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Extracellular carbonic anhydrase mediates hemorrhagic retinal and cerebral vascular permeability through prekallikrein activation


Excessive retinal vascular permeability contributes to the pathogenesis of proliferative diabetic retinopathy and diabetic macular edema, leading causes of vision loss in working-age adults. Using mass spectroscopy–based proteomics, we detected 117 proteins in human vitreous and elevated levels of extracellular carbonic anhydrase-I (CA-I) in vitreous from individuals with diabetic retinopathy, suggesting that retinal hemorrhage and erythrocyte lysis contribute to the diabetic vitreous proteome. Intravitreous injection of CA-I in rats increased retinal vessel leakage and caused intraretinal edema. CA-I–induced alkalinization of vitreous increased kallikrein activity and its generation of factor XIIa, revealing a new pathway for contact system activation. CA-I–induced retinal edema was decreased by complement 1 inhibitor, neutralizing antibody to prekallikrein and bradykinin receptor antagonism. Subdural infusion of CA-I in rats induced cerebral vascular permeability, suggesting that extracellular CA-I could have broad relevance to neurovascular edema. Inhibition of extracellular CA-I and kallikrein-mediated innate inflammation could provide new therapeutic opportunities for the treatment of hemorrhage-induced retinal and cerebral edema.

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Figure 1: Western blot analysis of proteins from vitreous samples.
Figure 2: Intravitreal CA-I induces retinal vascular permeability.
Figure 3: Intravitreal CA-I induces retinal vascular permeability (RVP) via the kallikrein-bradykinin receptor pathway.
Figure 4: Effect of CA-I on vitreous pH and effects of pH on RVP and activity of the components of the kallikrein system.
Figure 5: CA-I increases blood-brain barrier permeability to Evans blue dye.

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  1. Kempen, J.H. et al. The prevalence of diabetic retinopathy among adults in the United States. Arch. Ophthalmol. 122, 552–563 (2004).

    Article  Google Scholar 

  2. Williams, R. et al. Epidemiology of diabetic retinopathy and macular oedema: a systematic review. Eye 18, 963–983 (2004).

    Article  CAS  Google Scholar 

  3. The Diabetes Control and Complications Trial Research Group. The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. N. Engl. J. Med. 329, 977–986 (1993).

  4. Stratton, I.M. et al. Association of glycaemia with macrovascular and microvascular complications of type 2 diabetes (UKPDS 35): prospective observational study. Br. Med. J. 321, 405–412 (2000).

    Article  CAS  Google Scholar 

  5. UK Prospective Diabetes Study Group. Tight blood pressure control and risk of macrovascular and microvascular complications in type 2 diabetes: UKPDS 38. Br. Med. J. 317, 703–713 (1998).

  6. Klein, R., Klein, B.E., Moss, S.E. & Cruickshanks, K.J. The Wisconsin epidemiologic study of diabetic retinopathy: XVII. The 14-year incidence and progression of diabetic retinopathy and associated risk factors in type 1 diabetes. Ophthalmology 105, 1801–1815 (1998).

    Article  CAS  Google Scholar 

  7. Ferris, F.L., III, Davis, M.D. & Aiello, L.M. Treatment of diabetic retinopathy. N. Engl. J. Med. 341, 667–678 (1999).

    Article  Google Scholar 

  8. Klein, R., Klein, B.E., Moss, S.E., Davis, M.D. & DeMets, D.L. The Wisconsin epidemiologic study of diabetic retinopathy. IV. Diabetic macular edema. Ophthalmology 91, 1464–1474 (1984).

    Article  CAS  Google Scholar 

  9. Knudsen, S.T. et al. Macular edema reflects generalized vascular hyperpermeability in type 2 diabetic patients with retinopathy. Diabetes Care 25, 2328–2334 (2002).

    Article  Google Scholar 

  10. Krogsaa, B., Lund-Andersen, H., Mehlsen, J., Sestoft, L. & Larsen, J. The blood-retinal barrier permeability in diabetic patients. Acta Ophthalmol. (Copenh.) 59, 689–694 (1981).

