Ординатура / Офтальмология / Английские материалы / Retinal and Choroidal Angiogenesis_Penn_2008

angiogenesis, hypoxia is the initial stimulus that causes the upregulation of growth factors, integrins and proteinases that results in endothelial cell proliferation and migration, essential steps in new vessel formation.24

4.CELL-CELL INTERACTIONS IN THE FORMATION OF THE RETINAL VASCULATURE

The retina consists of three main cellular elements: the neurons, macroglia (including astrocytes and Müller cells), and the vasculature (including vascular endothelial cells, pericytes, and smooth muscle cells). Immune and phagocytic cells including retinal microglia, macrophages, and perivascular antigen presenting cells complete the cellular milieu. During the formation of the human retinal vasculature, these cellular elements interact in complex ways, resulting in the formation and then the remodeling of the vasculature to produce a vascular tree that is well matched to the metabolic needs of the tissue.

Cells of the astrocytic and vascular lineage interact closely during formation of the mammalian retinal vasculature (Figure 5). In a comparative study, Schnitzer has shown that occurrence of astrocytes in mammalian retinae coincides with the presence of blood vessels.25 We have shown the

close association of retinal astrocytes with the forming superficial vascular plexus in the developing kitten, rat and human retinae.1,7,26,27 More recent

studies have shown that the superficial vascular network in the neonatal mouse retina forms according to a pre-existing astrocytic template, and both the superficial and deep vascular layers use R-cadherin cell adhesion molecules as guidance cues.28

In the human fetal retina, Pax-2 expression is restricted to cells of the astrocytic lineage.29 Pax-2 is a member of the Pax family of transcription factors. Each member of the Pax family is expressed in a spatially and temporally restricted manner, which suggests that these proteins contribute to the control of tissue morphogenesis and pattern formation. Pax-2+/GFAP- astrocyte precursor cells (APCs) are first evident at the optic nerve head at 12 WG, preceding the appearance of Pax-2+/GFAP+ astrocytes. These immature astrocytes are seen immediately peripheral to the leading edge of vessel formation (approximately 20-40 μm) and at 18 WG loosely ensheath the newly formed vessels.7 With maturation, Pax2+/GFAP+ astrocytes extend toward the periphery, reaching the edge of the retina around 26 WG.29 The location of the astrocytes and APCs just ahead of the leading edge of vessel formation places them in an ideal position to mediate the angiogenic response to “physiological hypoxia” via upregulation of VEGF165 expression.17

Retinal vessels have blood-retina barrier (BRB) properties as soon as they become patent. Astrocytes have been shown to be responsible for inducing the blood-brain barrier properties in vascular endothelial cells30 and thus are thought to induce the BRB in the inner plexus. The processes of the Müller cells (the radial glia of the retina) ensheath the vessels of the outer plexus and are also capable of induction of the BRB.31

During normal human retinal vascularization, significant overproduction of vascular segments occurs, and the excess segments regress with maturation of the vasculature. Our earlier work has shown that endothelial cell apoptosis and macrophages do not initiate vessel retraction, but rather contribute to the removal of excess vascular endothelial cells throughout the immature retinal vasculature. Furthermore, our observations suggest that vessel retraction is mediated by endothelial cell migration and that endothelial cells derived from retracting vascular segments are re-deployed in the formation of new vessels.14 Mural cells (pericytes and smooth muscle cells) are an intrinsic part of blood vessel walls with broad functional activities, including blood flow regulation, and have been implicated in vessel stabilization.32 These cells are derived from a mural precursor cell which gives rise to pericytes on capillaries and smooth muscle cells on larger vessels.33 Immature mural cells, the ensheathing mural precursor cells, in cats, rats and mice, envelop newly formed vessels and have recently been shown to express VEGF165.3 The presence of these immature mural cells does not prevent vessel regression during normal development33 and hyperoxia-induced vessel regression.34 Because mature vasculatures with mature mural cells are considered stable, this suggests that mural cell maturation may be necessary for resistance to VEGF165 withdrawal-induced vessel regression. Macrophages are also part of the cellular milieu during formation of retinal vessels.35 Although their function is unclear, they are capable of expressing VEGF165 in the mouse model of hyperoxia-induced retinopathy36 and in ARMD.37

The endothelium in turn influences the development of astrocytes and mural cells. Retinal endothelial cells express PDGF-beta, which induces recruitment and proliferation of mural cells.38 It has also been shown that vascular endothelial cells can induce astrocyte differentiation.39,40 In addition, contact between mesenchymal precursor cells and vascular endothelial cells leads to mural cell differentiation in vitro.41 Taken together, these studies show that the formation and maturation of the human retinal vasculature is (1) concurrent with neuronal, astrocyte, and mural cell

differentiation and maturation, and (2) the result of complex cellular interactions in which the vasculature both takes its developmental cues from and also influences its cellular environment. During normal development, the retinal vasculature is remodeled, resulting in a vascular pattern that is well matched to the metabolic demands of the tissue. The retinal vasculature is under the constant influence of its environment, where neovascularization or regression is determined by the equilibrium between pro-angiogenic and anti-angiogenic signals, proteases42 and other molecular cues.

