Ординатура / Офтальмология / Учебные материалы / Retinal Vascular Disease Joussen Springer
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3.2 Vascular Endothelial Growth Factor in Retinal Vascular Disease |
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Fig. 3.2.7. Fundus photography and angiography of retina from vascular endothelial growth factor (VEGF)-injected eye. a Red-free fundus photograph of a VEGF-injected eye shows dilation and tortuosity of retinal vessels in the posterior pole. 35-degree field photograph. b Fluorescein angiogram of the same eye shown in a 54 s after dye injection shows non-perfusion of regions throughout the posterior pole including macula (arrows). There is leakage from the optic nerve and the retinal vessels end in bulbous microaneurysm-like structures. (Fig. 1 from [35])
culature. However, the fact that many of these growth factors are not regulated by tissue oxygenation suggests that they may play a subordinate role in coordinating vascularization in the retina. Recent studies suggest that many of their effects may be mediated through interactions with VEGF, a molecule whose production is regulated by tissue oxygenation, although other factors may also increase the expression of VEGF (Table 3.2.1).
Table 3.2.1. Interaction of VEGF and other growth factors
|
Angio- |
VEGF |
References |
|
genesis |
expression |
|
|
|
|
|
IGF |
+ |
+ |
[28, 17] |
Insulin |
+ |
+ |
[14] |
FGF |
++ |
+ |
[30, 5] |
TGF |
++ |
+ |
[24] |
CTGF |
+ |
? |
[34, 6] |
Estrogen |
+ |
+ |
[33] |
Leptin |
+ |
+ |
[31] |
Angiopoietin |
+ |
+ |
[32] |
Erythropoietin |
+ |
+ |
[39, 15] |
PEDF |
– |
? |
[9] |
|
|
|
|
CTGF connective tissue growth factor, PEDF pigment epitheli- al-derived factor
3.2.4.2 VEGF and Diabetic Retinopathy
Patients with diabetic retinopathy develop proliferative complications (secondary to neovascularization) and/or macular edema (secondary to increased vascular permeability). Recent work has implicated VEGF in human eye diseases characterized by both these conditions [1, 2, 12, 36]. Levels of ocular VEGF are tightly correlated with both growth and permeability of new vessels. Furthermore, introduction of VEGF into normal primate eyes induces similar pathological processes to those seen in diabetic retinopathy (Fig. 3.2.7). In patients with proliferative diabetic retinopathy in which tissue hypoxia promotes neovascularization, levels of VEGF are elevated in ocular tissues [2]. These elevated levels of VEGF decline when treatment with panretinal laser photocoagulation induces regression of neovascularization.
These observations suggest that VEGF may be an important therapeutic target in ocular neovascularization and macular edema secondary to diabetes. Results from a recent phase 2 trial demonstrate that patients with diabetic macular edema (DME) who are treated with an intravitreal anti-VEGF agent, pegaptanib sodium (Macugen), had better visual acuity outcomes, were more likely to show a reduction in macular edema, and were less likely to need focal laser photocoagulation for their edema than patients treated with a sham injection [8]. Addi-
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tional case series data suggest that other anti-VEGF agents, including bevacizumab, may have efficacy against diabetic neovascularization [29]. Currently, phase 2 and phase 3 clinical trials are ongoing using anti-VEGF agents to treat both diabetic neovascularization and DME.
