Roles of Endothelial Cell Migration and Apoptosis in Vascular ...

Microcirculation (2000) 7, 317–333 © 2000 Nature America Inc. 1073-9688/00 $15.00 www.nature.com/mn

Roles of Endothelial Cell Migration and Apoptosis in Vascular Remodeling during Development of the Central Nervous System SUZANNE HUGHES AND TAILOI CHAN-LING Department of Anatomy and Histology, Institute for Biomedical Research, University of Sydney, Sydney, Australia ABSTRACT Objective: To examine the roles of apoptosis, macrophages, and endothelial cell migration in vascular remodeling during development of the central nervous system. Methods: The terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL) technique was combined with Griffonia simplicifolia isolectin B4 histochemistry to detect apoptotic endothelial cells in retinal whole-mount preparations derived from rats at various stages of postnatal development as well as from rat pups exposed to hyperoxia. Macrophages were detected by immunohistochemistry with the monoclonal antibody ED1, and proliferating endothelial cells were identified by incorporation of bromodeoxyuridine. Results: Only small numbers of TUNEL-positive endothelial cells were detected during normal development of the retinal vasculature, with the apoptotic cell density in the inner plexus peaking during the first postnatal week and decreasing markedly during the second week, at a time when vessel retraction was widespread. Neither apoptotic endothelial cells nor macrophages were apparent at sites of initiation of vessel retraction. TUNEL-positive endothelial cells were observed in vessels destined to remain. Hyperoxia induced excessive vessel withdrawal, resulting in the generation of isolated vascular segments containing apoptotic endothelial cells. A topographical analysis showed low numbers of proliferating endothelial cells at sites of angiogenesis whereas vascular proliferation was increased in the adjacent inner plexus. Conclusions: Endothelial cell apoptosis and macrophages do not initiate vessel retraction, but rather contribute to the removal of redundant cells throughout the vasculature. We suggest that vessel retraction is mediated by endothelial cell migration and that endothelial cells derived from retracting vascular segments are redeployed in the formation of new vessels. Only when retraction results in compromised circulation and redeployment is not possible, does endothelial cell apoptosis occur. Microcirculation (2000) 7, 317–333. KEY

WORDS:

angiogenesis, apoptosis, inflammation, rat, retina, vessel retraction

INTRODUCTION

The central nervous system (CNS) consists of neuronal, glial, and vascular elements. The marked Supported by grants from the National Health and Medical Research Council of Australia, The RG Arnott Foundation, the Baxter Perpetual Charitable Trust, and The Clive and Vera Ramaciotti Foundations. For reprints of this article, contact Dr. Tailoi Chan-Ling, Department of Anatomy and Histology F13, University of Sydney, Sydney NSW 2006, Australia; e-mail: [email protected]. edu.au Received 6 January 2000; accepted 6 June 2000

overproduction of neuronal and glial cells during development and the subsequent death of the excess cells by apoptosis are well characterized (7,34,43). There is also a substantial overproduction of vascular segments during development of the CNS, after which the vascular tree is remodeled to match closely the metabolic demand of the tissue (12,15,51). However, the mechanisms of vascular remodeling in the developing CNS have remained unclear. Vascular remodeling entails both vessel regression and vessel growth, and results in changes in the density and morphology of the vascular tree. The cellu-

Endothelial cell migration and apoptosis S Hughes and T Chan-Ling 318

lar processes that contribute to vascular remodeling include changes in the basement membrane; vascular cell proliferation, migration, and death; and tube and lumen formation (27,33). Vascular regression encompasses both vascular involution and vascular pruning. Vascular involution occurs in adults when the metabolic demands of the surrounding tissue decrease markedly, such as during regression of the corpus luteum and wound healing, when the loss of myofibroblasts results in the formation of acellular scar tissue. It also occurs during development in situations in which entire vascular plexuses, such as the hyaloid vessels of the eye, are lost. In these instances, virtually all vessels regress, with only vestiges of the vasculature remaining. In contrast, vascular pruning is a more selective process and contributes, for example, to the remodeling of the retinal vasculature during development. Vascularization of the retina begins in the inner layers. Initially, a few large vessels radiate out from the center of the retina and feed directly into a dense and rapidly proliferating capillary mesh. As the retina develops and the outer layers become vascularized, a capillary-free zone forms around the major vessels and the immature mesh of the inner plexus is pruned into a sparser, differentiated vascular tree (3,12,18, 24,31). Pruning of the retinal vasculature is thought to be triggered by an increase in tissue oxygen tension, given that vascular retraction is most pronounced around large arterial vessels. Furthermore, hyperoxia induces massive vascular retraction in the developing retina, resulting in blood vessel loss and, in some animals, the obliteration of the inner retinal vasculature (2,13,14,15). This phenomenon is thought to represent an exaggeration of the normal process of developmental vascular retraction. Early investigators proposed that the endothelial cells of withdrawing retinal vessels either die or migrate away (2). Dying endothelial cells have been detected in the dense plexus of the developing human retinal vasculature (18). Endothelial cell apoptosis occurs during vascular involution accompanying wound healing (21), involution of the lactating breast (50), luteolysis (6), tumor regression (10,32), and the macrophage-dependent developmental loss of the pupillary membrane (36,39). These observations, together with the detection of vascular endothelial cells undergoing apoptosis during hyperoxiainduced regression of retinal vessels (1), led to the suggestion that apoptosis also contributes to vascular pruning (44); however, compelling evidence for this suggestion is lacking to date.

