Ординатура / Офтальмология / Английские материалы / Retinal and Choroidal Angiogenesis_Penn_2008

nuclear and outer plexiform layers (Figure 1B). The superficial plexus contains arterioles, venules, capillaries, and post-capillary venules, while the deep vascular bed consists pre-dominantly of capillary-sized vessels. Both the superficial and deep retinal plexuses reach almost to the edge of the human retina, except for a small avascular rim, where the thinness of the human retina likely permits adequate retinal oxygenation via the choroidal vasculature.1,5

A third intraretinal plexus, the radial peri-papillary capillaries (RPCs), is also located in the nerve fiber layer in a small rim surrounding the optic disc (see Figure 10 D-F in 6; Figure 6 E-F in 1). These RPCs are located superficially in a small region surrounding the optic nerve head where the nerve fiber bundles are thickest prior to exiting the retina. Their superficial location and the fact that these vessels lack smooth muscle actin ensheathment (personal observation) could make the nerve fiber bundles nourished by these vessels uniquely prone to ischemic damage as a consequence of reduced blood flow during periods of raised intraocular pressure.

1.2Two distinct mechanisms in the formation of the human retinal vasculature

Blood vessels in the human retina form by vasculogenesis, angiogenesis1,7-9 and intussusception.10 The term vasculogenesis describes the de novo formation of vessels from vascular precursor cells (VPCs), also called mesenchymal precursor cells,1,5,7 and angioblasts.9 Single spindle-shaped CD39+, Nissl stained VPCs (Figure 1C-F) stream in superficially from the optic nerve head into the avascular retina and differentiate at the location of future vessels, coalesce into cords (Figure 1F-H), differentiate into endothelial cells, and ultimately form patent vessels (Figure 1I-J and 1,6-9). These VPCs precede the leading edge of patent vessels by more than 1 mm (Figure 2A and 7). They differentiate to form a primordial vascular bed centered on the optic disk (Figure 1I-J). During human retinal vascular development, superficial inner retinal vessels form by vasculogenesis, starting at the optic nerve and developing along a gradient from the posterior to the anterior retina. Vasculogenesis is only responsible for formation of the primordial vessels that span the inner two-thirds of the superficial retinal plexus (Figure 2A).

The remaining retinal vessels form via angiogenesis, which produces increasing capillary density in the central retina, formation of the peripheral blood vessels of the superficial retinal plexus, formation of the deep vascular plexus, and formation of the RPCs. The term angiogenesis describes a different process of blood vessel formation in which proliferating endothelial cells from pre-existing blood vessels extend the vascular network. Angiogenesis can take place via budding or intussusception. Budding angiogenesis involves filopodial extension by proliferating and migrating vascular endothelial cells (Figure 2C-D and 1,11). The term intussusception describes the remodeling and expansion of new vessels by the insertion of interstitial tissue columns into the lumen of pre-existing vessels.10

2.TIMING AND TOPOGRAPHY OF HUMAN RETINAL VASCULAR FORMATION

The first event in retinal vascularization apparent before 14 weeks’ gestation (WG) is the appearance of large numbers of CD39+, Nissl stained VPCs centered around the optic nerve head (Figure 1C-H and 1,7). These VPCs are concentrated in 4 lobes of the future major artery-vein pairs of the human

Figure 6-1. (A-B) Developing human retina from an avascular undifferentiated neuroepithelium (A) to fully stratified adult retina (B). Two plexuses of retinal vessels are apparent. The superficial plexus is located predominantly in the ganglion cell and nerve fiber layers showing a range of vessel calibers from arterioles and venules to capillaries. The second deeper plexus is located at the junction of the inner nuclear layer and the outer plexiform layer with predominantly capillary-sized vessels. The formation and maturation of the human retinal vasculature is concurrent with neuronal differentiation and maturation. (C- D) Lowand high-magnification views of a Nissl-stained human retinal whole mount at 14 to 15 weeks’ gestation (WG). C shows a region that is immediately adjacent to the optic nerve head (lower left-hand corner). Large numbers of spindle-shaped cells (arrowheads in C), which are interspersed among other somas, stream in superficially from the optic nerve head. In D, spindle-shaped, presumably vascular precursor, cells join to form vascular cords of cells (arrowheads at top right). (E) 16.5-WG human retina labeled with CD39. Spindleshaped CD39+ vascular precursor cells streaming in superficially from the optic nerve head.

