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* Department of Developmental Biology, Harvard School of Dental Medicine, and Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA;
Angiogenesis Laboratory and Retina Service, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts, USA; and
Division of Microscopic Anatomy and Bioimaging, Niigata University Postgraduate School of Medical and Dental Sciences, Niigata City, Japan
1Correspondence: Department of Developmental Biology, Harvard School of Dental Medicine, 188 Longwood Ave., Boston, MA 02115, USA. E-mail: alexander_marneros{at}yahoo.com
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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3-fold larger than in control mice and showed increased vascular leakage. These differences were independent of age-related changes at the choroid-retina interface. Ultrastructural analysis of the choroidal vasculature in mutant mice excluded morphological vascular abnormalities as a cause for the larger CNV lesions. When recombinant endostatin was administered to collagen XVIII/endostatin-deficient mice, CNV lesions were similar to those seen in control mice. In control mice treated with recombinant endostatin, CNV lesions were almost undetectable. These findings demonstrate that endogenous endostatin is an inhibitor of induced angiogenesis and that administration of endostatin potently inhibits CNV growth and vascular leakage. Endostatin may have a regulatory role in the pathogenesis of CNV and could be used therapeutically to inhibit growth and leakage of CNV lesions.—Marneros, A. G., She, H., Zambarakji, H., Hashizume, H., Connolly, E. J., Kim, I., Gragoudas, E. S., Miller, J. W., Olsen, B. R. Endogenous endostatin inhibits choroidal neovascularization.
Key Words: angiogenesis • collagen XVIII • Bruch’s membrane • age-related macular degeneration
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3
4
5
6
7)
. Consequently, several experimental approaches to target CNV therapeutically have utilized inhibitors of VEGF expression or activity, and these approaches have resulted in significant clinical benefit in patients with CNV (8
9
10)
. In contrast, hypoxia down-regulates some antiangiogenic factors, such as pigment epithelium-derived factor (PEDF), and thus may promote new vessel formation (11
, 12)
. The primary causes of such a proposed imbalance of stimulators and inhibitors of angiogenesis in the pathogenesis of CNV remain largely unknown. Age-related changes of Bruch’s membrane and abnormal sub-RPE deposit formation may promote inflammation and cause abnormalities in RPE cell metabolism that lead to alterations in the transcriptional regulation of proangiogenic or antiangiogenic factors.
In the process of choroidal neovascularization, the vascular overgrowth of choroidal vessels is coupled with a localized proteolytic degradation of Bruch’s membrane and penetration into the subretinal space. This proteolytic degradation of the basement membrane is believed to result in release of antiangiogenic fragments derived from basement membrane components that may act to counterbalance excessive neovascularization locally (13)
. Thus, in addition to pro- and antiangiogenic factors expressed by RPE cells or inflammatory cells at the site of CNV lesion formation, release of antiangiogenic proteolytic fragments from basement membrane components is thought to contribute to the extent of CNV formation as well.