We have now investigated the possible roles of apoptosis, macrophages, and endothelial cell migration in vascular pruning during retinal development. The retina is an extension of the CNS and its vessels are representative of CNS vessels in their barrier properties, flow rates, venous pO2, ability to autoregulate, and glial ensheathment (14,29). Thus, any insights gained in the study of developing retinal vessels are applicable to the developing CNS. The thinness and laminar structure of the retina allow visualization of the cellular events that take place during vascular remodeling in a whole-mount preparation. The retinal whole-mount permits visualization of entire vascular segments with normal cell-cell interactions between neighboring cells, which is not possible in tissue culture and crosssectional studies of the developing CNS. An understanding of vessel regression during normal development of the CNS has important implications for the implementation of strategies inducing vessel regression in tumor biology and preventing vessel regression in hyperoxia-induced vaso-obliteration, as occurs in retinopathy of prematurity. The developing rat retinal vasculature was subjected to double-label histochemical staining with Griffonia simplicifolia isolectin B4 (GS lectin) and the terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL) technique to detect apoptotic endothelial and abluminal cells. Such an approach allows the detection of relatively small numbers of apoptotic cells in situ, as well as detailed investigations of the topography and density of vascular apoptosis. We show that apoptosis does not initiate capillary retraction during retinal development, but rather that it appears to be responsible for the removal of redundant endothelial cells within a vascular segment. We failed to detect any evidence that macrophages induce vessel withdrawal and endothelial cell apoptosis. We also conducted a detailed topographical analysis of the distribution of proliferating endothelial cells during active angiogenesis and vascular pruning. These data, combined with the lack of endothelial cell apoptosis in retracting vascular segments and weak CD34 expression during vascular remodeling, lead us to conclude that vessel withdrawal is mediated by endothelial cell migration, and that endothelial cells from retracting vessels are redeployed in the formation of new vessels in the immediate vicinity. Thus, this study has provided the first in situ evidence in support of endothelial migration and redeployment in vascular pruning.


MATERIALS AND METHODS Normal Rat Development

Adult Sprague-Dawley rats and animals on postnatal days (P) 1, 3, 5, 7, 8, 9, 10, 11, 12, 15, 17, 21, and 24 were studied. Retinal whole-mount preparations were subjected to a modified version of the TUNEL technique developed by Gavrieli and colleagues (26) in order to detect fragmented DNA of apoptotic bodies; they were subsequently labeled with GS lectin to allow visualization of vascular elements containing apoptotic bodies. Other wholemount preparations were processed for ED1 immunohistochemistry or for detection of bromodeoxyuridine (BrdU) incorporation. Experimental Hyperoxia

Rat pups at P2 to P3 were placed with their mother in a hyperoxic chamber (70% to 80% oxygen in air) for 10, 24, or 72 hours (15) with food and water ad libitum. The pups were killed immediately after exposure to hyperoxia. Age-matched controls were maintained in room air for equal periods. Retinas from both the experimental and control animals were subjected to combined TUNEL technique and GS lectin histochemistry. Combined TUNEL and GS Lectin Histochemistry

Rat pups and adults were anesthetized with sodium pentobarbitone (60 mg per kilogram of body mass, i.p.) and then perfused transcardially first with phosphate-buffered saline (PBS) and then with 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4) (PB). The retinas of all animals were dissected (16) and then fixed by immersion for 3–24 hours at 4 °C in 4% paraformaldehyde in PB. Retinas were permeabilized with 1% (v/v) Triton X-100 in PBS for 30 minutes at room temperature, washed, and incubated with 0.3% hydrogen peroxide in PBS for 30 minutes. After further washing, the retinas were subjected to the TUNEL technique with an in situ cell death detection kit POD (BoehringerMannheim, Mannheim, Germany). The horseradish peroxidase (HRP) label for TUNEL was detected by exposure to Tris-buffered saline containing diaminobenzidene (0.05%) and hydrogen peroxide (0.01%), which yielded an amber/red-brown colored reaction product. No labeling was detected if the TUNEL substrate was omitted. As a positive control, retinas were incubated with DNase I (26); this procedure resulted in the TUNEL labeling of all nuclei in the retinal ganglion cell layer, including those of the vasculature, as well as those of the outer nuclear layer (data not shown). Labeled nuclei were

not detected in the inner nuclear layers during postnatal development, even though programmed cell death is known to occur in these layers at this time (9,52); the fact that they were also not detected in the inner nuclear layers in positive controls suggests that the TUNEL reagents were not able to penetrate the middle layers of our retinal whole-mount preparations. GS lectin stains vascular endothelial cells and microglia in the rat CNS (4,48). Like von Willebrand factor (Factor VIII–related antigen), GS lectin labels endothelial cells in culture (42,47,49). It is able to detect the smallest capillary and endothelial cell extensions not always detectable by von Willebrand factor (35) and it does not label pericytes and astrocytes (48,49) To covisualize apoptotic bodies and the developing vasculature, we incubated retinas that had been subjected to the TUNEL technique first with hydrogen peroxide, in order to block any remaining HRP activity, and then overnight at 4 °C with HRP-conjugated GS lectin (Sigma Chemical Co., St. Louis, MO) at a concentration of 50 ␮g/mL in PBS containing 1% Triton X-100. Peroxidase activity was detected as described for the TUNEL technique but in the additional presence of nickel ammonium sulfate, which resulted in the generation of a brown to black reaction product (depending on the length of exposure to substrate). Thus, lectin labeling produced a brown to black reaction product whereas TUNEL labeling produced an amber/redbrown colored reaction product that could be easily distinguished. Washed whole-mount preparations were then mounted with the ganglion cell layer uppermost in glycerol-PBS (2:1, v/v) and viewed with Nomarski optics using a Leitz DMRBE research microscope. Combined TUNEL and GFAP or S100 Protein Immunohistochemistry

