Ординатура / Офтальмология / Английские материалы / Retinal and Choroidal Angiogenesis_Penn_2008

Figure 12-1. Relationship between adenosine (ADO) and its receptor signaling, hypoxia, and VEGF in retinal endothelial cells. Both adenosine and VEGF production are upregulated in hypoxia. For VEGF, this upregulation appears to be modulated through the transcription factor HIF-1α in endothelial cells,82,83 while adenosine upregulation is probably modulated through hypoxia induction of HIF-1α in Müller cells which upregulates adenosine nucleotide catabolism enzyme 5’ nucleotidase (5’N).8 There is evidence that adenosine, by signaling through the A2B receptor, can also upregulate VEGF production by endothelial cells in the absence of hypoxia, suggesting autocrine production by endothelial cells.52 Adenosine binding to either A2A or A2B receptor stimulates adenyl cyclase levels of cAMP. Grant and associates have demonstrated further that ligand binding to A2B activates extracellular signalrelated kinase (ERK) in a cAMP independent manner.32 Endothelial cell proliferation and migration and capillary tube formation are stimulated through this A2B/ERK pathway. Michiels et al. have suggested that ERK activation can result in HIF-1α activation84 but the complete pathway for this relationship has not been established. Therefore, autocrine production of VEGF by endothelial cells may be stimulated directly by hypoxia or indirectly through adenosine receptor signaling. The schematic was inspired by Grant et al.32,52

The angiogenic effect of adenosine may be more than a direct effect of adenosine binding its receptors and stimulating proliferation, migration, and tube formation of endothelial cells. Desai and associates found that adenosine binding the A2A receptor inhibited the secretion of thrombospondin-1, a potent inhibitor of angiogenesis.33 This would shift the balance between angiogenic and antiangiogenic agents toward angiogenesis.

4.ADENOSINE IN THE RETINA

As mentioned previously, the major source of adenosine in most tissues is ecto-5’N (CD73). Enzyme and immunohistochemical studies have demonstrated that the glycoprotein 5’N is localized in certain domains of Müller cells in several adult mammalian species34 and is also present during development in murine35 and canine retinas.36

Adenosine, the major product of 5’N, has been proposed as an intercellular communication molecule in retina.37 Histochemical studies have

demonstrated adenosine immunoreactivity in adult retinal neurons of several species.38,39 In retina, adenosine modulates blood flow in both adults and

neonates40-42 and is released in response to ischemia.43-45 Larsen et al. and Li et al. demonstrated that A1 receptor activation protected against the detrimental effects of ischemia/reperfusion (I/R), and the latter group demonstrated further that A2A stimulation may exacerbate the effects of I/R.45,46 Ischemic preconditioning (brief periods of ischemia) induces a state of ischemic tolerance, and this protective effect is modulated through A1 and A2A receptors.47 Ghiardi and associates have recently reviewed these aspects of adenosine’s action in retina.48

5.RELATIONSHIP BETWEEN ADENOSINE AND VEGF

Fisher et al. were the first group to demonstrate that adenosine stimulates production of vascular endothelial growth factor (VEGF).49 Takagi and associates then suggested that hypoxia-stimulated upregulation of VEGF mRNA happens via the cellular production of adenosine. They demonstrated that when adenosine agonists bind to A2A receptors on bovine retinal capillary endothelial cells, production of cAMP is elevated, activation of protein kinase A occurs, and then VEGF production is induced.50 Ironically, they have also found that binding of A2A receptor agonists inhibits the production of the VEGF receptor KDR.51

Grant and associates found that A2B activation specifically induced VEGF production.52 The nonselective agonist NECA (adenosine-5’N- ethylcarboxamide) stimulated production of VEGF mRNA and protein in addition to increasing proliferation (Figure 1). When anti-VEGF antibody was included, the increased proliferation was inhibited. A2B, and not A2A or A1, antagonists inhibited the effects of NECA. Feoktistov and associates have recently demonstrated that microvascular endothelial cells have A2B receptors and that agonist stimulation of these receptors increases expression of interleukin 8 (IL-8), basic fibroblast growth factor (bFGF), and VEGF.30

This effect was modulated by coupling to Gq and possibly G12/13 proteins.30 Agonist stimulation of HUVECs did not increase expression of any of these growth factors.