    Article  CAS  Google Scholar 

  11. Plehwe, W.E., Sleightholm, M.A. & Kohner, E.M. Does vitreous fluorophotometry reflect severity of early diabetic retinopathy? Br. J. Ophthalmol. 73, 255–260 (1989).

    Article  CAS  Google Scholar 

  12. Lattanzio, R. et al. Macular thickness measured by optical coherence tomography (OCT) in diabetic patients. Eur. J. Ophthalmol. 12, 482–487 (2002).

    Article  CAS  Google Scholar 

  13. Aiello, L.P. et al. Vascular endothelial growth factor in ocular fluid of patients with diabetic retinopathy and other retinal disorders. N. Engl. J. Med. 331, 1480–1487 (1994).

    Article  CAS  Google Scholar 

  14. Ogata, N., Nishikawa, M., Nishimura, T., Mitsuma, Y. & Matsumura, M. Unbalanced vitreous levels of pigment epithelium-derived factor and vascular endothelial growth factor in diabetic retinopathy. Am. J. Ophthalmol. 134, 348–353 (2002).

    Article  CAS  Google Scholar 

  15. Gragoudas, E.S., Adamis, A.P., Cunningham, E.T., Jr., Feinsod, M & Guyer, D.R. Pegaptanib for neovascular age-related macular degeneration. N. Engl. J. Med. 351, 2805–2816 (2004).

    Article  CAS  Google Scholar 

  16. Srinivas, S.P., Ong, A., Zhai, C.B. & Bonanno, J.A. Inhibition of carbonic anhydrase activity in cultured bovine corneal endothelial cells by dorzolamide. Invest. Ophthalmol. Vis. Sci. 43, 3273–3278 (2002).

    PubMed  Google Scholar 

  17. Wolfensberger, T.J. et al. Membrane-bound carbonic anhydrase in human retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci. 35, 3401–3407 (1994).

    CAS  PubMed  Google Scholar 

  18. Giusti, C., Forte, R., Vingolo, E.M. & Gargiulo, P. Is acetazolamide effective in the treatment of diabetic macular edema? A pilot study. Int. Ophthalmol. 24, 79–88 (2001).

    Article  CAS  Google Scholar 

  19. Miyamoto, K. et al. Prevention of leukostasis and vascular leakage in streptozotocin-induced diabetic retinopathy via intercellular adhesion molecule-1 inhibition. Proc. Natl. Acad. Sci. USA 96, 10836–10841 (1999).

    Article  CAS  Google Scholar 

  20. Wojtkowski, M. et al. Three dimensional retinal imaging with high-speed, ultrahigh resolution, optical coherence tomography. Ophthalmology 112, 1734–1746 (2005).

    Article  Google Scholar 

  21. Anderson, N.L. et al. The human plasma proteome: a nonredundant list developed by combination of four separate sources. Mol. Cell. Proteomics 3, 311–326 (2004).

    Article  CAS  Google Scholar 

  22. Wistrand, P.J., Schenholm, M. & Lonnerholm, G. Carbonic anhydrase isoenzymes CA I and CA II in the human eye. Invest. Ophthalmol. Vis. Sci. 27, 419–428 (1986).

    CAS  PubMed  Google Scholar 

  23. Carugati, A., Pappalardo, E., Zingale, L.C. & Cicardi, M. C1-inhibitor deficiency and angioedema. Mol. Immunol. 38, 161–173 (2001).

    Article  CAS  Google Scholar 

  24. Han, E.D., MacFarlane, R.C., Mulligan, A.N., Scafidi, J. & Davis, A.E., III. Increased vascular permeability in C1 inhibitor-deficient mice mediated by the bradykinin type 2 receptor. J. Clin. Invest. 109, 1057–1063 (2002).

    Article  CAS  Google Scholar 

  25. Shariat-Madar, Z., Mahdi, F. & Schmaier, A.H. Identification and characterization of prolylcarboxypeptidase as an endothelial cell prekallikrein activator. J. Biol. Chem. 277, 17962–17969 (2002).

    Article  CAS  Google Scholar 

  26. Houle, S., Molinaro, G., Adam, A. & Marceau, F. Tissue kallikrein actions at the rabbit natural or recombinant kinin B2 receptors. Hypertension 41, 611–617 (2003).