Retinal whole-mount preparations were subjected to the TUNEL technique and then to immunohistochemistry, performed essentially as described previously (38), with rabbit polyclonal antibodies to glial fibrillary acidic protein (GFAP) (Biogenex Laboratories, San Ramon, CA) or to S100 protein (␣ and ␤ subunits) (Silenus Laboratories, Melbourne, Australia). The HRP label was visualized in the presence of nickel ammonium sulfate as described above for GS histochemistry. Criteria for Counts of TUNEL-Positive Cells

In GS lectin–stained retinal whole-mount preparations, the vascular tree was clearly labeled. GS lectin


also stains microglia, but this was not a barrier to the analysis of endothelial cell death since microglia were readily distinguished on the basis of their amoeboid or ramified morphology. Using a ×40 objective, TUNEL-positive bodies in the nerve fiber and ganglion cell layers were classified as endothelial when present within the lectin-stained endothelium and in the same focal plane of the lectin-stained endothelium. TUNEL-positive bodies were classified as abluminal when present on the outer surface of a blood vessel or situated above or below the focal plane of the lectin-stained endothelium when using a ×40 objective. TUNEL-positive abluminal cells include astrocytes, microglia, perivascular cells, and mural cells. Lectin labeling revealed that some abluminal cells were microglia. An abluminal TUNELpositive body was classified as microglial if within the soma of a microglia or closely associated with microglial processes [Fig. 1(H)]. TUNEL-positive bodies surrounded by S100 labeling on a vascular structure were classified as TUNEL-positive astrocytes. Adjacent small TUNEL-positive bodies were counted as the remnants of one nucleus. Mapping of the Retina

The outline of the retina was mapped with a 1-mm grid incorporated into the ×10 eyepiece of an Olympus BH-2 microscope. The density of TUNELpositive bodies in the plane of the vasculature was sampled in 1-mm steps. The density of each type of TUNEL-positive body was determined in an area of 0.25 mm2. The statistical significance of differences in densities of TUNEL-labeled cells between normal development and hyperoxia was assessed with Student’s two-tailed t-test. ED1 Immunohistochemistry

The monoclonal antibody ED1, which is specific for macrophages and activated microglia in the rat, was used to identify cells with phagocytic activity, as previously described (30). Following enucleation of the globes, the retinas were dissected in ice-cold PBS and then fixed in 70% (v/v) ethanol at −20 °C. Fixed retinas were incubated overnight at 4 °C with the ED1 antibody (Serotec, Oxford, UK) diluted 1:200 with PBS containing 1% (w/v) bovine serum albumin. Antibody labeling was detected either with HRP following Hu and colleagues (30) or with Texas red–labeled sheep antibodies to mouse immunoglobulin (Amersham International, Little Chalfont, UK) diluted 1:50 for 4 hours at room temperature where ED1 labeling was to be combined with lectin labeling. In the latter case, the vasculature was then vi-

sualized by incubating the retinas overnight at 4 °C with biotinylated GS lectin (10 ␮g/mL) in Hanks’ balanced salt solution, and then with fluorescein isothiocyanate–conjugated streptavidin, as previously described (16). The retinal preparations were mounted in glycerol-PBS (2:1) and viewed with a Leitz DMRBE research microscope. CD34 Immunohistochemistry

CD34 is a glycoprotein expressed by human lymphoid and myeloid hematopoeitic progenitor cells and by endothelial cells; it is expressed to a much greater extent on the luminal surface of endothelial cells than on the abluminal surface (46). The developing vasculature of the human retina was subjected to immunohistochemical labeling with the CD34specific monoclonal antibody QBEND/10 as previously described (31). Briefly, eyes from human fetuses ranging in age from 14 to 38 weeks of gestation (WG) were collected in the People’s Republic of China in accordance with the guidelines set forth in the Declaration of Helsinki and was also approved by the human ethics committee of the University of Sydney. Combined BrdU Labeling and GS Lectin Histochemistry

Dividing cells in the rat retina were labeled with BrdU and covisualized with the vasculature by staining with GS lectin, as previously described (15,16). A photographic montage of a sector of a P11 rat retina double-labeled with GS lectin and BrdU was prepared to determine the location of proliferating endothelial cells in relation to the location of newly formed vascular sprouts in the outer vascular plexus. A tracing of the photographic montage was preppared, scanned with a flatbed scanner and Adobe Photoshop 5.0 used to produce a color representation of individual proliferating vascular endothelial nuclei within the vascular tree. RESULTS Timing and Topography of Vessel Retraction in the Inner Plexus

Marked vessel formation in the rat retina during the first few postnatal days results in the generation of exuberant plexuses of immature, broad capillaries [Fig. 1(A)] that are fed by pairs of larger radial vessels. These immature plexuses subsequently undergo substantial pruning. In the first postnatal week, major vessels are selected and capillaries retract from the major arteries, resulting in the formation of periarterial capillary-free spaces [Fig. 1(B)].