VEGF expression may also be increased in earlier stages of diabetic retinopathy when clear documentation of intraretinal hypoxia has not yet been clearly documented. Thus, other metabolites of glucose induced toxins such as oxidants, glycated proteins or activation of signaling pathways such as protein kinase C (PKC) have also been suggested to increase capillary permeability and to contribute to macular edema. Recently, the results of clinical trials using a selective inhibitors of the PKC 
isoforms (ruboxistaurin) have shown that ruboxistaurin to preserves vision in diabetic patients with vision threatening macular edema. The therapeutic effects of this PKC 
isoform inhibitor may be mediated by its effects by on both VEGF dependent and independent pathways.
neovascularization that is similar to that observed in diabetic retinopathy. As in diabetes, hypoxic induction of VEGF is likely the link between retinal ischemia in these clinical conditions and the development of neovascularization. Clinical studies analyzing VEGF concentrations in aqueous humor have established that there is a correlation between aqueous VEGF concentrations and the extent of non-perfu- sion in patients with central and branch retinal vein occlusions (CRVO and BRVO) [7, 22]. Increased VEGF levels are also correlated with the onset and persistence of neovascularization of the iris in cases of ischemic CRVO [7], and with increasing vascular permeability and severity of macular edema in cases of BRVO [22, 23]. Initial clinical studies appear to suggest that intravitreal anti-VEGF agents such as bevacizumab may be effective in improving clinical outcomes in CRVO patients, specifically in regard to decreasing macular edema and improving visual acuity [13].
3.2.4.3Hypertension As an Aggravating Factor in Diabetes-Induced Activation of VEGF
Epidemiological studies clearly identify hypertension as an independent risk factor for diabetic retinopathy. Hypertension increases the risk of retinopathy progression and the development of proliferative diabetic retinopathy (PDR) [16]. Patients with higher ranges of blood pressure are threefold more likely to develop PDR [26]. The incidence of macular edema is similarly increased with the concomitant presence of hypertension [11]. Patients with hypertension are 3.2-fold more likely to develop diffuse macular edema [19].
Severe hypertension itself can induce a retinopathy characterized by increased retinal vascular leakage [37, 38]. It is also known to independently increase levels of VEGF in the eye. The mechanism by which hypertension induces VEGF expression is by increasing mechanical cyclic stretch in vascular walls, an action that may also involve the enhancement of angiotensin actions. It is possible that the additional induction of VEGF is the reason that systemic hypertension exacerbates coexistent diabetic retinopathy and other ocular diseases that lead to neovascularization and increased vascular permeability.
3.2.4.4VEGF in Neovascularization Secondary to Retinal Vascular Occlusions
Patients with both branch and central retinal vascular occlusions are at risk for development of retinal
References
1.Adamis AP, Miller JW, Bernal MT, D’Amico DJ, Folkman J, Yeo TK, Yeo KT (1994) Increased vascular endothelial growth factor levels in the vitreous of eyes with proliferative diabetic retinopathy. Am J Ophthalmol 118:445 – 450
2.Aiello LP, Avery RL, Arrigg PG, Keyt BA, Jampel HD, Shah ST, Pasquale LR, Thieme H, Iwamoto MA, Park JE, Nguyen HV, Aiello LM, Ferrara N, King GL (1994) Vascular endothelial growth factor in ocular fluid of patients with diabetic retinopathy and other retinal disorders. N Engl J Med 331:1480 – 1487
3.Aiello LP, Northrup JM, Keyt BA, Takagi H, Iwamoto MA (1995) Hypoxic regulation of vascular endothelial growth factor in retinal cells. Arch Ophthalmol 113:1538 – 1544
