Ординатура / Офтальмология / Английские материалы / Retinal and Choroidal Angiogenesis_Penn_2008

The hallmark of Goldberg’s stage 3 is peripheral NV, which he termed the sea fan formation, and this defines the development of proliferative retinopathy. A sea fan is a tuft of neovascular tissue that resembles the marine invertebrate Gorgonia flabellum and occurs at the venous side of A/V anastomoses6 or, more commonly in our histopathological studies, at A/V crossings.33 Sea fans always grow from perfused retina toward peripheral nonperfused retina (Figure 3).

Figure 20-3. Superior retina from a 40-year-old sickle cell anemia subject (SS). The sample was incubated for ADPase activity. This image was taken when the retina was viewed with bright field illumination as a wet flat mount before embedding in glycol methacrylate, so the ADPase activity appears black. The entire peripheral retina is nonperfused (lacks ADPase+ vessels), and many neovascular structures (darkly stained blood vessels) are present at the border of perfused and nonperfused retina. The neovascular structure indicated by an arrow is a sea fan. This structure is shown in detail in Figure 5. From McLeod D.S. et al., Am. J. Ophthalmol. 124:455-472, 1997.

The ischemic peripheral retina may produce factors that initiate and promote the growth of the neovascular tissue.6 We have observed elevated basic fibroblast growth factor (bFGF) in peripheral nonperfused areas and increased vascular endothelial growth factor (VEGF) in a patient with sickle cell retinopathy.31 Ironically, we have also observed extremely high levels of the anti-angiogenic pigment epithelial growth factor (PEDF) associated with sea fans (Figure 4). Using densitometric analysis of the reaction products, we found that the ratio of VEGF to PEDF was reduced in the sea fans to 1:1, suggesting that an increase in VEGF shifted the balance toward angiogenesis.32 We also observed high levels of PEDF in autoinfarcted sea fans, whereas VEGF levels were very low.

Figure 20-4. PEDF and VEGF immunolocalization in retina, a feeder vessel (A-C) and a sea fan (D-F) in a 58-year-old SC patient. Heparan sulfate proteoglycan perlecan immunoreaction product (A and D) is only in viable retinal blood vessels, and a feeder vessel (double arrow, A, B, & C), which supplies the sea fans (sea fan vessels indicated with a single arrow in all panels). PEDF immunoreactivity (B and E) is present in the feeder vessel and the sea fan, but is also prominent in the matrix components of the sea fan (asterisk in E). VEGF immunoreactivity (C and F) is present predominantly in viable retinal blood vessels and the feeder vessel in (C) and viable vessels in the sea fan (F). Vessels were visualized using red AEC immunoreaction product followed by hematoxylin counterstaining. From Kim et al., Exp. Eye Res. 77:433-445, 2003.

Initially, the sea fan is flat and grows on the internal surface of the retina between the posterior hyaloid and the internal limiting membrane. It may be sustained by just one feeding arteriole and one draining venule. Further growth with the addition of more feeding and draining vessels may result in

Due to an inadequate blood-retina barrier, chronic transudation into the vitreous from the sea fans may result in early vitreous degeneration, collapse, and traction and prompt the onset of stages 4 and 5. Vitreous hemorrhage marks stage 6. Vitreous hemorrhage occurs most commonly in those patients with hemoglobin SC (23%) and less often in patients with hemoglobin SS (3%). Analysis of untreated eyes with sickle cell retinopathy suggested three risk factors for vitreous hemorrhage: hemoglobin SC, any vitreous hemorrhage in the eye on initial examination, and more than 60 total degrees of active neovascular lesions.35 If there is greater than 60 degrees of circumferential NV, then the risk of a vitreous hemorrhage is increased.35

Sea fans may grow into the vitreous, and traction on the delicate neovascular tissue from the vitreous may result in hemorrhage. Sea fans may bleed at irregular intervals for several years as a result of minor ocular trauma, vitreous movement, vitreous syneresis due to chronic transudation of serum, or contraction of vitreous bands from previous hemorrhages. The hemorrhage may be asymptomatic and remain localized to the area surrounding the sea fan or may break into the vitreous gel and interfere with visual function. Plasma and blood may also chronically leak from the neovascular tufts and stimulate vitreous strand and fibroglial membrane formation, possibly culminating in rhegmatogenous retinal detachment, or stage 5.

5.CHOROIDAL OCCLUSIONS

Choroidal occlusions have been described in patients with sickle cell

hemoglobinopathies14,36-38 and may have some temporal relationship to formation of choroidal NV23,24 and/or the black sunburst lesion.25,26 We have

observed that choroidal compromise is histopathologically associated with impacted erythrocytes, increased fibrin, and platelet-fibrin thrombi.39,40 Choroidal NV has also been observed at sites of compromise in periphery.