Figure 1. Combined TUNEL and GS lectin staining of retinal whole-mount preparations from postnatal rats. The vasculature predominantly comprises (A) a dense immature mesh at P3, whereas (B) vessel withdrawal is apparent along arteries by P5 and (C) in the capillary plexii by P11, resulting in the generation of a maturer vascular tree. TUNEL-positive cells were not detected or were extremely rare in these fields, although TUNEL reactivity was observed in scattered regions during the first postnatal week. (D) A region of a P5 retina containing a relatively large number of dark, round TUNEL-positive bodies. (E) A TUNEL-positive body on the abluminal surface of an apparently normal vessel at P5. (F) A TUNEL-positive body within the endothelium of a major radial vein at P3. (G) TUNEL-positive bodies on the abluminal surface of and within the endothelium of a newly formed capillary plexus, as well as in the parenchyma, at P5. (H) A GS lectin–labeled microglial cell engulfing a TUNEL-positive body at P5.

In the second postnatal week, when the outer plexus is forming, vessel retraction in the inner plexus is more widespread, occurring in the capillary networks and along veins and resulting in the production of a sparser vascular tree [Fig. 1(C)].

Identification of Apoptotic Endothelial Cells and Abluminal Cells by TUNEL

During the first postnatal weeks, scattered TUNEL reactivity was apparent in the nerve fiber and gan-


glion cell layers of the rat retina. All TUNEL reactivity was discrete and confined to membrane-bound bodies, indicative of apoptotic cells. TUNELpositive endothelial cells [Figs. 1(D), 1(F), and 1(G)] and TUNEL-positive abluminal cells [Figs. 1(D) and 1(E) were observed. Abluminal cells include astrocytes, microglia, perivascular macrophages, pericytes, and smooth muscle cells. Astrocyte TUNEL-labeling was confirmed by the combination of TUNEL and immunohistochemistry with antibodies to either GFAP or S100 [Figs. 2(A) and 2(B)]. At P3, 19.0% (n ⳱ 4) of TUNEL-labeled abluminal cells were astrocytes. TUNEL-positive bodies were also evident within the retinal parenchyma, consistent with the death of retinal ganglion

cells and amacrine cells by apoptosis during normal development. Microglia readily identifiable on the basis of lectin labeling and distinctive ramified morphology, were observed enveloping TUNEL-positive bodies [Fig. 1(H)], likely reflecting the phagocytic role of these cells rather than apoptosis of microglia (23). At P3, 15.4% (n ⳱ 4) of TUNEL-labeled abluminal cells were microglia. Role of Endothelial Cell Apoptosis and of Macrophages in Initiation of Vessel Retraction

Despite previous studies showing that significant endothelial cell apoptosis occurs during vascular involution (21,36,50), we rarely detected TUNEL-

Figure 2. (A) Combined GFAP immunohistochemical (brown) and TUNEL (amber) staining of a vascularized region of a whole-mount preparation of a rat retina at P3. (B) Combined S100 immunohistochemical (gray) and TUNEL (amber) staining of a vascularized region of a P5 rat retina. (C through F) Combined GS lectin (brown) and TUNEL (amber) staining of retinal whole-mount preparations from rats (C, D, and F) at P5 or (E) P17. In (C), the only TUNEL-positive body is weakly labeled and in the parenchyma. TUNEL-positive bodies are not present in (D). The slight variability in the color of the DAB reaction products in the different fields of view is due to the false colors introduced by Normarski optics.


positive endothelial cells in retracting retinal vessels [Figs. 1(B) and 1(C)]. Furthermore, we detected no evidence of TUNEL-positive endothelial cells in vessels undergoing the initial stages of retraction. To identify vessels at the earliest stage of retraction, we concentrated our analysis on retracting vessels in the region adjacent to arteries during the first postnatal week; vessels in this region undergo marked retraction at this time to produce the periarterial capillaryfree space. Vessel retraction was characterized initially by narrowing of the vessel lumen near the junction with the artery; TUNEL-positive endothelial cells were never detected at these sites [Figs. 2(C) and 2(D)], despite the presence of apoptotic bodies in the vicinity. Vessel narrowing was followed

by the apparent rounding of endothelial cells and their withdrawal into an adjacent vascular segment [Figs. 2(C) and 2(D)]. Whereas TUNEL-positive endothelial cells were not apparent in retracting vessels, they were occasionally detected in vascular segments that had lost functional circulation because of retraction at both ends of the segment [Figs. 2(E) and 2(F)]. Further evidence that apoptosis of endothelial cells is a rare event during CNS development and does not initiate vessel withdrawal was provided by a topographical analysis of the distributions of TUNELpositive endothelial cells and abluminal cells in the rat retina at various postnatal ages [Fig. 3(A)].

Figure 3. (A) Maps of the distribution of TUNEL-positive bodies within the endothelium (top row) and on the abluminal surface of the endothelium (bottom row) in the retina of rats at the indicated ages. The squares indicate the optic disk and the thickened line indicates the leading edge of vessel formation in the inner plexus. (B) A map of a P5 rat retina showing the location of TUNEL-positive endothelial cells (dots) and the location of regressing vessels (dashes). The inner line represents the leading edge of vessel formation.