A major constituent of Bruch’s membrane and the basement membranes of the choroidal vessels is collagen XVIII (14)
. This collagen is a heparan sulfate proteoglycan of vascular and epithelial basement membranes. Although it is found in almost all basement membranes, a particular requirement of collagen XVIII/endostatin for the eye has been shown by the identification of ocular abnormalities in mice and humans that either lack or have reduced expression of collagen XVIII (14
15
16
17
18
19)
. A proteolytic fragment of the C-terminal domain of collagen XVIII, termed endostatin, has been shown to possess antiangiogenic activity in vitro and in vivo when administered at high doses as a recombinant protein (20
, 21)
. Based on these findings, it has been suggested that collagen XVIII/endostatin may function as a physiological regulator of angiogenesis in vivo (17)
. However, a role for physiological levels of endostatin in regulating angiogenesis in vivo has not yet been shown. Lack of physiological concentrations of this collagen or its proteolytic fragment endostatin in Col18a1–/– mice does not lead to obvious vascular defects during development or postnatal growth (15)
, indicating that collagen XVIII/endostatin does not function as a critical negative regulator of angiogenesis. This collagen may be only one of many contributing factors involved in the dynamic interaction of proangiogenic and antiangiogenic components that regulate the complex process of new vessel formation, in which other regulators of angiogenesis may compensate for its absence. However, experiments with aortic explant cultures derived from Col18a1–/– mice showed increased vascular outgrowth compared with aortas derived from control littermates (22)
. Thus, a sensitive assay that allows for precise quantitation of neoangiogenesis in vivo may reveal a role for physiological levels of collagen XVIII/endostatin in regulating induced vessel growth. Therefore, we induced CNV lesions with a diode laser photocoagulator in Col18a1–/– mice and control littermates. Experimental CNV shows induced neoangiogenesis in a precise anatomic location with a clearly defined area of vascular remodeling, allowing for a precise quantitation of vessel growth. Although CNV lesions are induced experimentally in this acute injury model, they closely resemble neovascular lesions in the chronic disease AMD, and this acute laser injury model has been established as a valid surrogate model for neovascular AMD that has helped reveal several aspects of CNV pathogenesis.
Our data demonstrate a significant increase in the size of experimental CNV lesions in Col18a1–/– mice, with lesions forming large confluent areas; in contrast, such lesions remained small and clearly circumscribed in control mice. Ultrastructural analysis of the choroidal vasculature in mutant mice excluded the possibility that morphological defects in these vessels caused the increase in the size of CNV lesions. Furthermore, the increase of CNV lesion size observed in mutant mice was independent of age-related changes at the RPE-choroid interface. Notably, lack of collagen XVIII/endostatin also led to an increase in vascular permeability of CNV lesions, as assessed by fluorescein angiography. Administration of recombinant endostatin to Col18a1–/– mice rescued the observed phenotype, and CNV lesions were comparable to those seen in control mice. Notably, administration of recombinant endostatin to control mice resulted in CNV lesions that were hardly detectable.
These findings suggest that endogenous endostatin is a physiological inhibitor of induced angiogenesis in vivo and may act as a regulator of CNV growth in AMD. Endostatin’s antiangiogenic properties could be used therapeutically to target CNV growth and vascular leakage from these lesions. Col18a1–/– mice are the first mouse model to show increased CNV formation due to the absence of a single basement membrane component, and the data support the hypothesis that released antiangiogenic proteolytic fragments from basement membranes play a regulatory role during pathological angiogenesis, such as in CNV formation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The absence of collagen XVIII expression in Col18a1–/– mice was confirmed by a lack of immunostaining for collagen XVIII and endostatin using polyclonal antibodies, with positive staining in control eyes (14
, 15)
. Uniformity of genetic backgrounds of control (wild-type, or WT) and Col18a1–/– (KO) mice was ensured through backcrossing Col18a1–/– mice onto the C57BL/6J background over 15 generations. Mice were fed a regular chow diet. For all procedures, anesthesia was achieved by i.p. injection of 100 mg/kg body weight ketamine hydrochloride (Abbott, North Chicago, IL, USA) and 10 mg/kg body weight xylazine (Bayer, Shawnee Mission, KS, USA); pupils were dilated with topical 0.5% tropicamide (Alcon, Humacao, Puerto Rico). All animal experiments were in accordance with protocols reviewed and approved by the animal care committees of the Massachusetts Eye and Ear Infirmary and Harvard Medical School, and in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research.