6.ANIMAL MODELS FOR SICKLE CELL RETINOPATHY

Progress in realizing a treatment for sickle cell disease and the associated retinopathy has been hindered by a lack of animal models for sickle cell disease. Recently, transgenic animal technology has finally permitted the creation of animal models of sickle cell disease. Constructs of the different variants in human βs globin and the human α-globin gene have been inserted

into fertilized mouse eggs.42,43 Some of the animals expressed the human genes at low levels. By subsequently breeding the transgenic mice into a background of mouse βmajor deletion, the equivalent of β-thalassemia, the resultant mice expressed high levels of the human genes, but were not thalassemic. The RBCs of the mice sickled, and there were mildly elevated reticulocyte counts, enlarged spleens, and elevated mean cell hemoglobin concentrations.42,43 Recently, we have observed a severe form of sickle cell retinopathy in one of these transgenic mouse lines.41

The retinal pathological changes were observed in the αHβs[βMDD] transgenic mouse line of Fabry et al. that was established by simultaneously injecting µLCR-βs and µLCR-αH (LCR=locus control region) constructs

into C57BL/6J mice and breeding the transgenics with mice that were homozygous for the mouse β major deletion.42 The mice in this line have 80%

human βs-globin, spleen and lung pathologies, and many other hematological characteristics in common with human sickle cell disease.43 The retinas of these transgenic mice demonstrate vaso-occlusive processes, which result in the loss of precapillary arterioles, capillaries, and venules (Figure 6B). Intra and extra-retinal NV was observed (Figure 6F) and was associated mostly with veins, venules, and A/V anastomoses.41 Pigmented lesions resembling human black sunburst lesions were observed and consisted mostly of RPE-ensheathed blood vessels, which often appeared to be of choroidal origin (Figure 6G and H). Photoreceptor loss was observed in animals with severe chorioretinopathy (Figure 6F and H). Chorioretinopathy was bilateral and occurred in 30% of the animals examined, and its incidence increased with age. This model is probably the only genetically derived animal model for retinal and choroidal NV.41

The retinopathy that occurs in this mouse line is similar to human retinopathy in that occlusions, intraand extra-retinal NV, choroidal NV, and pigmented lesions occur. The retinopathy in the transgenic mice differs from human retinopathy, however, in that occlusions are not predominantly in peripheral retina and hemorrhages are observed infrequently. Also unlike the human was the frequency of NV: choroidal NV was most common, then intraretinal NV, and least common was preretinal NV. In the human, photoreceptor degeneration occurs in areas of nonperfusion only, while in advanced retinopathy in the mice all photoreceptors degenerate. In a second study from the Lutty laboratory, photoreceptor atrophy and choroidal NV were found to be associated with choroidal nonperfusion, as determined by using a vascular tracer.44

Figure 20-6. ADPase incubated retinas from a 12-month-old transgenic mouse with no retinopathy (A, C, E) and an 18-month-old sickle cell transgenic mouse with severe retinopathy (B, D, F-H). When the ADPase reaction product in the 12-month-old (A) is viewed en bloc with dark field illumination, the normal radial blood vessel pattern emanating from optic nerve head (O) is apparent, whereas the 18-month-old sickle mouse (B) has areas without viable blood vessels and abnormal vascular patterns that included A/V anastomoses (AV). When viewed with bright field illumination, no pigment is present in the 12-month-old retina (C), but there are many pigmented lesions in the 18-month-old retina (D). A section of the normal retina (E) shows inner and outer nuclear layers (on), photoreceptor outer segments (p), a vein in the superficial vascular system (v), and capillaries (arrowheads) in the deep capillary network. A section (F) through the IRMA-like (intraretinal microvascular abnormality) structure indicated by “F” in parts B and D shows a cluster of capillaries (c) at the base of an atrophic retina, suggesting that this was an IRMA formation. A section (G) through the area in parts B and D indicated by “G” appears to be an occluded major blood vessel that has retinal pigmented cells (R). A section (H) of the pigmented lesion indicated by “H” in parts B and D shows fibrillar material (L) surrounded by RPE cells (R) resembling the acinar formations shown in black sunburst lesions in human sickle cell retinopathy. From Lutty, G.A. et al., Am. J. Pathol. 145:490-497, 1994.