Figure 6-5. Astrocyte/endothelial relationship during formation of the superficial human retinal vasculature. (A–C) Human fetal retinal whole mounts triple labeled with Pax2/GFAP/CD34 at 14 WG. (A) At 14 WG, Pax2+(TR)/GFAP-(FITC) astrocyte precursor cells (APCs) extended in advance of the leading edge of CD34(FITC) blood vessels by a small but distinct margin. Arrows: some neonatal astrocytes that were just starting to express GFAP. (B) Representative field of view from mid-retina. Arrows: Pax2+/GFAP- APCs. Most cells in this area were Pax2+/GFAP+ immature astrocytes. CD34+ blood vessels (FITC) were also clearly evident. (C) A region near the optic nerve head (ONH) of a 14-WG fetus. Arrows: Pax2+/GFAP- APCs. Most of cells in this area were Pax2+/GFAP+ immature astrocytes. (D) Map of the 14-WG retina shown in A–C. Red somas: Pax2+/GFAP- APCs; yellow somas: Pax2+/GFAP+ immature astrocytes; green: CD34+ blood vessels. White boxes are the representative areas of the fields of view seen in A-C. (Modified from 7)(E-L) Pericyte/endothelial relationship during formation of the superficial rat retinal vasculature. (E) Representative fields of view of an E20 preparation of retinal vasculature, triple labeled with anti-SMA (green), anti-desmin (red), and GS lectin (blue). (Note: desmin was not detected).

(F) Postnatal day (P) 0 rat retinal vasculature triple labeled with anti-NG2 (green), antidesmin (red), and GS lectin (blue) showing ensheathing mural precursor cells (MPCs) with desmin filaments on undifferentiated vessels. (G) Embryonic day (E) 21 retinal vasculature triple labeled with anti-NG2 (green), anti-desmin (red), and GS lectin (blue) showing tips of vessels extending peripherally (Note: desmin was not detected in this field). (H) Capillaries in the adult retina triple labeled with GS lectin (blue), anti-desmin (red), and anti-NG2 (green). Shows an adult quiescent pericyte. (I) P0 preparation of rat retina, triple labeled with antiSMA (green), anti-desmin (red), and GS lectin (blue) showing differentiating central radial vessels immediately adjacent to the optic nerve head (Note: desmin was not detected in this field). (J) SMC differentiation in the rat retina. Central radial arteriole double labeled at P7 with anti-SMA (green), anti-desmin (red), and GS lectin (blue) showing an immature central radial arteriolar smooth muscle cell (SMC). (K) Central radial rat arterioles double labeled at P13 with anti-SMA (green), showing a juvenile central radial arteriolar SMC. (L) Adult radial and primary arterioles in the central retina double labeled with anti-SMA (green) and antidesmin (red). The abbreviated marker names are colored to represent the respective fluorescent dye in each micrograph. (Modified from 7,33)

5.CONCLUSION

Observations to date support the conclusion that the formation of primordial vessels of the superficial plexus in the central human retina is mediated by vasculogenesis, whereas angiogenesis is responsible for increasing vascular density and peripheral vascularization in the superficial retinal plexus. In contrast, the vessels in the perifoveal region and the deeper retinal plexus and the radial peripapillary capillaries are formed by angiogenesis only. Our understanding of retinal vessel formation is based on many sources, including the seminal works of Michaelson, Ashton, Dollery, Weiter, D’Amore, Schlingemann, Lutty, Penn, Das, Smith, Friedlander, Gariano, and our own review and analyses.

The fact that the human retina is vascularized through two distinct pathways with distinct molecular cues and cellular processes as highlighted in this review offers the attractive possibility of using distinct inhibitory and stimulatory methods for intervention. With a clear understanding of the cellular and molecular cues that drive normal retinal vascularization, we can gain clues to the mechanism underlying neovascularization. These insights could be of relevance to neovascularizing retinopathies of infancy and adulthood.