Experimental CNV model
Eyes of age- and gender-matched Col18a1–/– mice and control C57BL/6J littermates were exposed to laser photocoagulation for induction of experimental CNV. Laser photocoagulation was performed using a diode-pumped, frequency-doubled 532 nm laser (Oculight GLx Laser System, IRIDEX Corporation, Mountain View, CA, USA) attached to a slit lamp, and a coverslip was applied to the cornea to view the retina. Four lesions were induced using a power of 200 mW, a spot size of 50 µm, and a duration of 100 ms. The lesions were located at the 3, 6, 9, and 12 o’clock meridians centered on the optic nerve and located two or three disc diameters from the optic nerve. Laser-induced disruption of Bruch’s membrane was identified by the appearance of a bubble at the site of photocoagulation. Laser spots that did not result in the formation of a bubble were excluded from the studies.
Fluorescein angiography
Two weeks after laser photocoagulation, fluorescein angiography (FA) was undertaken by an operator masked to the genetic identity of the animals. FA was performed in anesthetized animals with dilated pupils using a digital fundus camera (Model TRC 50 IA; Topcon, Paramus, NJ, USA) and standard fluorescein filters. Fluorescein injections were administered by i.p. injection (0.2 ml of 2% fluorescein sodium; Akorn Inc., Decatur, IL, USA) and the timer was started as soon as the fluorescein bolus was injected. A PMMA contact lens (base curve 1.65 mm, power 7.0 D, size 2.5 mm; Unicon Corporation, Osaka, Japan) was placed on the mouse cornea to improve visualization and prevent corneal drying. Two masked retina specialists not involved in laser photocoagulation or angiography evaluated the fluorescein angiograms at a single sitting, and lesions were graded. Grade 0 lesions had no hyperfluorescence. Grade 1 lesions exhibited hyperfluorescence without leakage. Grade 2A lesions exhibited hyperfluorescence in the early or midtransit images and late leakage. Grade 2B lesions showed bright hyperfluorescence in the transit images and late leakage beyond treated areas (grade 2B lesions were defined as clinically significant). The relative distribution of FA grades for CNV lesions was determined within each experimental group of mice. For confluent CNV lesions, the same FA grade was assigned for all lesions within the confluent hyperfluorescent area.
Quantitation of the size of CNV lesions
Two weeks after laser injury, the size of CNV lesions was measured using choroidal flat mounts. Mice were anesthetized and perfused through the left ventricle with a 30 gauge blunt canula with 8 ml of lactated Ringer solution, followed by 5 ml of 5 mg/ml fluorescein-labeled dextran in 10% gelatin (2x106 MW; Sigma, St. Louis, MO, USA). The eyes were enucleated and fixed in 4% formalin for 24 h. The anterior segment and the lens were removed. The retina was separated from the underlying RPE and four relaxing radial incisions were made. The remaining RPE/choroid/sclera complex was flat mounted (Immu-Mount Vectashield Mounting Medium; Vector Laboratories, Burlingame, CA, USA) and coverslipped.
Choroidal flat mounts were analyzed by fluorescent microscopy using a Leica DMR microscope (Leica Microsystems, Wetzlar, Germany) equipped with epifluorescence illumination. Openlab software (Improvision, Boston, MA, USA) was used to measure the magnitude of the hyperfluorescent areas corresponding to CNV.
Administration of recombinant endostatin
Human recombinant endostatin expressed in Pichia pastoris was administered at a dose of 0.5 mg/kg/day via i.p. injections for 14 days (U.S. Biological, Swampscott, MA, USA). Age- and gender-matched control mice received PBS instead. The first dose was given on the day of laser photocoagulation; the last dose was administered 14 days later, prior to preparation of the choroidal flat mounts. This dose was calculated to result in endostatin serum levels that approximate physiological serum levels (23)
.
Scanning electron microscopy
The fixative for scanning electron microscopy (SEM) contained 2% glutaraldehyde in 100 mmol/L phosphate buffer. Eyes were enucleated and immersed in fixative for 2 days, then processed for SEM. Subsequently, eyes were treated with 30% potassium hydroxide in distilled water for 8 min at 60°C to remove the extracellular matrix around blood vessels. The specimens were stained with a solution of 2% tannic acid and 1% osmium tetroxide, dehydrated with a graded series of ethanol, transferred to isoamyl acetate, and critical point-dried in liquid CO2. Some areas were cracked with fine forceps under a dissection microscope to expose blood vessels within the specimen. Dried specimens were put on aluminum stubs, coated with osmium tetroxide in an Osmium Plasma Coater (Vacuum Device Corp, Japan), and examined with a Hitachi S-4300N scanning electron microscope (24)
.