There was no evidence of a central-peripheral gradient of endothelial cell apoptosis, or indeed of any such gradient related to the leading edge of vascular formation [shown as a thickened line throughout Fig. 3(A)]. The timing and distribution of endothelial cell apoptosis also did not coincide with those of vessel withdrawal throughout retinal development. Figure 3(B) is a map showing the distribution of every regressing vessel and all TUNEL-positive endothelial cells of a P5 rat retina and is representative of the lack of correlation between the exact locations of regressing vascular segments and TUNELpositive endothelial cells observed during postnatal development of the rat retina. In the first postnatal week, when peak endothelial cell apoptosis was observed, vascular retraction was largely restricted to the periarterial region [Fig. 1(B)]; conversely, in the second and third postnatal weeks, when vessel retraction was more extensive [compare Figs. 1(A) and 1(B) with Fig. 1(C)], the density of TUNEL-labeled endothelial cells had decreased markedly (Table 1). Thus, on the basis of both morphological evidence and topographical analysis, we conclude that apoptosis of endothelial cells does not play a major role in vessel withdrawal during normal retinal development. In particular, our data indicate that it does not initiate vessel withdrawal. Consistent with our observations suggesting that the mechanism of retinal vessel withdrawal differs markedly from that of involution of the pupillary membrane, we detected no evidence that ED1positive macrophages are associated with vessel withdrawal in the developing CNS. At P5, large numbers of ED1-positive cells were distributed throughout the nerve fiber layer in both the vascularized [Fig. 4(A)] and avascular [Fig. 4(B)] regions Table 1. Density (means ± SE) of TUNEL-positive endothelial cells and abluminal cells in retinas of rats at various ages Cell density (cells/mm2) Age P1 (n ⳱ 5) P3 (n ⳱ 11) P5 (n ⳱ 5) P7–P8 (n ⳱ 3) P9–P10 (n ⳱ 3) P11–P12 (n ⳱ 2) P15–P17 (n ⳱ 2) P21–P24 (n ⳱ 2) Adult (n ⳱ 1)

Endothelial

Abluminal

7.9 ± 0.9 6.3 ± 1.4 12.4 ± 2.3 6.9 ± 1.0 4.1 ± 1.0 1.5 ± 0.0 0.1 ± 0.1 0.2 ± 0.2 0.3

55.2 ± 10.3 31.3 ± 5.7 20.4 ± 2.8 11.0 ± 1.6 7.4 ± 1.5 4.9 ± 0.9 1.4 ± 0.3 1.4 ± 0.6 1.1

of the rat retina. The distribution of ED1-positive cells in the developing retinas showed a marked variability across the retina; in the same retina there are regions with clusters of ED1-positive cells and other regions with few or no ED1-positive cells. Lectinlabeled microglia were scattered throughout the inner retinal layers [Fig. 4(C)] and a subpopulation of these was ED1-positive [Fig. 4(D)]. Unlike the situation with the regressing pupillary membrane, ED1positive cells showed no correlation or close association with the location of regressing vascular segments [Figs. 4(C) and 4(D)]. A topographical examination of retinae at various postnatal ages showed a marked variation of distribution of ED1postive macrophages across the retina. Figures 5(A) and 5(B) were selected to show this variation. Furthermore, a consistent observation was a lack of colocalization of ED1-positive macrophages and regressing vascular segments throughout the retina. Regressing vessels were identified in these regions on the basis of vessel narrowing in the vicinity of a junction with another vessel [arrows in Figs. 5(A) and 5(B)]. Since the vascular plexus in these regions were undergoing active remodeling with significant removal of vascular segments, we could be confident that segments with these morphological criteria were regressing. Thus, ED1-positive macrophages do not appear to be responsible for inducing vessel retraction in the rat retina. Endothelial Cell Apoptosis in Vessels Destined To Remain

Whereas our observations led us to conclude that apoptosis is not responsible for vessel withdrawal in the normal retina, we did detect substantial evidence that apoptosis contributes to the removal of endothelial cells during vascular remodeling. The peak density of TUNEL-positive endothelial cells in the rat retina was 12.4 cells/mm2 at P5 (n ⳱ 5, Table 1). These apoptotic endothelial cells were often present in normal, healthy vessels not destined to regress, including the major radial veins [Fig. 1(F)] and radial arteries (data not shown). TUNELpositive endothelial cells were also detected in the immature capillaries of newly formed plexuses [Fig. 1(G)]. These observations suggest that endothelial cell apoptosis is a normal feature of vessel growth and maturation during retinal development. Apoptosis of Endothelial Cells and Abluminal Cells are Well Matched

We also investigated whether vessel retraction is associated with TUNEL labeling of abluminal cells, which include astrocytes, microglia, perivascular


Figure 4. (A and B) ED1-positive phagocytic cells detected using HRP-labeled conjugates, in the (A) vascularized and (B) avascular regions, of the inner layers of a P5 rat retina. Using Nomarski optics, it was possible to determine the outer limit of vascularization by visualization of red blood cells within the lumens of vessels (arrowheads). (C and D) Show the same field of view in a P5 rat retina double-labeled (C) with GS lectin and (D) for ED1 antigen. (C) GS lectin labeled the vasculature including a retracting vessel (arrow), and microglia (arrowheads). (D) The same region of the retina shown in (C) double-labeled with the ED1 antibody showing that a subpopulation of activated GS lectin positive labeled microglia is ED1-positive (arrowheads) and are not closely associated with regressing vessels (arrow).

cells, pericytes, and smooth muscle cells. Our combined GS lectin and TUNEL protocol allows us to detect apoptotic abluminal cells, but not to identify the constituent cell types. Similar to apoptotic endothelial cells, substantial numbers of TUNEL-positive abluminal cells were detected in association with normal vessels not destined to regress, including major radial veins (data not shown) and radial arteries [Fig. 1(D)], in the rat retina. TUNEL-positive abluminal cells were also observed in association with the immature capillaries of newly formed plexuses [Fig. 1(E)]; however, they were not apparent during the initial stage of vessel retraction [Figs. 2(C) and 2(D)]. In addition, topographical analysis of the distribution of TUNEL-positive abluminal cells during postnatal development of the rat retina showed that the timing and topography of TUNEL-labeling of these cells were similar to those of vascular endothelial cells [Fig. 3(A), Table 1]. TUNEL-labeling of abluminal cells was maximal during the first postnatal week, gradually decreasing to adult levels by the third postnatal week. These observations thus suggest that the numbers of abluminal cells and of endothelial cells in a vascular bed are well matched. The absence of TUNEL-positive abluminal cells on retracting vascular segments supports our conclusion above that apoptosis does not initiate vessel withdrawal.