ACKNOWLEDGMENT

This contribution would not have been possible without the generous assistance provided by Ruth-Ann Sterling, Suzanne Hughes, and Louise Baxter. This work was supported by grant #153789 and #402824 from the National Health and Medical Research Council of Australia and the Financial Markets Foundation for Children.

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33.S. Hughes and T. Chan-Ling, Characterization of smooth muscle cell and pericyte differentiation in the rat retina in vivo. Invest. Ophthalmol. Vis. Sci. 45 (8), 2795-2806, (2004).

34.T. Chan-Ling, M. P. Page, T. Gardiner, L. Baxter, E. Rosinova, and S. Hughes, Desmin ensheathment ratio as an indicator of vessel stability: evidence in normal development and in retinopathy of prematurity. Am. J. Pathol. 165 (4), 1301-1313, (2004).

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Chapter 7

IGF-1 AND RETINOPATHY

Lois E. H. Smith, MD, PhD

Department of Ophthalmology, Children’s Hospital, Harvard Medical School, Boston,

Massachusetts

Abstract: Retinopathy continues to be a major cause of blindness in children (retinopathy of prematurity, ROP), in adults (diabetic retinopathy), and in the elderly (age-related macular degeneration), despite current therapy. Although ablation of the retina reduces the incidence of blindness by suppressing the neovascular phase of ROP and diabetic retinopathy, the visual outcomes after treatment are often poor. Preventive therapy is required and will likely come from a better understanding of the pathophysiology of the disease.

1.TWO PHASES OF ROP AND DIABETIC RETINOPATHY

Retinopathy of prematurity (ROP) was first recognized in the late 1940’s and was associated with excessive oxygen use.1 Despite controlled oxygen delivery, the number of infants with ROP has increased further, probably because of the increased survival of very low birth weight infants,2 indicating the likely association of ROP with both oxygen-related and non oxygen-related growth factors.

Both ROP and diabetic retinopathy occur in two phases. In the first phase of ROP, there is cessation of the normal retinal vascular growth, which would normally occur in utero, as well as loss of some of the developed vessels. As the infant matures, the resulting non-vascularized retina becomes increasingly metabolically active and increasingly hypoxic. Similarly, the first phase of diabetic retinopathy consists of slow loss of capillaries associated most prominently with poor control of hyperglycemia. The second phase of ROP and diabetic retinopathy, retinal neovascularization or

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© Springer Science+Business Media B.V. 2008

proliferative retinopathy, is hypoxia-induced.3 In ROP, the onset occurs at about 32 weeks post-menstrual age, and the progression of neovascularization is similar to that in adult diabetic retinopathy.

There are no rodent models available for studying proliferative or neovascular diabetic retinopathy. We developed a mouse model of ROP to take advantage of the genetic manipulations possible in the murine system. The eyes of some animals, though they are born full-term, are incompletely vascularized at birth and resemble the retinal vascular development of premature infants. Exposure of these animals to hyperoxia causes vasoobliteration and cessation of normal retinal blood vessel development, which mimics Phase I of ROP.4-6 When mice return to room air, the nonperfused portions of the retina become hypoxic, which in turn causes retinal neovascularization similar to Phase II of ROP and of other retinopathies.

2.VEGF AND PHASE II OF ROP

Because hypoxia is a driving force for retinal neovascularization or proliferative retinopathy, we first searched for a hypoxia-regulated factor during Phase II of ROP.

Vascular endothelial growth factor (VEGF) is a hypoxia-inducible cytokine7 and is a vascular endothelial cell mitogen.8 In the mouse5, retinal hypoxia stimulates an increase in the expression of VEGF before the development of neovascularization.9 Furthermore, inhibition of VEGF decreases the neovascular response,10,11 indicating that VEGF is a critical factor in retinal neovascularization. Other investigators have also shown that VEGF is associated with ocular neovascularization in other animal models, confirming the central role of VEGF in neovascular eye disease.12-15 These results have been corroborated clinically. VEGF is elevated in the vitreous of patients with retinal neovascularization.16 In a patient with ROP, VEGF was found in the retina in a pattern consistent with mouse results.14

3.VEGF AND PHASE I OF ROP

In animal models, the first phase of ROP is also VEGF-dependent. VEGF is required for normal blood vessel growth. VEGF is found anterior to the developing vasculature, in what has been described as a wave of physiological hypoxia that precedes vessel growth.17,18 As the retina develops anterior to the vasculature, there is increased oxygen demand, which creates localized hypoxia. VEGF is expressed in response to the hypoxia, and blood vessels grow toward the VEGF stimulus. As the hypoxia