4.Aiello LP, Pierce EA, Foley ED, Takagi H, Chen H, Riddle L, Ferrara N, King GL, Smith LE (1995) Suppression of retinal neovascularization in vivo by inhibition of vascular endothelial growth factor (VEGF) using soluble VEGFreceptor chimeric proteins. Proc Natl Acad Sci U S A 92: 10457 – 10461
5.Asahara T, Bauters C, Zheng LP, Takeshita S, Bunting S, Ferrara N, Symes JF, Isner JM (1995) Synergistic effect of vascular endothelial growth factor and basic fibroblast growth factor on angiogenesis in vivo. Circulation 92:II365 – 371
6.Babic AM, Chen CC, Lau LF (1999) Fisp12/mouse connective tissue growth factor mediates endothelial cell adhesion and migration through integrin alphavbeta3, promotes endothelial cell survival, and induces angiogenesis in vivo. Mol Cell Biol 19:2958 – 2966
7.Boyd SR, Zachary I, Chakravarthy U, Allen GJ, Wisdom GB, Cree IA, Martin JF, Hykin PG (2002) Correlation of increased vascular endothelian growth factor with neovascularization and permeability in ischaemic central vein occlusion. Arch Ophthalmol 120:1644 – 1650
8.Cunningham ET, Jr, Adamis AP, Altaweel M, Aiello LP, Bressler NM, D’Amico DJ, Goldbaum M, Guyer DR, Katz B, Patel M, Schwartz SD (2005) A phase II randomized doublemasked trial of pegaptanib, an anti-vascular endothelial
3.2 Vascular Endothelial Growth Factor in Retinal Vascular Disease
growth factor aptamer, for diabetic macular edema. Ophthalmology 112:1747 – 1757
9.Dawson DW, Volpert OV, Gillis P, Crawford SE, Xu H, Benedict W, Bouck NP (1999) Pigment epithelium-derived factor: a potent inhibitor of angiogenesis. Science 285: 245 – 248
10.Duh E, Aiello LP (1999) Vascular endothelial growth factor and diabetes: the agonist versus antagonist paradox. Diabetes 48:1899 – 1906
11.El-Asrar AM, Al-Rubeaan KA, Al-Amro SA, Kangave D, Moharram OA (1998) Risk factors for diabetic retinopathy among Saudi diabetics. Int Ophthalmol 22:155 – 161
12.Funatsu H, Yamashita H, Noma H, Mimura T, Yamashita T, Hori S (2002) Increased levels of vascular endothelial growth factor and interleukin-6 in the aqueous humor of diabetics with macular edema. Am J Ophthalmol 133: 70 – 77
13.Iturralde D, Spaide RF, Meyerle CB, Klancnik JM, Yannuzzi LA, Fisher YL, Sorenson J, Slakter JS, Freund KB, Cooney M, Fine HF (2006) Intravitreal bevacizumab (Avastin) treatment of macular edema in central retinal vein occlusion: a short-term study. Retina 26:279 – 284
14.Jiang ZY, He Z, King BL, Kuroki T, Opland DM, Suzuma K, Suzuma I, Ueki K, Kulkarni RN, Kahn CR, King GL (2003) Characterization of multiple signaling pathways of insulin in the regulation of vascular endothelial growth factor expression in vascular cells and angiogenesis. J Biol Chem 278:31964 – 31971
15.Kertesz N, Wu J, Chen TH, Sucov HM, Wu H (2004) The role of erythropoietin in regulating angiogenesis. Dev Biol 276:101 – 110
16.Klein R, Klein BE, Moss SE, Cruickshanks KJ (1998) 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
17.Kondo T, Vicent D, Suzuma K, Yanagisawa M, King GL, Holzenberger M, Kahn CR (2003) Knockout of insulin and IGF- 1 receptors on vascular endothelial cells protects against retinal neovascularization. J Clin Invest 111:1835 – 1842
18.Leung DW, Cachianes G, Kuang WJ, Goeddel DV, Ferrara N (1989) Vascular endothelial growth factor is a secreted angiogenic mitogen. Science 246:1306 – 1309
19.Lopes de Faria JM, Jalkh AE, Trempe CL, McMeel JW (1999) Diabetic macular edema: risk factors and concomitants. Acta Ophthalmol Scand 77:170 – 175
20.Lu M, Kuroki M, Amano S, Tolentino M, Keough K, Kim I, Bucala R, Adamis AP (1998) Advanced glycation end products increase retinal vascular endothelial growth factor expression. J Clin Invest 101:1219 – 1224
21.Neufeld G, Cohen T, Gengrinovitch S, Poltorak Z (1999) Vascular endothelial growth factor (VEGF) and its receptors. FASEB J 13:9 – 22