Secondary Vascular Endothelial Apoptosis Is Associated with Compromised Circulation

Hyperoxia induces widespread vessel retraction and endothelial cell apoptosis in the developing retina (1,3). We therefore examined the effects of hyperoxia in the rat retina to gain further insights into the mechanism of vessel retraction. The retinas of rat pups maintained for 3 days in a hyperoxic environment were characterized by a marked decrease in vascular density, widening of the capillary-free space surrounding arteries, an increased percentage of constricted vessels, and obliterated vascular segments [Figs. 6(A) and 6(B)]. Hyperoxia also resulted in a significant increase in endothelial and abluminal cell TUNEL-labeling within 10 hours, indicating that hyperoxia-induced apoptosis of vascular cells is a very rapid event. The number of TUNEL-labeled endothelial cells remained significantly higher after 24 hours of hyperoxia but declined after 72 hours of hyperoxia, with densities not significantly different from those of age-matched controls (Table 2). Following 10 hours of hyperoxia, TUNEL-positive endothelial and abluminal cells were not observed at the sites of initial vessel withdrawal [Fig. 6(C)]. The retinas of rats exposed to hyperoxia for 10 hours showed an increase in vascular retraction in the central retina [compare Figs. 6(C) and 6(D)], and con-


Figure 5. (A and B) Combined ED1 immunohistochemical (red) and GS lectin (green) staining of rat retinal wholemount preparations at P10. These are regions of similar eccentricity from the same retina where vessel regression (arrowheads) is widespread and illustrate the variation in ED1-positive cell density within a developing retina. Regressing vessels were identified by vessel narrowing in the vicinity of a junction with another vessel. These micrographs showed that there is no correlation between vessel regression and the presence of ED1-positive macrophages. (C through E) Combined GS lectin (green) and BrdU (red) labeling of (C and E) vessels forming in the outer plexus of P11 retinas and of (D) those immediately central to the leading edge of inner plexus formation in a P4 retina. (F) CD34 immunohistochemical staining of human retinal vessels at 21 WG, showing vessels withdrawing from an artery.

sequent increases in the numbers of both vascular segments isolated from the circulation [Fig. 6(E)] and terminal vascular segments in which perfusion is severely compromised [Figs. 6(F) and 6(G)]. Endothelial cell apoptosis was clearly associated with these isolated vascular segments. Similar to the situation for normal development, the number of TUNEL-positive abluminal cells in the retinas of rats exposed to 10 hours of hyperoxia was well matched to that of TUNEL-positive endothelial cells [Fig. 6(H), Table 2]. These observations indicate that, in the retina, the same cellular processes contribute to the vessel regression associated with normal development and to that induced by hyperoxia, with the exception that

hyperoxia induces marked narrowing of selected vascular segments at their junctions with neighboring vessels, resulting in impaired blood flow. This lack of blood flow results in the trapping of endothelial cells and significant apoptosis along the capillary segment. Thus, endothelial cell apoptosis does not underlie vascular pruning in the retina, but rather is a consequence of the vascular segment isolation and compromised circulation induced by excessive pruning. Roles of Migration and Redeployment of Endothelial Cells in Vascular Retraction and Angiogenesis

If the endothelial cells of retracting vessels do not undergo apoptosis, what is their fate? Several obser-


Figure 6. Combined TUNEL and GS lectin staining of retinal whole-mount preparations from rats maintained under normoxic or hyperoxic conditions. (A) A P5 rat raised under normoxic conditions. (B) A P5 rat exposed to hyperoxia for 3 days from P2. (C through H) P3 rats (C, E through H) exposed to hyperoxia for 10 hours or (D) maintained under normoxic conditions. A central region of the retina is shown in (C) through (G). Endothelial TUNEL-positive bodies are not present in (C) or (D).

vations lead us to suggest that migration and redeployment of endothelial cells play an important role in formation of the outer vascular plexus of the retina. Both endothelial cell proliferation and migration are involved in the formation of vessels by angiogenesis (5). Figure 5(D) shows the large numbers of proliferating vascular endothelial cells at the leading edge of formation of the inner plexus at P4, com-

pared with the small numbers of proliferating vascular endothelial cells associated with the formation of the outer plexus at P11 [Figs. 5(C) and 5(E)]. The low numbers of proliferating vascular endothelial cells observed in the outer vascular plexus suggest that the density of BrdU+ endothelial cells within the outer vascular plexus was inadequate to account for the formation of these vessels.