6.DEVELOPMENT OF THE RETINAL VASCULATURE

There is controversy over whether retinal vascular development occurs via vasculogenesis or angiogenesis. Vasculogenesis is the development of the vasculature by differentiation and organization of endothelial cell precursors, or angioblasts, while angiogenesis is the formation of blood vessels from an existing blood vessel by migration and proliferation of endothelial cells. In the dog, the retina is only 60% vascularized at birth, so development of the primary or inner retinal vasculature and secondary or deep vasculature can be observed.53 Our observations in dog support the view that the primary retinal vasculature forms by vasculogenesis. These observations were possible because angioblasts and endothelial cells have adenosine diphosphatase (ADPase, now known as CD39) activity54 and menadionedependent alpha glycerophosphate dehydrogenase activity.55 Both enzymes are exclusively found in angioblasts and endothelial cells in newly formed blood vessels in the neonatal canine retina. Angioblasts differentiate from a spherical morphology into spindle-shaped cells as they migrate anteriorly through the cell free spaces formed by the inner Müller cell processes.56 Angioblasts then organize to form cords and eventually lumens in the cell free spaces, essentially happening in the absence of proliferation, suggesting the primary or superficial retinal vasculature forms by vasculogenesis.56 The deep or secondary retinal vasculature starts forming in the dog by angiogenesis at about 15 days of age. We have recently demonstrated that human retinal angioblasts also express ADPase (CD39) and development of the initial human retinal vasculature happens in similar manner to dog.57

6.1Adenosine and vascular development

Considering the integral morphological role of Müller cells during primary vasculogenesis and their capacity for producing vasogenic adenosine via 5’N, we examined the localization and relative levels of 5’N and adenosine during normal development of the retinal vasculature. The adenosine A2A receptor was also examined immunohistochemically, viable blood vessels were labeled with anti-von Willebrand’s factor (vWf) (Figure 2A), and angioblasts and newly formed blood vessels were labeled with αGPDH (Figure 2B). Microdensitometric image analysis was applied to provide

semiquantitative information about enzymes and their immunohistochemical reaction products36,58 and how they change with age and with oxygen-

induced retinopathy.

5’N histochemical activity in inner Müller cell processes was elevated in areas of vasculogenesis and declined in periphery toward ora serrata at postnatal day 1 (Figure 2C, E). This activity was inhibited by α,β-methylene- adenosine 5’-diphosphate,36 a specific inhibitor of 5’N.59 Adenosine immunoreactivity was highest near the edge of the forming superficial vasculature (Figure 2D, F). A2A immunoreactivity was prominent in angioblasts and endothelial cells in forming blood vessels (data not shown).58 We have been unable to study A1, A2B, or A3 receptors as yet because the antibodies

available do not react with dog.

At postnatal day 5, 5’N activity and adenosine immunoreactivity are still greatest in inner retina, but adenosine is also elevated in the inner nuclear layer (Figure 3). 5’N activity is still most prominent in inner Müller cell processes, which surround developing blood vessels (Figure 3C). A2A receptor localization is also still confined to inner retina.58 By 15 days of age, inner retina still has the greatest level of 5’N activity and adenosine immunoreactivity (Figure 4). At day 22, when the inner retinal vasculature is complete and the secondary network continues to form in the inner nuclear layer, 5’N activity is greatest in the synaptic zones of the two plexiform layers. At this time (22-28 days postnatal), adenosine immunoreactivity is greatest in ganglion cells and also prominent in inner nuclear layer and photoreceptor inner segments (data not shown). This is the localization of adenosine that Braas and associates observed in adult retina.38 A2A immunoreactivity is still prominent in peripheral blood vessels in inner retina and in the secondary or deep retinal vasculature. However, A2A immunoreactivity is also now prominent in nerve fibers of inner retina and in optic nerve head.58

In summary, during development of the primary retinal vasculature in the nerve fiber layer, 5’N activity and adenosine and A2A immunoreactivity are highest in inner retina where vasculogenesis occurs. When the secondary or deep retinal vasculature forms, A2A immunoreactivity is also associated with the new deep capillaries, and adenosine immunoreactivity and 5’N activity shift toward outer retinal layers. As development of the retinal vasculature nears completion, 5’N in inner retina declines while levels in both plexiform layers increases. Coordinately, the level of adenosine in inner retina declines between day 15 and 28, except for that associated specifically with ganglion cell bodies, which remains elevated through adulthood. Levels in the developing photoreceptor inner segments and inner nuclear layer increase between 15 and 28 days of age. Therefore, 5’N at day 28 is most prominent in both plexiform layers, and adenosine is elevated in the inner nuclear layer, where A2A expression is increased at this time.