Animal models to study the mechanism of vaso-occlusion in sickle cell disease have also recently been developed. Initially, vasculatures like mesocecum and mesoappendix were used so that vital examination of sickle erythrocyte (sRBC) adhesion and vascular obstruction could be performed.45

We have introduced a model to evaluate the retention of sRBCs in retina and choroids.46 Rats are administered fluorescently labeled human sRBCs intravenously, and retention of the cells in retina is evaluated in flatmounts of retinas from the rat after sacrifice. This model has demonstrated two mechanisms for retention of sRBCs in retina and choroid: mechanical

retention of dense sRBCs during hypoxia46 and adhesion of reticulocytes after cytokine exposure.47,48

With the advent of animal models, the mechanisms of vaso-occlusion in the sickle cell retina can be determined. Therapies addressing these mechanisms will be evaluated in the transgenic mouse lines to determine if the treatments can prevent or allay vaso-occlusive processes in the sickle cell retina. Like diabetic retinopathy,49 if the vaso-occlusive processes can be prevented, all subsequent vasculopathy can be avoided.

ACKNOWLEDGMENTS

The authors acknowledge their collaborators Carol Merges, M.E.S., Michaela Kunz Matthews, M.D., and Jingtai Cao, M.D., who contributed substantially to the studies discussed in this manuscript. This work was supported by NIH grants EY 01765 (Wilmer Institute) and HL45922 (GL), the Reginald F. Lewis Foundation (GL), and Research to Prevent Blindness (Wilmer). Gerard A. Lutty is an American Heart Association Established Investigator and the recipient of a Research to Prevent Blindness Lew Wasserman Merit Award.

REFERENCES

1.W. A. Eaton and J. Hofrichter, Hemoglobin S gelation and sickle cell disease. Blood 170, 1245-1266, (1987).

2.T. F. Necheles, D. M. Allen, and P. S. Gerald, The many forms of thalassemia: definition and classification of the thalassemia syndromes. Ann. NY Acad. Sci. 165 (1), 5-12, (1969).

3.G. R. Serjeant, Sickle cell disease. Oxford: Oxford University Press; 1985.

4.M. F. Goldberg, Natural history of untreated proliferative sickle retinopathy. Arch. Ophthalmol. 85, 428-437, (1971).

5.M. F. Goldberg, Sickle cell retinopathy. In: Clinical Ophthalmology. Hagerstown, MD: Harper and Row, Publishers, Inc; 1976. p. 1-44.

6.M. F. Goldberg, Retinal neovascularization in sickle cell retinopathy. Trans. Am. Acad. Ophthalmol. Otolaryngol. 83, 409-431, (1977).

7.J. G. Clarkson, The ocular manifestations of sickle-cell disease: a prevalence and natural history study. Tr. Am. Ophth. Soc. 90, 481-504, (1992).

8.D. Paton, The conjunctival sign of sickle cell disease. Arch. Ophthalmol. 68, 627-632, (1962).

9.D. Paton, The conjunctival sign of sickle cell disease. Arch. Ophthalmol. 66, 90-94, (1961).

10.M. K. Horne, III, Sickle cell anemia as a rheologic disease. Am. J. Med. 70 (2), 288-298, (1981).

11.S. Charache and C. L. Conley, Rate of Sickling of Red Cells During Deoxygenation of Blood from Persons with Various Sickling Disorders. Blood 24, 25-48, (1964).

12.T. Jensen, J. Bjerre-Knudsen, B. Feldt-Rasmussen, and T. Deckert, Features of endothelial dysfunction in early diabetic nephropathy. Lancet 1, 461-463, (1989).

13.R. B. Welch and M. F. Goldberg, Sickle-cell hemoglobin and its relation to fundus abnormality. Arch. Ophthalmol. 75 (3), 353-362, (1966).

14.D. McLeod, M. Goldberg, and G. Lutty, Dual perspective analysis of vascular formations in sickle cell retinopathy. Arch. Ophthalmol. 111, 1234-1245, (1993).

15.M. Kunz Mathews, D. McLeod, C. Merges, J. Cao, and G. Lutty, Neutrophils and leukocyte adhesion molecules in sickle cell retinopathy. Brit. J. Ophthalmol. 86, 684-690, (2002).

16.M. H. Goldbaum, Retinal depression sign indicating a small retinal infarct. Am. J. Ophthalmol. 86 (1), 45-55, (1978).

17.M. Raichand, R. V. Dizon, K. C. Nagpal, M. F. Goldberg, M. F. Rabb, and M. H. Goldbaum, Macular holes associated with proliferative sickle cell retinopathy. Arch. Ophthalmol. 96 (9), 1592-1596, (1978).

