Parmar R, Kamalska M, Kim YJ, Lovett D, Grant MB, Chaqour B (2012) Connective tissue growth factor regulates retinal neovascularization through p53 protein-dependent transactivation of the matrix metalloproteinase (MMP)-2 gene. J Biol Chem 287:40570–40585

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Hasan A, Pokeza N, Shaw L, Lee HS, Lazzaro D, Chintala H, Rosenbaum D, Grant MB, Chaqour B (2011) The matricellular protein cysteine-rich protein 61 (CCN1/Cyr61) enhances physiological adaptation of retinal vessels and reduces pathological neovascularization associated with ischemic retinopathy. J Biol Chem 286:9542–9554

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Yi X, Ogata N, Komada M, Yamamoto C, Takahashi K, Omori K, Uyama M (1997) Vascular endothelial growth factor expression in choroidal neovascularization in rats. Graefes Arch Clin Exp Ophthalmol 235:313–319

    Article  CAS  PubMed  Google Scholar 

  27. Booij JC, Baas DC, Beisekeeva J, Gorgels TG, Bergen AA (2010) The dynamic nature of Bruch's membrane. Prog Retin Eye Res 29:1–18

    Article  CAS  PubMed  Google Scholar 

  28. Caballero S, Yang R, Grant MB, Chaqour B (2011) Selective blockade of cytoskeletal actin remodeling reduces experimental choroidal neovascularization. Invest Ophthalmol Vis Sci 52:2490–2496

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Singh SR, Grossniklaus HE, Kang SJ, Edelhauser HF, Ambati BK, Kompella UB (2009) Intravenous transferrin, RGD peptide and dual-targeted nanoparticles enhance anti-VEGF intraceptor gene delivery to laser-induced CNV. Gene Ther 16:645–659

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Eleazu CO, Eleazu KC, Chukwuma S, Essien UN (2013) Review of the mechanism of cell death resulting from streptozotocin challenge in experimental animals, its practical use and potential risk to humans. J Diabetes Metab Disord 12:60

    Article  PubMed  PubMed Central  Google Scholar 

  31. Sullivan KA, Hayes JM, Wiggin TD, Backus C, Su Oh S, Lentz SI, Brosius F 3rd, Feldman EL (2007) Mouse models of diabetic neuropathy. Neurobiol Dis 28:276–285

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Szkudelski T (2001) The mechanism of alloxan and streptozotocin action in B cells of the rat pancreas. Physiol Res 50:537–546

    CAS  PubMed  Google Scholar 

  33. Shin ES, Sorenson CM, Sheibani N (2014) Diabetes and retinal vascular dysfunction. J Ophthalmic Vis Res 9:362–373

    PubMed  PubMed Central  Google Scholar 

  34. Hu B, Zhang Y, Zeng Q, Han Q, Zhang L, Liu M, Li X (2014) Intravitreal injection of ranibizumab and CTGF shRNA improves retinal gene expression and microvessel ultrastructure in a rodent model of diabetes. Int J Mol Sci 15:1606–1624

    Article  PubMed  PubMed Central  Google Scholar 

  35. Van Geest RJ, Leeuwis JW, Dendooven A, Pfister F, Bosch K, Hoeben KA, Vogels IM, Van der Giezen DM, Dietrich N, Hammes HP et al (2014) Connective tissue growth factor is involved in structural retinal vascular changes in long-term experimental diabetes. J Histochem Cytochem 62:109–118

    Article  PubMed  PubMed Central  Google Scholar 

  36. Prabhakar P, Zhang H, Chen D, Faber JE (2015) Genetic variation in retinal vascular patterning predicts variation in pial collateral extent and stroke severity. Angiogenesis 18:97–114

    Article  CAS  PubMed  Google Scholar 

  37. Grossniklaus HE, Kang SJ, Berglin L (2010) Animal models of choroidal and retinal neovascularization. Prog Retin Eye Res 29:500–519

    Article  PubMed  PubMed Central  Google Scholar 

  38. Weerasekera LY, Balmer LA, Ram R, Morahan G (2015) Characterization of retinal vascular and neural damage in a novel model of diabetic retinopathy. Invest Ophthalmol Vis Sci 56:3721–3730

    Article  CAS  PubMed  Google Scholar 

  39. Nerup J, Mandrup-Poulsen T, Helqvist S, Andersen HU, Pociot F, Reimers JI, Cuartero BG, Karlsen AE, Bjerre U, Lorenzen T (1994) On the pathogenesis of IDDM. Diabetologia 37(Suppl 2):S82–S89

    Article  PubMed  Google Scholar 

  40. Yang Y, Santamaria P (2006) Lessons on autoimmune diabetes from animal models. Clin Sci (Lond) 110:627–639

    Article  CAS  Google Scholar 

  41. Yoshioka M, Kayo T, Ikeda T, Koizumi A (1997) A novel locus, Mody4, distal to D7Mit189 on chromosome 7 determines early-onset NIDDM in nonobese C57BL/6 (Akita) mutant mice. Diabetes 46:887–894