(F) Appearance of the edge of vasculature using Nissl-stained preparation from an 18-WG specimen. Nissl staining showed spindle-shaped cells in advance of the vasculature. (G-H) CD39+/CD34-/+ solid vascular cords at the leading edge of vessel formation at 17 WG. (I-J) Lowand high-magnification views of the first primordial vascular arcades labeled with CD34, evident in the region of the optic nerve head at 15 WG. These vascular arcades show the four-lobed topography of formation that is indicative of the future superior and inferior (temporal and nasal) artery vein pairs. Morphologically, they are straight and lack significant capillary density. (Modified from 1)

retina. Figure 2A shows the distribution of these cells at 14-15, 18 and 21 WG (modified from 1). Formation of the patent superficial vascular plexus begins by 14 to 15 WG. These primordial vessels are centered on the optic disc and show a four-lobed topography (Figure 2A). In the following weeks, the inner vascular plexus extends peripherally, curving around the location of the incipient fovea (Figure 2A). By 32 WG, the inner plexus reaches its outer limits, leaving a narrow rim of avascular tissue at the periphery of the retina.

In contrast, the formation of the outer vascular plexus begins in the perifoveal region between 25 and 26 WG, coincident with the peak period of eye opening, when the visually evoked potential, indicative of a functional visual pathway and photoreceptor activity, is first detectable in the human infant.12 Formation of the deeper vascular plexus subsequently spreads with an elongated topography along the horizontal meridian (Figure 2B) and is centered around the fovea, rather than the optic disc, thus mimicking the topography of photoreceptor maturation.13 The timing and topography of formation of the deeper plexus supports the conclusion that angiogenesis is driven by increasing metabolic demand as a result of neuronal maturation. The outer plexus forms via extension of capillary-sized buds from the existing superficial vessels. This deeper plexus reaches the edge of the human retina by birth.

2.1Angiogenesis results in increasing capillary density followed by vascular regression and remodeling

In addition to its contribution to the spread of vessels peripherally, angiogenesis also is responsible for increasing the vascular density of the primordial plexus formed by vasculogenesis. Initially, capillary networks are rare (Figure 1I-J). However, by 18 WG, regions of active sprouting begin to give rise to substantial capillary networks within the existing vascular tree (Figure 2C). As the retina increases in area and the radial vessels spread peripherally, the distance between these vessels becomes greater, and it is in these avascular spaces that sprouting angiogenesis is most pronounced. Filopodia extend, establish contact with other filopodia or vessels, and subsequently dilate to form vascular segments. Moreover, sprouting is evident even from the edges of larger preformed vessels and is especially marked near and along veins. By 21 WG, exuberant immature capillary plexuses are apparent throughout the vascular tree. Thus, angiogenesis augments the initial radial vessels by increasing capillary density.

Figure 6-2. (A) The distribution of the spindle cells shown at 14.5, 18, and 21 WG. The stippled regions show the distribution of the Nissl stained spindle cells at each age; the white regions show the areas with vascular cords. Both the spindle-shaped vascular precursors and the vascular cords were more extended in the temporal and superior directions than in the nasal direction. With increasing maturity, the outer limit of the vascular cords expanded markedly, whereas that of the vascular precursor cells did not. At 21 WG, no spindle cells were evident in the retina. It is clear from these maps that the area formed by vasculogenesis is not circular in the developing human retina. The X indicates the location of the incipient fovea. (B) Maps of the outer limits of the inner and outer vascular plexuses as well as that of the RPCs at various times during development of the human fetal retina. (C-D) CD34-stained human retina from an 18 WG specimen. Red blood cells were apparent in patent vessels utilizing Normaski optics. Angiogenic budding as evidenced by filopodial extensions was abundant. (Modified from 1)