18.M. S. Roy, P. Gascon, and D. Giuliani, Macular blood flow velocity in sickle cell disease: relation to red cell density. Br. J. Ophthalmol. 79 (8), 742-745, (1995).

19.D. Gagliano and M. F. Goldberg, Evolution of the salmon patch in sickle cell retinopathy. Arch. Ophthalmol. 107, 1814-1815, (1989).

20.N. Romayananda, M. F. Goldberg, and W. R. Green, Histopathology of sickle cell retinopathy. Trans. Am. Acad. Ophthalmol. Otolaryngol. 77, 652-676, (1973).

21.G. Asdourian, K. C. Nagpal, M. Godlbaum, D. Patrianakos, M. F. Goldberg, and M. Rabb, Evolution of the retinal black sunburst in sickling haemoglobinopathies. Br. J. Ophthalmol. 59, 710-716, (1975).

22.J. C. van Meurs, Evolution of a retinal hemorrhage in a patient with sickle cellhemoglobin C disease. Arch. Ophthalmol. 113 (8), 1074-1075, (1995).

23.J. C. Liang and L. M. Jampol, Spontaneous peripheral chorioretinal neovascularization in association with sickle cell anaemia. Br. J. Ophthalmol. 67, 107-110, (1983).

24.G. A. Lutty, C. Merges, S. Crone, and D. S. McLeod, Immunohistochemical insights into sickle cell retinopathy. Curr. Eye Res. 13 (2), 125-138, (1994).

25.G. N. Wise, C. T. Dollery, and P. Henkind, The retinal circulation. New York, NY: Harper and Row Publishers; 1971.

26.D. G. Cogan, Ophthalmic manifestations of systemic vascular disease. Philadelphia, PA: W.B. Saunders Co.; 1974.

27.M. F. Goldberg, Retinal vaso-occlusion in sickling hemoglobinopathies. Birth Defects 12, 475-515, (1976).

28.A. S. G. Curtis and G. M. Seehar, The control of cell division by tension or diffusion. Nature 274, 52-53, (1978).

29.J. C. van Meurs, Ocular findings in sickle cell disease on Curacao [Thesis]: Catholic University Nijmegen; 1990.

30.M. Raichand, M. F. Goldberg, K. C. Nagpal, M. H. Goldbaum, and G. K. Asdourian, Evolution of neovascularization in sickle cell retinopathy. Arch. Ophthalmol. 95, 1543-1552, (1977).

31.J. Cao, M. Kunz Mathews, D. S. McLeod, C. Merges, L. M. Hjelmeland, and G. A. Lutty, Angiogenic factors in human proliferative sickle cell retinopathy. Br. J. Ophthalmol. 83, 838-846, (1999).

32.S. Y. Kim, C. Mocanu, D. S. McLeod, I. A. Bhutto, C. Merges, M. Eid, et al. Expression of pigment epithelium-derived factor (PEDF) and vascular endothelial growth factor (VEGF) in sickle cell retina and choroid. Exp. Eye Res. 77, 433-445, (2003).

33.D. S. McLeod, C. Merges, A. Fukushima, M. F. Goldberg, and G. A. Lutty, Histopathological features of neovascularization in sickle cell retinopathy. Am. J. Ophthalmol. 124, 473-487, (1997).

34.P. I. Condon and G. R. Serjeant, Behaviour of untreated proliferative sickle retinopathy. Br. J. Ophthalmol. 64, 404-411, (1980).

35.P. I. Condon, R. A. F. Whitelock, A. C. Bird, J. F. Talbot, and G. R. Serjeant, Recurrent visual loss in homozygous sickle cell disease. Br. J. Ophthalmol. 69, 700-706, (1985).

36.P. I. Condon, G. R. Serjeant, H. Ikeda, Unusual chorioretinal degeneration in sickle cell disease. Br. J. Opthalmol. 57, 81-88, (1973).

37.R. V. Dizon, L. M. Jampol, M. F. Goldberg, C. Juarez, Choroidal occlusive disease in sickle cell hemoglobinopathies. Surv. Ophthalmol. 23, 297-306, (1973).

38.M. R. Stein and A. J. Gay, Acute chorioretinal infarction in sickle cell trait. Arch. Opthalmol. 84, 485-490, (1970).

39.G. Lutty, C. Merges, S. Crone, and D. McLeod, Immunohistochemical insights into sickle cell retinopathy. Curr. Eye Res. 13, 125-138, (1994).

40.G. Lutty and M. Goldberg, Ophthalmologic complications. In: Embury SD, Hebbel RP, Mohandas N, Steinberg MH, editors. Sickle cell disease: basic principles and clinical practice. New York: Raven Press, Ltd.; 1994. p. 703-724.