Figure 12-2. Relationship between developing blood vessels (A-B) and 5’N (C) and ADO (D) in a one-day-old dog retina. (A) Forming blood vessels in inner retina have vWf immunoreactivity. The edge of the forming blood vessels is indicated by an arrow in this and all other images at the internal limiting membrane of retina. (B) Alpha glycerophosphate dehydrogenase (αGPDH) enzyme histochemical reaction product is present in the forming blood vessels and in angioblasts (arrowheads). (C) 5’N enzyme histochemical activity is most prominent in inner Müller cell processes. (D) ADO immunoreactivity is most prominent in inner retina. (E) Relative grayscale values for 5’N in inner Müller cell processes are shown from central retina to ora serrata. The density from image analysis is greatest in areas with blood vessels and just in advance of the forming blood vessels and declines peripherally beyond the edge of the forming blood vessels. (F) ADO relative gray scale values are greatest just beyond the edge of the forming vasculature.

Figure 12-3. Blood vessels (vWf labeling), 5’N activity, and ADO in 5-day-old normal dog retina (A, C, E) and in 5-day-old oxygen-exposed animals (B, D, F). (A) At 5 days of age, only the superficial retinal vasculature is formed (arrows). (B) After exposure to hyperoxia, blood vessels are limited in inner retina and surviving channels are extremely constricted (arrows). (C) 5’N activity is greatest in the inner Müller cell processes. (D) Hyperoxia has yielded a severe reduction in 5’N activity. (E) ADO immunoreactivity in the air control is prominent in inner retina and in the anterior half of the neuroblastic layer. (F) ADO immunoreactivity is significantly lower in all areas of the hyperoxia-exposed retina. (Republished with permission of the Association for Research in Vision and Ophthalmology, Inc. Fig. 2 from Taomoto M, McLeod DS, Merges C, Lutty GA, Localization of adenosine A2a receptor during retinal vasculogenesis and oxygen-induced retinopathy. Invest. Opthalmol. Vis. Sci. 2000;41:230-243; permission conveyed through Copyright Clearance Center, Inc.)

7.ADENOSINE IN OIR, A MODEL

FOR RETINOPATHY OF PREMATURITY

Retinopathy of prematurity can be modeled in several species of neonatal animals by exposure of the neonates to high oxygen levels.60 The time of hyperoxic insult and level of oxygen used varies in the models of different species.61-65 The end result of hyperoxic insult, however, is that retinal vascular development ceases and many formed blood vessels degenerate, a process called vaso-obliteration or vaso-attenuation.54 It is of note that the choroidal vasculature, which is completely formed at birth, is unaffected by the hyperoxic insult.54 Once the animals are returned to room air, oxygen levels return to normal, but the retina becomes hypoxic because of its attenuated vasculature and because neurogenesis has progressed at some level, putting demands on the ambient retinal oxygen available.66-68 At this point, the vasoproliferative stage of the disease occurs, resulting in intraretinal and extraretinal neovascularization.

Our studies have focused on the canine model of oxygen-induced retinopathy (OIR). One-day-old dogs are placed in 95-100% oxygen for four days and then removed to room air. The hyperoxic insult results in 70% decrease in capillary density of the forming retinal vasculature with little morphological effect on retinal neurons.54 The insult also results in a significant decline in levels of both 5’N activity and adenosine immunoreactivity (Figure 3D, F), which may be due to oxygen radical damage to 5’N during exposure to hyperoxia. This has already been demonstrated by Kitakaze in heart and in polymorphonuclear leukocytes.69,70 However, Chen and associates found that 5’N activity in kidney increased after exposure to superoxide.71 It is also possible that Müller cells, which are in contact with virtually every cell type in developing retina, act as sensors of retinal oxygen levels. During the initial hyperoxic insult in OIR, when it is thought that the inner retina becomes hyperoxygenated by diffusion of oxygen from the unaffected choriocapillaris,54 Müller cells may downregulate 5’N and, therefore, decrease adenosine concentrations, favoring vasoconstriction, which occurs during the first 24 hours of hyperoxia. The recent evidence identifying a HIF-1-dependent regulatory pathway for 5’N expression suggests the latter scenario.8