22.Noma H, Funatsu H, Yamasaki M, Tsukamoto H, Mimura T, Sone T, Jian K, Sakamoto I, Nakano K, Yamashita H, Minamoto A, Mishima HK (2005) Pathogenesis of macular edema with branch retinal vein occlusion and intraocular levels of vascular endothelial growth factor and interleukin-6. Am J Ophthalmol 140:256 – 261
23.Noma H, Minamoto A, Funatsu H, Tsukamoto H, Nakano K, Yamashita H, Mishima HK (2006) Intravitreal levels of vascular endothelial growth factor and interleukin-6 are correlated with macular edema in branch retinal vein occlusion. Graefes Arch Clin Exp Ophthalmol 244:309 – 315
24.Pertovaara L, Kaipainen A, Mustonen T, Orpana A, Ferrara N, Saksela O, Alitalo K (1994) Vascular endothelial growth factor is induced in response to transforming growth fac- tor-beta in fibroblastic and epithelial cells. J Biol Chem 269:6271 – 6274
25.Pierce EA, Avery RL, Foley ED, Aiello LP, Smith LE (1995) Vascular endothelial growth factor/vascular permeability factor expression in a mouse model of retinal neovascularization. Proc Natl Acad Sci U S A 92:905 – 909
26.Roy MS (2000) Diabetic retinopathy in African Americans with type 1 diabetes: The New Jersey 725: II. Risk factors. Arch Ophthalmol 118:105 – 115
27.Senger DR, Perruzzi CA, Feder J, Dvorak HF (1986) A highly conserved vascular permeability factor secreted by a variety of human and rodent tumor cell lines. Cancer Res 46:5629 – 5632
28.Smith LE, Shen W, Perruzzi C, Soker S, Kinose F, Xu X, Robinson G, Driver S, Bischoff J, Zhang B, Schaeffer JM, Senger DR (1999) Regulation of vascular endothelial growth factor-dependent retinal neovascularization by insulin-like growth factor-1 receptor. Nat Med 5:1390 – 1395
29.Spaide RF, Fisher YL (2006) Intravitreal bevacizumab (Avastin) treatment of proliferative diabetic retinopathy complicated by vitreous hemorrhage. Retina 26:275 – 278
30.Stavri GT, Zachary IC, Baskerville PA, Martin JF, Erusalimsky JD (1995) Basic fibroblast growth factor upregulates the expression of vascular endothelial growth factor in vascular smooth muscle cells. Synergistic interaction with hypoxia. Circulation 92:11 – 14
31.Suganami E, Takagi H, Ohashi H, Suzuma K, Suzuma I, Oh H, Watanabe D, Ojima T, Suganami T, Fujio Y, Nakao K, Ogawa Y, Yoshimura N (2004) Leptin stimulates ischemiainduced retinal neovascularization: possible role of vascular endothelial growth factor expressed in retinal endothelial cells. Diabetes 53:2443 – 2448
32.Suri C, Jones PF, Patan S, Bartunkova S, Maisonpierre PC, Davis S, Sato TN, Yancopoulos GD (1996) Requisite role of angiopoietin-1, a ligand for the TIE2 receptor, during embryonic angiogenesis. Cell 87:1171 – 1180
33.Suzuma I, Mandai M, Takagi H, Suzuma K, Otani A, Oh H, Kobayashi K, Honda Y (1999) 17{beta}-estradiol increases VEGF receptor-2 and promotes DNA synthesis in retinal microvascular endothelial cells. Invest Ophthalmol Vis Sci 40:2122 – 2129
34.Suzuma K, Naruse K, Suzuma I, Takahara N, Ueki K, Aiello LP, King GL (2000) Vascular endothelial growth factor induces expression of connective tissue growth factor via KDR, Flt1, and phosphatidylinositol 3-kinase-Akt-depen- dent pathways in retinal vascular cells. J Biol Chem 275:40725 – 40731
35.Tolentino MJ, McLeod DS, Taomoto M, Otsuji T, Adamis AP, Lutty GA (2002) Pathologic features of vascular endothelial growth factor-induced retinopathy in the nonhuman primate. Am J Ophthalmol 133:373 – 385
36.Tolentino MJ, Miller JW, Gragoudas ES, Jakobiec FA, Flynn E, Chatzistefanou K, Ferrara N, Adamis AP (1996) Intravitreous injections of vascular endothelial growth factor produce retinal ischemia and microangiopathy in an adult primate. Ophthalmology 103:1820 – 1828
37.Tso MO, Jampol LM (1982) Pathophysiology of hypertensive retinopathy. Ophthalmology 89:1132 – 1145
38.Walsh JB (1982) Hypertensive retinopathy: description, classification, and prognosis. Ophthalmology 89:1127 – 1131