41.G. Lutty, D. McLeod, A. Pachnis, F. Costantini, M. Fabry, and R. Nagel, Retinal and choroidal neovascularization in a transgenic mouse model of sickle cell disease. Am. J. Pathol. 145, 490-497, (1994).

42.M. E. Fabry, F. Constantini, A. Pachnis, S. M. Suzuka, N. Bank, H. S. Aynedjian, et al. High expression of human bs- and a-genes in transgenic mice: erythrocyte abnormalities, organ damage, and the effect of hypoxia. Proc. Natl. Acad. Sci. USA 89, 12155-12159, (1992).

43.M. E. Fabry, E. Fine, V. Rajanayagam, S. M. Factor, J. Gore, M. Sylla, et al. Demonstration of endothelial adhesion sickle cells in vivo: a distinct role for deformable sickle cell discocytes. Blood 79, 1602-1611, (1992).

44.G. A. Lutty, C. Merges, D. S. McLeod, S. D. Wajer, S. M. Suzuka, M. E. Fabry, et al. Nonperfusion of retina and choroid in transgenic mouse models of sickle cell disease. Curr. Eye Res. 17 (4), 438-444, (1998).

45.D. K. Kaul, M. E. Fabry, R. L. Nagel, Microvascular sites and characteristics of sickle cell adhesion to vascular endothelium in shear flow conditions: Pathophysiological implications. Proc. Natl. Acad. Sci. USA 86, 3356-3360, (1989).

46.G. A. Lutty, A. Phelan, D. S. McLeod, M. E. Fabry, and R. L. Nagel, A rat model for sickle cell-mediated vaso-occlusion in retina. Microvascular Res. 52, 270-280, (1996).

47.G. A. Lutty, M. Taomoto, J. Cao, D. S. McLeod, P. Vanderslice, B. W. McIntyre, et al. Inhibition of TNFa-induced sickle RBC retention in retina by a VLA-4 antagonist. Invest. Ophthalmol. Vis. Sci. 42, 1349-1355, (2001).

48.G. A. Lutty, T. Otsuji, M. Taomoto, D. S. McLeod, P. Vanderslice, B. McIntyre, et al. Mechanisms for sickle RBC retention in choroid. Curr. Eye Res. 25, 163-171, (2002).

49.E. Kohner and M. Porta, Vascular abnormalities in diabetes and their treatment. Trans. Ophthalmol. Soc. UK 100, 440-444, (1980).

Chapter 21

DIABETIC RETINOPATHY

Clinical Applications of Angiogenesis Research

Robert N. Frank, MD

Kresge Eye Institute, Wayne State University School of Medicine, Detroit, Michigan

Abstract: This chapter will discuss the potential applications of research into angiogenesis, and particularly retinal angiogenesis, for the treatment of patients with diabetic retinopathy. Vascular endothelial growth factor (VEGF) is the angiogenic growth factor that has received the most attention in retinal angiogenesis research. Several anti-VEGF strategies either have been tested or

are currently being tested for the treatment of retinal neovascularization either clinically in humans or experimentally in laboratory animals. These include: anti-VEGF antibodies, an anti-VEGF aptamer, a VEGF “trap,” and a VEGF receptor blocker. Other approaches currently under investigation include studies of protein kinase C inhibitors and, for diabetic macular edema, which is

thought to involve the increased vascular permeability induced by VEGF, intravitreal or periocular steroid injections or periocular injections of a steroidlike molecule. Animal models of retinal neovascularization have shown only about 40% inhibition following employment of various anti-VEGF strategies. Therefore, more generalized anti-angiogenic therapies, or multi-drug therapies to block multiple growth factors, merit further investigation. Pigment epithelium-derived factor (PEDF) is inhibited by hypoxia and inhibits neovascularization. Gene therapy to increase endogenous PEDF production is therefore being attempted. Finally, there is evidence that diabetic retinopathy is worsened by “oxidative stress.” Further studies of antioxidant therapies for diabetic retinopathy are therefore merited. Hypoxia is thought to be a major effector of neovascularization, producing upregulation of angiogenic growth factors and downregulation of molecules that inhibit angiogenesis. There is some evidence that oxygen supplementation is beneficial for individuals with diabetic macular edema. “Pan-retinal” laser photocoagulation may also ameliorate hypoxia by reducing metabolism in hypoxic regions of midperipheral retina.

407

J.S. Penn (ed.), Retinal and Choroidal Angiogenesis, 407–418.

© Springer Science+Business Media B.V. 2008