A remarkable feature of angiogenesis is the exuberance of the initial vessels during formation of the superficial human retinal plexus. Our earlier work in the rat retina has shown that this significant overproduction with subsequent vascular remodeling, or pruning, involves a combination of apoptosis of vascular endothelial cells and withdrawal of endothelial cells from excess vascular segments into neighboring vascular segments, where they are utilized to form new vessel segments.14

2.2Formation of the Perifoveal and Temporal Raphe Vessels by Angiogenesis

The incipient fovea is avascular at 25 WG (see Figure 6A-B in 1). The avascular zone is oval in shape, with a diameter of 500 to 600 mm. Because no spindle cells were observed in the region of the temporal raphe or the perifoveal region of the human retina, these areas must be vascularized by angiogenesis alone.

2.3Formation of the Deep Vascular Plexus by Angiogenesis

Capillary sprouting from the inner vascular plexus is first evident at the fovea between 25 and 26 WG. Capillary-sized buds descend into the inner nuclear and outer plexiform layers, giving rise to small vascular segments in a deeper plane (Figure 3B-C). With maturation, a confluent outer plexus becomes apparent.

2.4Formation of RPCs by Angiogenesis

Fine RPCs were evident in the nerve fiber layer, extending from the inner vasculature, from 21 WG. RPCs in the region of the optic nerve head of 25and 26-WG retinas (see Figures 6E-F in 1) were located superficially in the nerve fiber layer and extended radially from the optic nerve head. The timing and extent of their formation suggest that their formation is driven by hypoxia-mediated VEGF resulting from the need to satisfy the metabolic requirements of the thick nerve fiber layer that surrounds the optic nerve head.

3.TWO DISTINCT PATTERNS OF MITOTIC ACTIVITY ASSOCIATED WITH RETINAL VASCULOGENESIS AND ANGIOGENESIS IN THE KITTEN AND HUMAN RETINA

Bromodeoxy-uridine (BrdU) is an analog of thymidine and can be used to identify proliferating cells. We have previously combined BrdU with endothelial cell-specific markers to demonstrate the distinct pattern of cell division associated with vasculogenesis and angiogenesis in the kitten retina.15,16 Vasculogenesis and angiogenesis are associated with markedly different patterns of mitotic activity. The formation of vessels by vasculogenesis is preceded by low mitotic activity among the vascular precursor cells some millimeters peripheral to the edge of the patent vessels (Figure 3A). More centrally, mitotic activity increases significantly during formation of patent vessels (Figure 3A), continues at a lower level as large vessels differentiate from the initial capillary plexus, and then falls to zero in the adult cat. Central to this leading edge of vessel formation, dividing vascular endothelial cells were frequently evident in the vascular tree as they remodeled and selected major channels. Possibly as a result of the higher oxygen tension in arteries than in veins, the density of mitotically active vascular cells was markedly higher in veins than in arteries.16

In contrast, the formation of vessels by angiogenesis is not preceded by migration and division of vascular precursor cells. During angiogenesis,

division occurs only in the endothelial cells close to, but not at the tips of, growing vessels (Figure 3B-C and 15,16). A third vascular plexus that we have

previously described as being formed by vascular budding or angiogenesis comprises the RPCs. These vessels are fine capillaries that radiate out from the region of the optic nerve head in the nerve fiber layer. Their formation is likely the result of the fact that the nerve fiber layer is thickest in this region, so that its metabolic requirements are not adequately satisfied by the superficial retinal plexus. Mitotic activity associated with the formation of RPCs is similar to that typical of angiogenesis (Figure 3D), with one or two mitotic nuclei evident close to the tip of the forming capillaries.

We have recently developed a new in vitro technique for identifying vascular proliferation in human retina and choroid. Utilizing triple-label immunohistochemistry for CD39/CD34/BrdU, it was possible to demonstrate that both CD39+/BrdU+ vascular precursor cells and CD34+/BrdU+ vascular endothelial cells proliferate in situ in the human retina (Figure 4). A comparison of 4E and 4F shows that a higher proportion of CD30+ cells are proliferative than CD34+ cells.

Figure 6-3. (A-D) BrdU/Griffonia isolectin B4 double label histochemistry to visualize proliferating vascular endothelial cells in kitten retina. New vessel formation at the outer vascular plexus occurs by a budding process. (A) Small numbers of proliferating vascular precursor cells are evident preceding the leading edge of vessel formation, toward the right of the field of view. Large numbers of mitotic endothelial cells are evident within the leading edge of patent vessel formation. (B-C) Mitotic cells are evident close to the tips of angiogenic buds, but not at the tips of angiogenic buds (15, 16). Fields of view show new vessel growth in the outer vascular plexus of a P8 retina. Note the clear absence of mitotic vascular precursor cells in the region preceding the growing vessel bud. (D) Low numbers of mitotic cells associated with the formation of RPCs of a P28 kitten. (E-F) Desmin and Griffonia simplicifolia isolectin B4 double-labeled P8 rat retina showing desmin+ mural precursor cells

are present concurrently with the presence of newly formed vascular segments. (Modified from 33)

Figure 6-4. Vascular precursor cells and vascular endothelial cells in human retina proliferate in situ. (A-G) An 18-WG human retinal whole mount labeled with CD39 (Cy5), CD34 (Alexa 488) and BrdU (Cy3). Panel D shows overlapping expression between CD39+ and CD34+ cells as the cells mature along the vascular endothelial cell lineage. (E-F) Significant proportions of both CD39+ and CD34+ cells were also BrdU+. A comparison between E and F shows that a greater proportion of CD39+ vascular precursor cells are proliferative than the CD34+ vascular endothelial cells. (G) Triple labeling of 18-WG human retina showing CD39+/CD34+/BrdU+ vascular precursor cells (arrowheads), CD39-/CD34-/BrdU+ soma, likely an astrocyte or neuron (arrow), and nonproliferative vascular endothelial cells lining a lumen (double arrows). Scale bar = 50µm

3.1Formation of retinal vessels via vasculogenesis is independent of VEGF165

A review of the literature and our own observations in the developing human retina led us to conclude that formation of retinal vessels via vasculogenesis is independent of metabolic demand and hypoxia-induced VEGF expression.1 Evidence for this conclusion includes the observations that (1) substantial vascularization in the human retina occurs prior to detection of VEGF mRNA,17 (2) vasculogenesis is well established by 14 to 15 WG, before the differentiation of most retinal neurons, and (3) the topography of formation of vessels by vasculogenesis does not correlate at all with the topography of neuronal density and maturation. Formation of the outer plexus begins around the incipient fovea between 25 and 26 WG, coincident with the peak period of eye opening and the first appearance of the visually evoked potential, indicative of a functional visual pathway and photoreceptor activity.1

Further, comparative analysis of retinal vascularization in other species has shown that VEGF expression, tissue oxygen levels, and vascularization are not always correlated.18 The guinea pig retina is virtually anoxic and yet remains avascular,19 whereas overexpression of VEGF in the avian retina did not induce vascularization.20 Further evidence of the independence of vasculogenesis from VEGF is provided by VEGF knockout mice. In these animals, in which not only paracrine but also autocrine VEGF production is lost, vessels still form by vasculogenesis but are highly abnormal.21 Reduced VEGF expression in mice heterozygous for the VEGF null mutation is associated with the formation of vessels in the forebrain mesenchyme but not in the neuroepithelium.22 Given that the formation of vessels in the forebrain mesenchyme is thought to occur by vasculogenesis, whereas that within the neuroepithelium is thought to take place by angiogenesis, these observations provide further evidence that vasculogenesis is not dependent on hypoxiainduced VEGF expression.

3.2Retinal angiogenesis in human retina is mediated by

hypoxia-induced VEGF165 expression by astrocytes,

Müller cells and pericytes

In marked contrast to vasculogenesis, the timing and topography of angiogenesis in the human retina supports the conclusion that angiogenesis is induced by “physiological hypoxia,” a transient but physiological level of hypoxia induced by the increasing activity of retinal neurons.15,23 The formation of retinal vessels via angiogenesis is mediated by hypoxia-induced

VEGF expression by astrocytes, Müller cells2,17 and pericytes.3 In retinal