    Article  CAS  PubMed  Google Scholar 

  42. Park CJ, Zhao Z, Glidewell-Kenney C, Lazic M, Chambon P, Krust A, Weiss J, Clegg DJ, Dunaif A, Jameson JL et al (2011) Genetic rescue of nonclassical ERalpha signaling normalizes energy balance in obese Eralpha-null mutant mice. J Clin Invest 121:604–612

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Clausen BE, Burkhardt C, Reith W, Renkawitz R, Forster I (1999) Conditional gene targeting in macrophages and granulocytes using LysMcre mice. Transgenic Res 8:265–277

    Article  CAS  PubMed  Google Scholar 

  44. Sorensen I, Adams RH, Gossler A (2009) DLL1-mediated Notch activation regulates endothelial identity in mouse fetal arteries. Blood 113:5680–5688

    Article  PubMed  Google Scholar 

  45. Wirth A, Benyo Z, Lukasova M, Leutgeb B, Wettschureck N, Gorbey S, Orsy P, Horvath B, Maser-Gluth C, Greiner E et al (2008) G12-G13-LARG-mediated signaling in vascular smooth muscle is required for salt-induced hypertension. Nat Med 14:64–68

    Article  CAS  PubMed  Google Scholar 

  46. Ganat YM, Silbereis J, Cave C, Ngu H, Anderson GM, Ohkubo Y, Ment LR, Vaccarino FM (2006) Early postnatal astroglial cells produce multilineage precursors and neural stem cells in vivo. J Neurosci 26:8609–8621

    Article  CAS  PubMed  Google Scholar 

  47. Dhillon H, Zigman JM, Ye C, Lee CE, McGovern RA, Tang V, Kenny CD, Christiansen LM, White RD, Edelstein EA et al (2006) Leptin directly activates SF1 neurons in the VMH, and this action by leptin is required for normal body-weight homeostasis. Neuron 49:191–203

    Article  CAS  PubMed  Google Scholar 

  48. Zhang XM, Ng AH, Tanner JA, Wu WT, Copeland NG, Jenkins NA, Huang JD (2004) Highly restricted expression of Cre recombinase in cerebellar Purkinje cells. Genesis 40:45–51

    Article  PubMed  Google Scholar 

  49. Roesch K, Jadhav AP, Trimarchi JM, Stadler MB, Roska B, Sun BB, Cepko CL (2008) The transcriptome of retinal Muller glial cells. J Comp Neurol 509:225–238

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Nakazawa K, Quirk MC, Chitwood RA, Watanabe M, Yeckel MF, Sun LD, Kato A, Carr CA, Johnston D, Wilson MA et al (2002) Requirement for hippocampal CA3 NMDA receptors in associative memory recall. Science 297:211–218

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Mori M, Gargowitsch L, Bornert JM, Garnier JM, Mark M, Chambon P, Metzger D (2012) Temporally controlled targeted somatic mutagenesis in mouse eye pigment epithelium. Genesis 50:828–832

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported in whole by grants from the National Eye Institute of the National Institutes of Health, EY022091 (to B.C.).

Author information

Authors and Affiliations

  1. Department of Cell Biology, SUNY Downstate Medical Center, College of Medicine and SUNY Eye Institute, State University of New York, Brooklyn, NY, USA

    Sangmi Lee, Menna Elaskandrany, Afruja Ahad & Brahim Chaqour

  2. Department of Ophthalmology, SUNY Downstate Medical Center, College of Medicine and SUNY Eye Institute, State University of New York, 450 Clarkson Avenue, MSC 5, Brooklyn, NY, 11203, USA

    Sangmi Lee, Menna Elaskandrany, Afruja Ahad & Brahim Chaqour

Authors
  1. Sangmi Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Menna Elaskandrany
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Afruja Ahad
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Brahim Chaqour
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Brahim Chaqour .

Editor information

Editors and Affiliations

  1. Advanced Research Center for Oral and Craniofacial Sciences, Okayama University Dental School/Graduate School of Medicine Dentistry and Pharmaceutical Sciences, Okayama, Japan

    Masaharu Takigawa

Rights and permissions

Reprints and permissions

Copyright information

© 2017 Springer Science+Business Media New York

About this protocol

Cite this protocol

Lee, S., Elaskandrany, M., Ahad, A., Chaqour, B. (2017). Analysis of CCN Protein Expression and Activities in Vasoproliferative Retinopathies. In: Takigawa, M. (eds) CCN Proteins. Methods in Molecular Biology, vol 1489. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6430-7_46

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-6430-7_46

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6428-4

  • Online ISBN: 978-1-4939-6430-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation