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1 From the Department of Anatomy and Histology and Institute for Biomedical Research, University of Sydney, Sydney, NSW, Australia; 2 Department of Anatomy, Western China University of Medical Sciences, Chengdu, China.
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Abstract |
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METHODS. The vasculature of human eyes obtained from fetuses ranging in age from 14 to 38 weeks of gestation (WG) was examined in Nissl-stained, whole-mount preparations and by anti-CD34 immunohistochemistry.
RESULTS. The first event in retinal vascularization, apparent before 15 WG, was the migration of large numbers of spindle-shaped mesenchymal precursor cells from the optic disc. These cells proliferated and differentiated to produce cords of endothelial cells. By 15 WG, some cords were already patent and formed an immature vascular tree in the inner retinal layers that was centered on the optic disc. These processes are consistent with vessel formation by vasculogenesis. Angiogenesis then increased the vascular density of this immature plexus and extended it peripherally and temporally. Maturation of the plexus was characterized by substantial remodeling, which involved the withdrawal of endothelial cells into neighboring vascular segments. The outer plexus was formed as a result of the extension of capillary-sized buds from the existing inner vessels, a process that began around the incipient fovea between 25 and 26 WG.
CONCLUSIONS. These observations suggest that the formation of primordial vessels in the central retina is mediated by vasculogenesis, whereas angiogenesis is responsible for increasing vascular density and peripheral vascularization in the inner retina. In contrast, the outer plexus and the radial peripapillary capillaries are formed by angiogenesis only. These mechanisms of retinal vascularization appear similar to those of vascularization of the central nervous system during development.
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Introduction |
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The retina, embryologically an extension of the diencephalon, is an excellent model in which to study vascular development in the CNS. The retinal vasculature consists of inner and outer layers that are joined by fine capillaries. The thin laminar structure of the retina renders it suitable for whole-mount preparations that allow visualization of the entire forming vasculature in situ. An understanding of normal retinal vascularization is particularly important, given that several retinopathies are caused by abnormal vessel growth. The developing retinal vasculature of premature infants is extremely vulnerable, and perturbation of the normal developmental processes can result in retinopathy of prematurity (ROP), the leading cause of infant blindness in the Western world. The incidence of blindness resulting from ROP increases markedly for infants born before 26 weeks of gestation (WG).2 However, relatively little is known about the normal state of human retinal vascularization at this developmental stage or of the reasons for its pronounced susceptibility. Such information would be invaluable to ophthalmologists and neonatologists treating premature infants, as well as to clinicians participating in the National Institutes of Health multicenter trial "Stop ROP" for evaluation of the benefits of supplemental oxygen therapy as a noninvasive treatment for ROP.3 4
Vascular development in the retina has been examined in several species. Initially, spindle-shaped cells are apparent migrating ahead of the developing inner vasculature. These cells are characterized by their labeling with Griffonia simplicifolia isolectin and their distribution as revealed by Nissl staining in the cat5 as well as by their ATPase6 and ADPase7 activities in the dog. They subsequently coalesce to form solid vascular cords, which in turn give rise to patent vessels,8 9 suggesting that vasculogenesis contributes to formation of the inner retinal plexus.5 Further vascularization of the cat retina occurs by angiogenesis.5 Our previous studies led us to suggest that formation of retinal vessels is promoted by the increased metabolic demand of neurons, which results in local tissue hypoxia, or "physiological hypoxia,"5 10 11 and that this effect is mediated by vascular endothelial growth factor (VEGF), a potent angiogenic protein induced by hypoxia.12
Despite insights gained from animal studies, our knowledge of the normal development of the human retinal vasculature is incomplete. Previous human studies have examined ink-perfused retinas (in which only patent vessels are apparent), digested tissue (in which the normal relations of vessels with neighboring structures are destroyed), or transverse sections (which provide little information on the topography of vessel formation) or were restricted to small numbers of specimens.13 14 15 16 17 18 19 20 The importance of studying the human retina is highlighted by comparative data showing that, although retinal vascularization in humans resembles that in other mammals, there are significant differences.13 20 21 In addition, formation of the human retinal vasculature occurs in utero, where arterial oxygen tension is <30 mm Hg, whereas substantial portions of the cat and rat retinal vasculatures are formed after birth, at markedly higher arterial oxygen tensions.
We have now examined a series of Nissl-stained, retinal whole-mount preparations from human fetuses at 14 to 26 WG for evidence of vascular precursor cells and have mapped changes in their topographical distribution with maturation. In addition, we have applied immunohistochemistry with an antibody to human CD34, a protein that is expressed by hematopoietic precursor cells and capillary endothelial cells, to visualize the formation of blood vessels from 15 to 38 WG. Our study thus represents the most complete description to date of both the cellular and topographical features of the forming retinal vasculature in humans.
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Methods |
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After enucleation of the eyes, the anterior segment and vitreous were removed, and the eyecup was fixed at 4°C for a minimum of 2 days with 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4). The retina was then dissected as previously described.23 A small series of Nissl-stained (1% cresyl violet), human retinal whole-mounts prepared previously by J. Provis24 also was examined.
Anti-CD34 and Anti-GFAP Immunohistochemistry
The vasculature was visualized by immunohistochemistry with a
monoclonal antibody (QBEND/10, 1:50 dilution; Serotec, Oxford,
UK) to CD34, a single-chain transmembrane glycoprotein with a
molecular mass of 110 kDa that binds L-selectin and is selectively
expressed on human lymphoid and myeloid hematopoietic progenitor cells
as well as on the filopodial extensions and the luminal membrane of
endothelial cells.25
Astrocytes were visualized with
rabbit polyclonal antibodies (1:2 dilution; Biogenex, San Ramon,
CA) to glial fibrillary acidic protein (GFAP). Retinas were
labeled with these antibodies as previously described.26
Mapping of the Outer Limit of the Retinal Vasculature
The outer limit of spindle-shaped presumed vascular precursor
cells and the outer limit of vascular cords were determined in three
specimens aged 14 to 15, 18, and 21 WG that had been subjected to Nissl
staining. In addition, maps of the extent of retinal vascularization at
various ages were prepared from a series of retinas subjected to
anti-CD34 immunohistochemistry. Retinal boundaries, the outer limit of
CD34+ vessels in both inner and outer layers of
the vasculature, and the radial peripapillary capillaries (RPCs) were
mapped for each retina with a 1-mm grid incorporated into the 10x
eyepiece of an Olympus Vanox microscope (Tokyo, Japan), as
previously described.5
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Results |
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Role of Angiogenesis in Vascular Spread and in Increasing Vascular
Density in the Inner Retinal Plexus
Contribution of Angiogenesis to Vascular Spread.
The antibody to CD34 labeled the vasculature but did not label the
spindle cells. At 15 WG, patent radial vessels, with few
interconnecting segments, were apparent emanating from the optic disc
(Fig. 4A) . This vascular pattern suggests the existence of only a low
level of metabolite exchange. As the retina matured, capillary networks
became apparent, linking the radial vessels. At 17 to 18 WG, discrete
netlike vascular formations (Figs. 4B
4C)
were observed at the leading
edge of vessel formation. Localized exuberant capillary meshes also
were evident (Fig. 4D)
both nasally and temporally, and these became
more widespread by 21 WG (data not shown). From 25 WG, as the
vasculature approached the retinal periphery, the leading edge of
vessel formation was characterized by the presence of vascular shunts
between terminal artery–vein pairs (Fig. 4E)
.
The antibody to CD34 binds to the filopodial extensions of vascular endothelial cells,25 which are indicative of angiogenesis. From 18 to 30 WG, filopodia often were present at the leading edge of vessel formation (Figs. 4F 4G) . They were apparent extending into regions where spindle cells were never observed, indicating that angiogenesis is responsible for the vascularization of these regions. The presence of red blood cells at the outer limit of CD34 immunoreactivity (Fig. 4G) revealed that these newly formed vessels were patent.
Contribution of Angiogenesis to the Increase in Vascular Density.
In addition to its contribution to the spread of vessels peripherally,
angiogenesis also was responsible for increasing the vascular density
of the primordial plexus formed by vasculogenesis. Initially, capillary
networks were rare (Figs. 4A
5A
). However, by 18 WG, regions of active sprouting began to give rise to
substantial capillary networks within the existing vascular tree (Fig. 5B)
. As the retina increased in size and the radial vessels spread
peripherally, the distance between these vessels became greater, and it
was in these avascular spaces that sprouting was most pronounced.
Filopodia extended, established contact with other filopodia or
vessels, and subsequently dilated to form vascular segments (Figs. 5C
5D) . Moreover, sprouting was evident even from the edges of larger
preformed vessels (Fig. 5E)
and was especially marked near and along
veins. By 21 WG (Fig. 5F)
, exuberant immature capillary plexuses were
apparent throughout the vascular tree. Thus, angiogenesis augments the
initial radial vessels by increasing capillary density. Such a
prominent role for angiogenesis in increasing the vascular density of
the inner plexus of the retina has not been described in other species.
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Retraction of Excess Vascular Segments
The immature vascular meshes of the inner plexus are subsequently
remodeled to form sparser vascular trees, a process that is not
complete at birth. Vessel retraction was especially marked in regions
of higher tissue oxygenation, such as along arteries, contributing to
the formation of a peri-arterial capillary-free space (Figs. 5F 7A
7B
). Retraction also was apparent in close proximity to newly formed
vessels, in regions where functional circulation recently had been
established, including those immediately central to the terminal
vascular shunts (Fig. 4E)
, and in areas where the outer plexus was
formed or forming (Fig. 7C)
. The proximity of angiogenesis and vascular
retraction (Fig. 7A)
indicated just how localized these processes can
be. Anti-CD34 immunohistochemistry revealed the rounding up of vascular
endothelial cells, resulting in the severance of their normal junctions
with neighboring cells (Figs. 7B
7C
7D)
. A basement membrane was all that
remained after retraction of such endothelial cells into a neighboring
capillary segment (Figs. 7A
7B
7C
7D)
.
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Topography of Formation of the Inner and Outer Vascular Plexuses
Formation of the inner vascular plexus had begun by 14 to 15 WG.
These early vessels were centered on the optic disc and showed a
four-lobed topography (Figs. 4A
8)
. In the following weeks, the inner vascular plexus extended
peripherally, curving around the location of the incipient fovea (Fig. 8)
. By 32 WG, the inner plexus had reached 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 began in the
perifoveal region at about 25 to 26 WG and subsequently spread with an
elongated topography along the horizontal meridian (Fig. 8)
.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The earliest vessels detected in the human retina were radial vessels that were present at low density. The early vasculature exhibited the same four-lobed pattern as that of the spindle cells and vascular cords, leading us to conclude that it is formed by vasculogenesis. Angiogenesis was responsible for increasing vascular density in regions previously pioneered by vasculogenesis, and, given that spindle cells were not detected after 21 WG, it also appeared to be solely responsible for formation of the vessels at the raphe and in the perifoveal and peripheral regions as well as for that of the outer vascular plexus and the RPCs. We propose that vasculogenesis provides a mechanism for rapid formation of a rudimentary vascular plexus in the regions previously invaded by vascular precursor cells and that this plexus is then expanded by angiogenesis to satisfy the increasing metabolic needs of the developing retina.
The mechanism of retinal vascularization is thus similar to that of vascularization of the brain during development.27 28 29 30 The primordial vascular bed on the surface of the neuroepithelium is derived from migratory vascular precursor cells and is thus formed, at least in part, by vasculogenesis.29 30 New vessel segments sprout from these preexisting vessels and grow tangentially by angiogenesis into the neuroepithelium.28 These similarities are not unexpected given that the retina is an extension of the CNS during embryological development.
Roles of "Physiological Hypoxia" and VEGF in Angiogenesis in
the Human Retina
We previously proposed that the "physiological hypoxia" that
results from the increasing metabolic demands of maturing neurons is
the driving force for retinal vascularization.10
11
Earlier evidence suggests that the formation of vessels in response to
physiological hypoxia is mediated by VEGF expressed by neuroglia: VEGF
expression is spatially and temporally correlated with ocular
neovascularization,31
is closely associated with vessel
formation during retinal development,12
32
and is
downregulated by hyperoxia,12
which inhibits retinal
vessel formation.11
However, several observations in our
present study have led us to refine our original hypothesis: We now
propose that, rather than all retinal vascular formation being driven
by hypoxia-induced VEGF expression, only vessel formation by
angiogenesis (not that by vasculogenesis) is mediated in this manner.
Angiogenesis in the developing human retina was localized and was coincident with unmet metabolic demand: (1) Angiogenesis was the only means of vessel formation in the raphe and perifoveal regions of the human retina, both of which are areas of high metabolic activity, given that they coincide with peak ganglion cell density and maturity. Angiogenesis also was responsible for the peripheral spread of the vasculature after 21 WG, which followed the central–peripheral gradient of retinal ganglion cell maturation.24 VEGF is expressed in both these regions of angiogenesis.32 (2) Formation of the outer vascular plexus began 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.33 Formation of the outer plexus also was centered around the fovea, rather than around the optic disc, thus mimicking the topography of photoreceptor maturation.34 (3) Formation of RPCs by angiogenesis was apparent by 21 WG, when the nerve fiber layer in the region of the optic disc becomes too thick to be adequately supplied by the inner retinal vessels. These observations are consistent with the hypothesis that physiological hypoxia induced by the metabolic demands of neurons stimulates angiogenesis.
Lack of Dependence of Vasculogenesis in the Retina on
Hypoxia-Induced VEGF Expression
Although retinal angiogenesis appears to be driven by
hypoxia-induced VEGF, several observations in the present study lead us
to conclude that vasculogenesis in the human retina is independent of
metabolic demand and hypoxia-induced VEGF expression: (1) Substantial
vascularization in the human retina occurs in the absence of VEGF
expression. At 18 WG, the inner plexus covered ~54% of the retinal
area. However, VEGF mRNA was not detected in the human retina by in
situ hybridization until 20 WG.32
(2) Given that formation
of the inner plexus by vasculogenesis is well established by 14 to 15
WG, this process occurs before the differentiation of most retinal
neurons.24
(3) Ganglion cell density and neuronal
maturation are greatest at the perifoveal region of the human retina
between 15 and 18 WG.24
35
36
If retinal vascularization
were driven only by the metabolic needs of neurons, one would expect
that vascular density and the extent of vascular spread would be
maximal in this area. Instead, the raphe and perifoveal regions of the
human retina were avascular between 15 and 18 WG. (4) Formation of the
primordial vessels by vasculogenesis is centered around the optic disc,
whereas neuronal maturation is centered around the
fovea.24
35
36
A comparative analysis of retinal vascularization in other species has shown that VEGF expression, tissue oxygen levels, and vascularization are not always correlated.37 The guinea pig retina is virtually anoxic and yet remains avascular,38 whereas overexpression of VEGF in the avian retina did not induce vascularization.39 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.40 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.41 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 hypoxia-induced VEGF expression.
Uniqueness of the Foveal Region
Spindle cells were not detected in the foveal region, leading us
to conclude that vessels in this region could only be formed by
angiogenesis. The fovea contains a high density of cones with a high
concentration of mitochondria in their outer segments. If physiological
hypoxia resulting from the increase in retinal metabolic activity is
the main impetus for angiogenesis, then why does the fovea remain
avascular? The absence of foveal vessels might be attributable to one
of two distinct mechanisms: the lack of a stimulus for vascular
formation or the retraction of vascular segments. We have previously
shown that VEGF expression by astrocytes contributes to the formation
of the inner plexus during retinal development.12
We have
now shown that astrocytes do not enter the fovea during embryonic
development of the human retina. It is therefore possible that
angiogenesis does not take place in the fovea because of an absence of
VEGF expression by retinal astrocytes.
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Acknowledgements |
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Footnotes |
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Submitted for publication June 28, 1999; revised October 14, 1999; accepted November 8, 1999.
Commercial relationships policy: N.
Corresponding author: Tailoi Chan-Ling, Department of Anatomy and Histology (F13), University of Sydney, Sydney, NSW 2006, Australia. tailoi{at}anatomy.usyd.edu.au
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