Table 2. Densities (means ± SE) of TUNEL-positive endothelial cells and abluminal cells in the retinas of rats maintained under hyeroxic or normoxic conditions for 10 hours from P3, or 24 hours or 72 hours from P2 Cell density (cells/mm2) Treatment

Endothelial

Abluminal

10 h hyperoxia (n ⳱ 6) P3 normoxic littermates (n ⳱ 7) 24 h hyperoxia (n ⳱ 8) P3 normoxic controls (n ⳱ 11) 72 h hyperoxia (n ⳱ 2) P5 normoxia controls (n ⳱ 5)

11.46 ± 1.1*

67.9 ± 7.1*

3.0 ± 0.6 12.5 ± 1.5*

41.4 ± 6.4 26.9 ± 2.8

6.3 ± 1.4 5.0 ± 0.6

31.3 ± 5.7 17.2 ± 2.0

12.4 ± 2.7

20.4 ± 2.8

*p < 0.05 versus corresponding value for normoxia controls.

We examined the contribution of endothelial cell proliferation to the formation of the outer plexus in the rat retina at P11. Using a combination of GS lectin histochemistry with BrdU labeling of proliferating cells, the number of BrdU-labeled vascular endothelial cells for the retinal sector shown in Fig. 7 was 227 cells in the inner retinal plexus, compared with 57 cells in the outer vascular plexus. However, when normalized for vessel density, the proliferation index was similar for the inner and outer plexus (inner ⳱ 15.9 cells/mm2 vessel area, outer ⳱ 14.8 cells/ mm2 vessel area). Remarkably, our analysis shows that the proliferation index was the same in a plexus that is undergoing remodeling with significant vessel loss as a forming vascular plexus. A detailed topographical analysis revealed marked endothelial cell division in the capillary beds of the inner plexus located immediately above the newly forming vessels of the outer plexus [Figs. 7(A) and Fig. 7(B)]. These results, together with the lack of endothelial cell apoptosis within retracted vascular segments in the vicinity of newly formed vessels, suggest that substantial numbers of endothelial cells may be redeployed from the inner plexus to contribute to the formation of the outer plexus. Further evidence for this model of endothelial cell redeployment comes from studies in the human retina using the QBEND/10 monoclonal antibody, which recognizes a CD34 epitope on endothelial cells. Vessel retraction was particularly pronounced in the inner plexus in regions where the outer plexus was actively forming [Figs. 8(B) and 8(C)]. This close proximity of vessel regression to angiogenesis is consistent with the hypothesis that the endothelial

cells of retracting vessels migrate away and are redeployed in growing vessels in the immediate vicinity. CD34 expression decreases during angiogenesis (28) and is correlated with loss of cell contact with neighboring cells (20); thus, weak CD34 labeling is suggestive of endothelial cells with reduced contact and consistent with a migratory phenotype. CD34 immunohistochemistry in the human retina revealed marked heterogeneity in the intensity of CD34 staining in regressing vascular segments [Fig. 5(F)], whereas the endothelial cells of established vessels were strongly labeled [Fig. 8(A)]. This weak labeling within retracting vascular segments was prominent in regions of the inner plexus located above areas of active vessel formation in the outer plexus [Figs. 8(B) and 8(C)]. In addition, substantial numbers of weakly labeled endothelial cells were also present in regions of active vascular sprouting as indicated by filopodial extensions [Fig. 8(D)]. These observations show heterogeneity of CD34 intensity is associated with both angiogenesis and vascular regression and are consistent with the model of endothelial redeployment. DISCUSSION

Our results show that endothelial cell apoptosis is not associated with the initial retraction of vascular segments during development of the CNS. This indicates that the mechanism of vascular pruning during normal retinal development differs from those of the involution of vascular beds in wound healing and in the breast after lactation, which involve significant endothelial cell apoptosis. Neither endothelial cell apoptosis nor ED1-positive macrophages were associated with the initiation of vessel withdrawal in the retina. Our observations in the retina thus also differ from those of Lang and colleagues in their studies of the pupillary membrane. Endothelial cell apoptosis in the pupillary membrane is widespread, is initiated by macrophages in apparently normal vessels, and results in vessel narrowing, flow stasis, and consequent secondary endothelial cell apoptosis (22,36,39). These differences likely reflect the fact that involution involves the loss of virtually the entire vascular tree and the elimination of virtually all endothelial cells, whereas the retinal vasculature is merely pruned. Furthermore, the environment of an involuting vasculature is often characterized by the loss of surrounding cells, which results in a marked decrease in metabolic demand; in contrast, pruning of the vasculature in the developing CNS occurs in an environment with a substantial metabolic demand.


Exposure of rat pups to experimental hyperoxia resulted in a significant increase in endothelial cell apoptosis in the retina. However, even under hyperoxic conditions, endothelial cell apoptosis was not observed at sites of initiation of vessel retraction, but rather occurred in vessel segments that had become isolated from the circulation as a result of excessive vessel withdrawal. Such endothelial cell apoptosis induced by compromised circulation is analogous to the secondary apoptosis detected in the pupillary membrane, which results from a loss of circulation. Vascular endothelial growth factor (VEGF) has been suggested to act as a survival factor that prevents endothelial cell apoptosis in the retina in response to hyperoxia (1,11). Our observations that endothelial cell apoptosis is not the initiating event of retinal vessel withdrawal, either during normal development or under conditions of experimental hyperoxia, suggest that VEGF does not act as a survival factor in preventing hyperoxia-induced vaso-obliteration, but rather that it may act primarily as a vasodilator to prevent vessel withdrawal and to maintain adequate plasma perfusion, thus minimizing secondary endothelial cell apoptosis (39).