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39.Watanabe D, Suzuma K, Matsui S, Kurimoto M, Kiryu J, Kita M, Suzuma I, Ohashi H, Ojima T, Murakami T, Kobayashi T, Masuda S, Nagao M, Yoshimura N, Takagi H (2005) Erythropoietin as a retinal angiogenic factor in proliferative diabetic retinopathy. N Engl J Med 353:782 – 792
40.Williams B, Gallacher B, Patel H, Orme C (1997) Glucoseinduced protein kinase C activation regulates vascular permeability factor mRNA expression and peptide production
by human vascular smooth muscle cells in vitro. Diabetes 46:1497 – 1503
41.Xia P, Aiello LP, Ishii H, Jiang ZY, Park DJ, Robinson GS, Takagi H, Newsome WP, Jirousek MR, King GL (1996) Characterization of vascular endothelial growth factor’s effect on the activation of protein kinase C, its isoforms, and endothelial cell growth. J Clin Invest 98:2018 – 2026
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3.3 Involvement of the Ephrin/Eph System in |
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Angioproliferative Ocular Diseases |
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H. Agostini, G. Martin |
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Core Messages
Ephrin and their Eph receptors both are membrane bound and signal upon cell-cell contact Ephrin/Ephs are essential for axonal guidance
during neuronal development and are involved in angiogenic cell recruitment EphrinB2 is expressed in arteries, EphB4 in veins
Expression of ephrinB2, EphB2, and EphB3 is increased in diabetic retinopathy, and expression of EphA7 in RPE is increased in age-related macular degeneration (AMD)
The potential for a therapeutic use of recombinant ephrins or Ephs remains to be determined
3.3.1First Studies: Ephrins in Retinotectal Projection
The ephrins and Eph receptors are a large subgroup of the receptor tyrosine kinases (RTK, Fig. 3.3.1). They are bound to the cell membrane and mediate signals of cell contact and interaction between cells. This way they provide positional information during cell growth and cell migration, especially if they are
expressed in a gradient. Their role in a developing neuronal network was intensively studied in axon guidance (reviewed in [15, 17, 19, 20, 24, 27, 28]). The differentiating murine retina and its projection in the brain is a well studied example of the biological function of the ephrin/Eph system during axonal growth. The essentials are summarized in Fig. 3.3.2.
Topographic mapping of axons of retinal ganglion cells (RGCs) at the neuronal target site occurs along
Fig. 3.3.1. Human Eph receptors and ephrins. Angiogenic ephrins are highlighted. EphrinAs interact with EphA receptors, and ephrinBs interact with EphB receptors. Only EphA4 interacts with EphB receptors, too. The sequence of EphA6 is not yet fully determined
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Fig. 3.3.2. Ephrins in mouse retinotectal projection. Retinal ganglion cells form axons which grow through the optic nerve head to the optic chiasm where they are projected contralaterally or ipsilaterally to the colliculus superior. Ephrin gradients (indicated by graded triangles) in the optic chiasm or the colliculus superior guide the growing axons depending on their load of Eph receptors. Note that the portion of the non-crossing temporal axons is much smaller in mice than in humans. The known interactors of the ephrins are indicated in parentheses
two orthogonal axes: the nasal-temporal axis of the retina maps along the anterior-posterior axis (A-P) of the corpus geniculatum laterale (man) or colliculus superior (mouse) and the dorsal-ventral retinal axis along the lateral-medial axis (L-M). During eye development in the embryo, RGCs express EphA5 and EphA6 in a high temporal to low nasal gradient
[11].At the superior colliculus (SC) in the midbrain, they reach an area with a low anterior to high posterior gradient expression of ephrinA5 and ephrinA2
[12].The more Eph an RGC expresses on the axon tip the earlier it generates a signal upon interaction with the ephrin, resulting in a stop of growth.