After three days return to room air, the level of 5’N increased well beyond normal throughout the retina, and the level of adenosine immunoreactivity was similarly elevated in inner retina where the vasculature is reforming.36 A2A receptor expression is also significantly elevated in inner retina where the retinal vasculature becomes dilated as it is reforming.58 Patz et al., Ashton, and more recently, Chan-Ling et al. have suggested that the retina is in a state of hypoxia after vaso-obliteration and during retinal vascular development.66-68 Adenosine levels increase after induction of retinal ischemia in other animal models,44 suggesting that Müller cells could upregulate 5’N production in ischemic environments, like retina during the vasoproliferative stage of OIR. It would, therefore, be logical for 5’N activity to be high, as we have shown, during vascular development and during the period of hypoxia that follows vasoobliteration,36 since 5’N expression is HIF-1-mediated. Furthermore, the levels of immunoreactive adenosine (a product of 5’N) were elevated at the same time that 5’N activity increased, as Kitakaze has shown in heart.70

By day 15, 10 days after hyperoxic insult and return to room air, preretinal neovascularization is prominent in canine OIR. At this time, 5’N activity is greatly elevated throughout retina but is not present in preretinal neovascularization (Figure 4). Adenosine and A2A receptor immunoreactivities are greatly elevated in both inner retina and preretinal neovascularization at this time. The preretinal neovascularization is still morphologically immature and consists of angioblastic-like masses with poorly defined lumens.72 Evidence for the immaturity of these formations is the high A2A immunoreactivity (Figure 4D) and high αGPDH enzyme activity.58

By days 22-28 in dog OIR, the neovascular formations have matured, and there is a 1:1 ratio of endothelial cells to pericytes in some preretinal vessels.63 The mature formations express less A2A receptor than the immature formations at 15 days of age, and proliferation in these formations is reduced (data not shown). 5’N activity and adenosine immunoreactivity remain elevated in retina at this time, and adenosine is still prominent in the preretinal neovasculature. During this period, astrogliosis has occured throughout the inner retina, even in avascular regions where astrocytes are not present during normal vasculogenesis.63

Our results suggest that A2A receptor is associated with normal vasculogenesis and angiogenesis in OIR in the dog. However, we have been unable to examine the localization of A2B receptors in dog because of the lack of an appropriate antibody. Mino and associates used a mouse model of OIR to study the effects of adenosine receptor antagonists on angiogenesis.1 Antagonists selective for A1, A2A, and A2B receptors were evaluated by

intraperitoneal administration of the antagonists daily for five days starting immediately after hyperoxia. Only the A2B antagonist significantly inhibited neovascularization in this model of OIR. The preretinal neovascularization is not only robust but persists up to P-45, 40 days after hyperoxia, and tractional retinal folds can form as early as P-28.63

Adenosine may also contribute to other aspects of OIR and retinopathy of prematurity. Vasodilation is prominent during the proliferative stage in canine OIR and during vascular development in dog. Adenosine and VEGF could be responsible for this as well. Adenosine is a potent vasodilator, and the A2A receptor, specifically, is an important modulator of vascular tone.74,75 Binding of the A2A receptor on endothelial cells and smooth muscle cells induces vasodilation by stimulating L-arginine transport and nitric oxide (NO) production.76,77 Gidday and Park have demonstrated that A2 receptors can specifically modulate vasodilation in the neonatal pig.42 Extreme vasodilation associated with increased adenosine and A2A receptors in oxygen-treated animals may contribute to the tortuousity of arteries and hemorrhage we have observed in the canine model of OIR.63 The importance of NO in OIR was recently demonstrated by Brooks et al.73 They found that vaso-obliteration in the mouse model of OIR was significantly reduced by inhibiting NO synthase with NG-nitro-L-arginine (L-NNA). Furthermore, both vaso-obliteration and vasoproliferation were significantly reduced in endothelial NO synthase (eNOS) knockout mice.

8.CONCLUSIONS

There is an intimate relationship between retinal vascular development and adenosine. During vasculogenesis in inner retina, 5’N produces high levels of adenosine in the region where vasculogenesis is occurring (Figure 5). Both angioblasts and endothelial cells have adenosine A2A receptors in the dog. When inner retinal vasculature development is complete, 5’N activity and elevated adenosine are present in more posterior retina, where the secondary or deep capillary network is forming. Adenosine is also associated with angiogenesis in the canine model of OIR. After exposure to hyperoxia, retinal vascular development ceases and vaso-obliteration occurs. This is accompanied by significant reductions in 5’N activity and adenosine (Figure 5). When the vasoproliferative stage of OIR begins, 5’N activity and adenosine levels are elevated well beyond normal. Angiogenesis at this stage is accompanied by elevated levels of A2A receptors in the retinal vasculature and in preretinal neovascular formations. Astrogliosis also occurs in inner retina at this time, which may retard anterior vascular growth (Figure 5).