We have shown that the endothelial cells of regressing retinal vessels in the rat round up and withdraw into neighboring vessels. Immunohistochemical staining of the human fetal retina with an antibody to CD34 revealed a subset of endothelial cells that were weakly labeled and consistently associated with regions of active angiogenesis and vascular regression. Our preferred interpretation of these observations is that the weakness in CD34 expression is due to endothelial cell migration. Evidence in support of this include the fact that CD34 expression decreases during angiogenesis, and is well correlated with the loss of cell contact (20,28) consistent with a migratory phenotype. Furthermore, the CD34 epitope recognized by QBEND/10 is preferentially expressed on the luminal surface of endothelial cells (46), and thus a weaker expression in a particular vessel segment suggests that this segment no longer possess a lumen, a likely occurrence during vascular remodeling. However, an alternative explanation for the weakness in CD34 expression could merely reflect changes in leukocyte adhesion properties, since CD34 binds L-selectin (8) and is likely to be involved with cell adhesion (25).

TUNEL-labeling of endothelial cells and abluminal cells were well correlated during normal development of the rat retina as well as during experimental hyperoxia. Apoptotic endothelial and abluminal cells were apparent in the rat retina during the first postnatal week, at a time of pronounced vessel formation, and in many vessels destined to remain. Survival of endothelial cells is dependent on specific growth factors and on the ability of the cells to attach to and spread on specific substrata. We propose that competition for such survival factors and suitable substrata during periods of rapid cell proliferation and vessel formation is responsible for the regulation of cell numbers in a vascular bed.

Similarities between regressing and forming vessels have been noted in earlier in vivo studies (17,45), leading to the suggestion that similar processes are involved in angiogenesis and regression. Given that angiogenesis involves endothelial cell migration (5), our data are consistent with the notion that endothelial cell migration contributes to vascular retraction. There is existing evidence in the literature supporting our model of endothelial migration and redeployment. In the early stages of vessel retraction in the corpus luteum, apparently normal endothelial cells are seen transversing capillary lumens (6). In vitro studies have shown that endothelial cells of retracting vascular sprouts withdraw, often migrate along capillary-like structures, and are redeployed in new and existing vessels (41). Furthermore, ultrastructural evidence suggests that endothelial cell migration occurs during vessel regression in the developing limb (37).

If, as our observations suggest, the endothelial cells of retracting vessels in the developing CNS do not undergo apoptosis, what is their fate? In the corpus luteum, in which the vasculature undergoes almost complete involution, most endothelial cells of retracting vessels appear to be shed into the vessel lumen before apoptosis can be detected (40). Such a scenario is unlikely in the retina; given that scattered apoptotic endothelial cells were detected still attached to the vessel wall throughout the vascular tree. Our observations of the intact developing CNS vasculature suggest that the endothelial cells of retracting segments are redeployed during angiogenesis.

Our results suggest that endothelial cell migration may play an important role in formation of the outer retinal plexus. Endothelial cell proliferation was marked in the inner plexus in regions located above sites of outer plexus formation, but was much less intense in the forming outer vessels themselves. Thus, existing vascular beds appear to be a source of endothelial cells for the formation of new vessels by angiogenesis. Furthermore, in the inner plexus of the human retina, substantial vessel withdrawal coin-


Figure 7. Tracings of photographic montages showing the distribution of BrdU+ vascular endothelial cells (red) in the (A) inner and (B) outer vascular plexii of the rat retina at P11. (Number of BrdU+ endothelial cells in the inner plexus ⳱ 227, outer plexus ⳱ 57).

cides with the onset of formation of the outer plexus. An association of vessel withdrawal in the inner plexus with the formation of the outer plexus has also been observed in the pig retina (19). The close proximity between retracting vessels and angiogenesis has been noted in vivo (17,45) and is consistent with the conclusion that the endothelial cells of withdrawing vessels are redeployed in growing vessels. The weakness of CD34 expression in vascular segments in the immediate vicinity of newly forming vessels, together with the evidence that endothelial cell death is not present during the initiation of ves-

sel regression, and the fact that the density and topography of proliferating endothelial cells do not appear to account for the formation of all new vessels, lead us to suggest the migration and redeployment model of angiogenesis, which states that during normal development, the endothelial cells of retracting vessels migrate into neighboring vascular segments and are redeployed in growing vessels. These detailed topographical analyses were only possible because of the advantages offered by the retinal wholemount technique. We have shown that the mechanism of vascular pruning during development of the CNS differs markedly from those of vascular invo-


Figure 8. CD34 immunohistochemical staining of vessels of the inner vascular plexus in retinal whole-mount preparations derived from human fetuses. (A) The periphery of a retina, where the outer plexus does not form, from a full-term fetus. (B and C) The inner plexus of a retina at 31 WG (B) in a region immediately peripheral to the sprouting that gives rise to the outer plexus and (C) in a region where the vessels of the outer plexus are just starting to form. (D) A region of angiogenesis in a retina at 21 WG, prior to outer plexus formation.

lution, and suggest that endothelial cell migration and redeployment play a more important role in vessel withdrawal and the formation of new vessels than was previously appreciated. We have thus provided evidence that vascular endothelial cells in the developing CNS have three possible fates: 1) to line the lumen of vascular segments destined to remain throughout adult life; 2) for endothelial cells located within vascular segments that retract, to migrate and be redeployed in the formation of neighboring angiogenic sprouts; or 3) to undergo apoptosis where it is in excess of the needs of a particular vascular segment.

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ACKNOWLEDGMENTS

We thank A. Saleh for assistance in mapping the density of apoptotic bodies, C. Jeffrey and R. Smith for assistance with photography, and P. Kent for general technical assistance.

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