Similarly, EphB2 and EphB3 are expressed in a high ventral to low dorsal gradient in the mouse retina. This corresponds to a low lateral to high medial gradient of ephrinB1 in the SC resulting in lateral innervation of dorsal RGCs in the SC and in medial innervation of ventral RGCs. EfnB1 seems to be attractive rather than repulsive for EphB2 or EphB3 expressing RGCs. Mutants without EphB2 or EphB3 show additional innervation of ventral RGCs in the lateral SC [14].
The EphB1 receptor is expressed in a high ventral
to low dorsal gradient on RGCs during axon pathfinding in the mouse. At the optic chiasm, axons of the ventral RGCs are repelled by ephrinB2 to their own side while axons from dorsal RGCs cross to the other side. Loss of EphB1, but not EphB2 or EphB3, in mutant mice reduced ipsilateral projection as did injection into the chiasm of soluble EphB4-Fc, which blocks ephrinB2 [35].
In humans, the retinotectal development seems to follow comparable molecular mechanisms [18]. However, species specific differences exist. As binocular vision is much more prominent in humans than in mice, EphB1 is expressed in the whole temporal retina compared to the small border in mice, resulting in a higher part of ipsilateral projection. The center of the EphA5 and EphA6 expression is shifted to the foveal area, and this shift is related to the lateral orientation of the mouse eyes.
3.3.2 Ephrins in Vascular Development
The pattern of the peripheral nervous system is similar to the vascular system. Therefore, it was asked if the factors involved in axon guidance are also
3.3 Involvement of the Ephrin/Eph System in Angioproliferative Ocular Diseases
α?
α 
Fig. 3.3.3. Signal transduction of angiogenic ephrins. The EphB4 gene is activated by HoxA9 while the ephrinB2 gene is activated by Notch4 and the ephrinA1 gene by TNF . One way of EphB4 signaling is through PI3K and Akt while signal transduction of ephrinB2 also uses PI3K in a different way. The same is true for EphA2 signaling. Data are not yet complete
involved in angiogenesis. Indeed, all major groups of axon guidance factors like ephrin/Eph, netrin/DCC/ Unc5, semaphorin/neuropilin/plexin, and slit/robo were also found to be involved in angiogenesis [6, 10, 34]. VEGF, which is produced by nerve cells, guides arterial formation through VEGF receptors expressed on endothelial cells [25].
The role of ephrins in angiogenesis has been reviewed [2, 3, 8]. EphrinB2 is expressed in arterial angioblast and endothelial and perivascular mesenchymal cells, while EphB4 is expressed in endothelial cells of veins. The knockout mutants have severe defects in vascular development and die at embryonic stages [33]. Similar defects are observed in EphB2/ EphB3 double knockout mutants, while the single EphB2 or EphB3 mutants have no vascular phenotype [1]. EphrinB1, EphB1, EphB2, and EphB3 are expressed both in arteries and veins. EphB4 signals through PI3K and Akt [30] and is activated by HoxA9 [5]. EphrinB2 is activated by Notch4 and Dll4 in cell culture experiments with human endothelial cells [16, 29] (Fig. 3.3.3).