    Article  CAS  Google Scholar 

  27. Veloso, D., Silver, L.D., Hahn, S. & Colman, R.W. A monoclonal anti-human plasma prekallikrein antibody that inhibits activation of prekallikrein by factor XIIa on a surface. Blood 70, 1053–1062 (1987).

    CAS  PubMed  Google Scholar 

  28. Yang, Z. et al. Mutant carbonic anhydrase 4 impairs pH regulation and causes retinal photoreceptor degeneration. Hum. Mol. Genet. 14, 255–265 (2005).

    Article  CAS  Google Scholar 

  29. Xi, G. et al. Mechanisms of edema formation after intracerebral hemorrhage: effects of extravasated red blood cells on blood flow and blood-brain barrier integrity. Stroke 32, 2932–2938 (2001).

    Article  CAS  Google Scholar 

  30. Qureshi, A.I. et al. Spontaneous intracerebral hemorrhage. N. Engl. J. Med. 344, 1450–1460 (2001).

    Article  CAS  Google Scholar 

  31. Yamane, K. et al. Proteome analysis of human vitreous proteins. Mol. Cell. Proteomics 2, 1177–1187 (2003).

    Article  CAS  Google Scholar 

  32. Nakanishi, T., Koyama, R., Ikeda, T. & Shimizu, A. Catalogue of soluble proteins in the human vitreous humor: comparison between diabetic retinopathy and macular hole. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 776, 89–100 (2002).

    Article  CAS  Google Scholar 

  33. Ouchi, M., West, K., Crabb, J.W., Kinoshita, S. & Kamei, M. Proteomic analysis of vitreous from diabetic macular edema. Exp. Eye Res. 81, 176–182 (2005).

    Article  CAS  Google Scholar 

  34. Henricsson, M., Sellman, A., Tyrberg, M. & Groop, L. Progression to proliferative retinopathy and macular oedema requiring treatment. Assessment of the alternative classification of the Wisconsin Study. Acta Ophthalmol. Scand. 77, 218–223 (1999).

    Article  CAS  Google Scholar 

  35. Svastova, E. et al. Hypoxia activates the capacity of tumor-associated carbonic anhydrase IX to acidify extracellular pH. FEBS Lett. 577, 439–445 (2004).

    Article  CAS  Google Scholar 

  36. Iwaki, T. & Castellino, F.J. Plasma levels of bradykinin are suppressed in factor XII-deficient mice. Thromb. Haemost. 95, 1003–1010 (2006).

    Article  CAS  Google Scholar 

  37. Shariat-Madar, Z., Mahdi, F. & Schmaier, A.H. Recombinant prolylcarboxypeptidase activates plasma prekallikrein. Blood 103, 4554–4561 (2004).

    Article  CAS  Google Scholar 

  38. Joseph, K. & Kaplan, A.P. Formation of bradykinin: a major contributor to the innate inflammatory response. Adv. Immunol. 86, 159–208 (2005).

    Article  CAS  Google Scholar 

  39. Nagelhus, E.A. et al. Carbonic anhydrase XIV is enriched in specific membrane domains of retinal pigment epithelium, Muller cells, and astrocytes. Proc. Natl. Acad. Sci. USA 102, 8030–8035 (2005).

    Article  CAS  Google Scholar 

  40. Rassam, S.M., Patel, V. & Kohner, E.M. The effect of acetazolamide on the retinal circulation. Eye 7, 697–702 (1993).

    Article  Google Scholar 

  41. Vorstrup, S., Henriksen, L. & Paulson, O.B. Effect of acetazolamide on cerebral blood flow and cerebral metabolic rate for oxygen. J. Clin. Invest. 74, 1634–1639 (1984).

    Article  CAS  Google Scholar 

  42. Stefansson, E. et al. Optic nerve oxygen tension in pigs and the effect of carbonic anhydrase inhibitors. Invest. Ophthalmol. Vis. Sci. 40, 2756–2761 (1999).