EphrinA1 is expressed in vascular tissues during embryonic development [23]. Its gene is activated by TNF [7]. The ephrinA1 receptor, EphA2, has no expression in embryonic vasculature. Mutants are viable and fertile but show reduced angiogenesis in adult animals, for example in wound healing or tumor growth [4]. EphA2 signals through PI3K and Rac1 GTPase, which is a rather common intracellular signaling pathway [3] (Fig. 3.3.3). Inhibition of both results in reduced tumor growth, and both are involved in tumor vascularization [26].
3.3.3 Ephrins and Ocular Angiogenesis
Immunohistochemical staining for ephrinB2, EphB2, and EphB3 expression was found in endothelial cells of fibroproliferative membranes from human eyes [32]. Membranes were derived from patients with proliferative diabetic retinopathy and from retinopathy of prematurity. The staining pattern was equal for all factors and correlated with that of factor VIII, a marker for endothelial cells. Although detectable in pathological specimens, the biological functions of these factors are not yet clear in this context.
EphA7 was found in cells cultured from choroidal neovascularization membranes (CNV-RPE) as well as in paraffin sections of CNV membranes by immunohistochemistry [21]. It is thought that EphA7 is involved in the transdifferentiation process of RPE cells into CNV matrix cells, especially during late stages of age-related macular degeneration (AMD).
Human retinal endothelial cells (HRECs) express ephrinB2 but only low levels of EphB4 [31]. Stimulation of ephrinB2 by a soluble EphB4/Fc fusion protein resulted in phosphorylation of PI3K, Src, and ERK1/2, indicating that MAPK signaling may be involved.
These data show that ephrins are present in normal and pathologic tissues of the eye. But at the moment, their role in pathogenesis needs further clarification.
3.3.4Ephrins in Retinal and Subretinal Animal Models
EfnB2 as well as its inducer Dll4 is not expressed in retinal arteries of young mice if they are treated with mild hypoxia (10 % oxygen) directly after birth for up to 6 days [9]. This corresponds with a 2.5-fold increase of ephrinB2 expression during treatment of 7-day-old mice with 75 % oxygen [22]. EphrinB2 may be induced by oxygen via HIF1.
EphrinB2 and EphB4 were found to be expressed in choroidal endothelial cells and in laser-induced choroidal neovascularizations (CNV) in rat [13]. CNV formation was inhibited by intravitreal injection of monomeric sEphB4 consisting of the extracellular part of EphB4 only. A system for testing retinal neovascularization is the mouse model of oxy- gen-induced retinopathy. Seven-day-old mice are placed in 75 % oxygen and returned to normal air after 5 days. During the next 5 days, they develop retinal neovascularization prompted by relative hypoxia. This retinal neovascularization is enhanced after intravitreal injection of dimeric EphB4-Fc or eph- rinB2-Fc and reduced by injection of monomeric
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sEphB4 or s-ephrinB2 [22]. Phosphorylated forms of Eph receptors are detected in preretinal blood vessel tufts in histological eye sections stained with a pan- anti-phospho-Eph antibody [36]. Expression of EphB4 as well as of VEGF, VEGF-R1, and VEGF-R2, but not of ephrinB2, is enhanced 2 days after return of the mice to normal oxygen. This indicates that EphB4 and ephrinB2 influence angiogenesis by independent signaling pathways.
3.3.5 Therapeutic Potential
Ephrins are involved in late stages of capillary formation. Since ephrins themselves have no known direct proliferative effect on endothelial cells, intervention at the level of ephrin action is expected to have an indirect effect on angiogenesis by changing cell recruitment.