    CAS  PubMed  Google Scholar 

  43. Weber, A. et al. Unexpected nanomolar inhibition of carbonic anhydrase by COX-2-selective celecoxib: new pharmacological opportunities due to related binding site recognition. J. Med. Chem. 47, 550–557 (2004).

    Article  CAS  Google Scholar 

  44. van Doorn, M.B. et al. A phase I study of recombinant human C1 inhibitor in asymptomatic patients with hereditary angioedema. J. Allergy Clin. Immunol. 116, 876–883 (2005).

    Article  CAS  Google Scholar 

  45. Kakoki, M., Takahashi, N., Jennette, J.C. & Smithies, O. Diabetic nephropathy is markedly enhanced in mice lacking the bradykinin B2 receptor. Proc. Natl. Acad. Sci. USA 101, 13302–13305 (2004).

    Article  CAS  Google Scholar 

  46. Early Treatment Diabetic Retinopathy Study Research Group. Grading diabetic retinopathy from stereoscopic color fundus photographs–an extension of the modified Airlie House classification. ETDRS report number 10. Ophthalmology 98, 786–806 (1991).

  47. Aiello, L.P. et al. Vascular endothelial growth factor-induced retinal permeability is mediated by protein kinase C in vivo and suppressed by an orally effective beta-isoform-selective inhibitor. Diabetes 46, 1473–1480 (1997).

    Article  CAS  Google Scholar 

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We thank X. Chen and D. Bursell for technical assistance. This work was supported in part by the US National Institutes of Health (grants DK 60165, DK 36836, EY011289 and EY014106), the Juvenile Diabetes Research Foundation (1-2005-1047), the Massachusetts Lions Eye Research Fund, the Adler Foundation and the Air Force Office of Scientific Research Medical Free Electron Laser Program (FA9550-040-1-0046).

Author information

Authors and Affiliations



B.-B.G. conducted the proteomic and bioinformatic analysis; performed all western blot and in vitro enzymatic analyses, and cerebral vascular permeability experiments; and contributed to manuscript writing. A.C. and S.-E.B. performed retinal permeability analyses, intravitreal injections, and vitreous pH analyses; and assisted in designing the in vivo experiments. S.R. assisted with retinal permeability experiments and coordinated vitreous collection. S.J.F. performed proteome statistical analysis and assisted in editing the manuscript. V.S., M.W. and J.G.F. conducted the OCT measurements, and analyzed and interpreted the results. R.L.A. and P.G.A. provided vitreous samples and conducted the clinical diagnoses. L.P.A. provided vitreous samples and clinical information, and contributed to manuscript writing. E.P.F. designed the entire study, supervised all components of the study, and wrote the manuscript.

Corresponding author

Correspondence to Edward P Feener.

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Competing interests

J.G.F. receives royalties from intellectual property licensed by MIT to Carl Zeiss Meditec and LightLab Imaging. E.P.F. and L.P.A. have a pending patent application that includes data from this report.

Supplementary information

Supplementary Fig. 1

Effect of C1-INH on PK activation by FXII in the presence of HK. Kallikrein activity was measured as cleavage of synthetic fluorogenic kallikrein substrate (0.4 mM) on a microplate reader at 37 °C at 15 min. (PDF 23 kb)

Supplementary Fig. 2

Effect of anti-PK antibody on PK activation by FXII in the presence of HK. (PDF 22 kb)

Supplementary Fig. 3

Effect of pH, CA-I and HCO3 on PK activation by FXII in the presence of HK. (PDF 16 kb)

Supplementary Fig. 4

Effect of pH on PK activation by FXII in the presence of HK. **P < 0.01 vs pH 7.4. (PDF 17 kb)

Supplementary Fig. 5

Effect of pH on FXII autoactivation in the absence of PK and HK. (PDF 14 kb)

Supplementary Table 1

Demographics of Study Subjects (PDF 31 kb)

Supplementary Table 2

Vitreous proteome in individuals with NDM, noDR or PDR (PDF 317 kb)

Supplementary Methods (PDF 20 kb)

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Gao, BB., Clermont, A., Rook, S. et al. Extracellular carbonic anhydrase mediates hemorrhagic retinal and cerebral vascular permeability through prekallikrein activation. Nat Med 13, 181–188 (2007).

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