In order to manipulate the Ephrin/Eph system it is helpful to visualize the mode of how cell-bound ephrins or Ephs are activated. Upon binding of a corresponding ligand, clustering is thought to be essential before the signaling cascade is activated. So far, two different experimental approaches with therapeutic potential were pursued by different groups by modulating angiogenesis with monomeric or dimeric ephrinB2 and EphB4, respectively. Monomeric ligands or receptors are thought to inhibit clustering whereas dimeric recombinant proteins could be potential activators of cluster formation. The results are still conflicting. Monomeric EphB4 seems to be the strongest inhibitor of vessel formation followed by monomeric ephrinB2. With regard to the dimeric forms both inhibition and enhancement of angiogenesis are described. Further studies will be necessary before a clear recommendation for clinical use of ephrin/Eph inhibitors can be given.
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20.Mann F, Harris WA, Holt CE (2004) New views on retinal axon development: a navigation guide. Int J Dev Biol 48:957 – 964
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I 3
78 I Pathogenesis of Retinal Vascular Disease
4 I |
4 Hematopoietic Stem Cells in Vascular Development |
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and Ocular Neovascularization |
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N. Sengupta, M.B. Grant, S. Caballero, M.E. Boulton
Core Messages
Hematopoietic stem cells (HSCs) have defined developmental origins
HSCs reside in the bone marrow and repopulate the blood and vascular components
HSCs and their differentiated progeny can be identified and isolated by specific surface markers
Adult neovascularization is now recognized to occur by recruitment and incorporation of pre-
cursor and stem cells as well as by the growth of mature resident cells
Endothelial progenitor cells (EPCs) are major contributors to aberrant ocular neovascularization associated with proliferative diabetic retinopathy and age-related macular degeneration EPC recruitment, migration and differentiation are dependent on systemic and local mediators EPCs can be manipulated in culture
HSCs are involved in ocular neovascularization
4.1 Background
Neovascular diseases of the eye include retinopathy of prematurity (ROP), proliferative diabetic retinopathy (PDR), and the exudative or “wet” form of agerelated macular degeneration (ARMD). Together these diseases affect all age groups and are the leading causes of vision impairment in developed nations [77].
Pathologies of the vascular system, including ocular neovascularization, involve the endothelium. Endothelial cells comprise the innermost layer of all blood vessels, cardiac valves, and several other body cavities. They are responsible for maintaining vessel patency, preventing thrombosis, and relaxation and contraction of vessels [80]. The migration, proliferation, and organization of endothelial cells and their precursors into tubules defines the process of neovascularization, whether it be during development or during adult reparative or pathological processes.
Vasculogenesis has been used to characterize de novo formation of blood vessels from less differentiated precursor cells, and is considered to occur solely during embryonic development. During embryogenesis the so-called hemangioblast gives rise to the vasculature as well as to erythrocytes and blood leukocytes. Postnatal neovascularization is classically thought to involve angiogenesis, i.e., the creation of new vessels by the proliferation, migration and tubule formation of resident, mature endothelial
cells. With the knowledge that endothelial progenitor cells (EPCs) and their undifferentiated antecedents, hematopoietic stem cells (HSCs), participate in postnatal neovascularization, it is now recognized that both pathological and physiological postnatal neovascularization consists of a combination of both angiogenesis and vasculogenesis.
4.2 Developmental Origins of HSCs
During mammalian development, the primitive hematopoietic cells consist of precursors with erythroid and/or myeloid potential and, in the mouse, appear in the blood islands of the yolk sac beginning 7 days after fertilization [10]. Progenitors endowed with long-term reconstitution (LTR) activity are generated solely in the intraembryonic para-aortic splanchnopleura and not in the yolk sac [27]. After circulation is established, multipotent precursors with LTR activity can then be found in the blood [31, 92] and these cells then go to the yolk sac [45]. The para-aortic splanchnopleura becomes the aorta, gonads, and mesonephros (AGM). The hematopoietic precursors present in the AGM at 10.5 days postfertilization have LTR capacity when transferred to adult normal recipient mice [92].
The AGM, also called the splanchnic mesoderm, is the site of origin of intraembryonic HSCs [29, 46]. These HSCs exist as clusters of basophilic cells located within the floor of the aorta from 10 to 10.5 days