Immunohistochemistry
For immunohistochemical analysis, whole eyes were fixed in 4% paraformaldehyde, followed by infiltration in 30% sucrose. Eyes were embedded in OCT (Tissue-Tek, Sakura, Japan) and 7 µm cryostat sections were made. Sections were incubated with a monoclonal rat anti-mouse CD31 (BD PharMingen, San Diego, CA, USA) or a monoclonal rat antiperlecan (Lab Vision, Fremont, CA, USA) antibody. Sections were incubated with FITC-conjugated secondary IgG antibodies (Vector Laboratories) and a mounting medium containing DAPI was used (Vector Laboratories). For histological analysis, sections were stained with hematoxylin and eosin according to standard procedures.
Electron and light microscopy
For morphological examination of CNV lesions, eyes were enucleated and fixed for 24 h in 2.5% formaldehyde and 1.5% glutaraldehyde in 0.1M cacodylate buffer (pH 7.4). After postfixation in 4% osmium tetroxide and dehydration steps, the eyes were embedded in TAAB epon (Marivac, Inc., St. Laurent, Quebec, Canada) overnight. For light microscopy, serial sections of 0.5 µm were stained with toluidin blue or azure II, and 85 nm-thin sections were used for standard transmission electron microscopy.
Statistical analysis
A two-sample Student’s t test with unequal variance was used for statistical analyses of the quantitative CNV flat mount data. Statistical significance was set at P < 0.05. The hyperfluorescent areas of CNV lesions in each eye were measured, and the total area of CNV lesions per eye was determined as their sum. For confluent lesions, the entire hyperfluorescent area was measured and the number of initial CNV lesions within confluent lesions was counted.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Here we performed ultrastructural analysis of the choroidal vasculature by scanning EM, and found no significant difference between the choroidal vessels in Col18a1–/– and control littermate mice (Fig. 1
A, B). Choroidal vessels in mutant mice contained erythrocytes and showed a fenestrated endothelium (Fig. 1C, D
). There were no observable major structural alterations of the choroidal vasculature in Col18a1–/– mice, and thus their choroidal vessels can be directly compared with the choroidal vasculature of control mice of same genetic strain, age, and gender in acute laser injury CNV models. Scanning EM analysis of RPE cell morphology in Col18a1–/– mice (after removal of Bruch’s membrane) demonstrated reduced basal infoldings (Fig. 1F
).
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Fluorescein angiography shows increased vascular leakage from CNV lesions in mice lacking collagen XVIIII/endostatin
FA was performed in 2-month-old Col18a1–/– (n=10) and control mice (n=9) 2 wk after laser photocoagulation (Figs. 2
and 3
). Significant fluorescein leakage, defined as grade 2B lesions, was observed in both 2-month-old Col18a1–/– mice (93% of all lesions) and control mice (81% of all lesions, Fig. 3
).
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In control mice, laser photocoagulation resulted in well-circumscribed hyperfluorescent areas at the sites of injury to the Bruch’s membrane (Fig. 2E, F
), indicating the formation of subretinal CNV as confirmed by histological examination (Fig. 4
). These subretinal CNV lesions represent outgrowths of the choroidal vessels at the site of laser injury, as demonstrated by immunohistochemistry with the endothelial cell marker CD31 or the basement membrane marker perlecan (Fig. 5
). In contrast, laser photocoagulation in Col18a1–/– mice induced the formation of large hyperfluorescent areas, which occupied most of the visible posterior pole (Fig. 2A, B
). The hyperfluorescent areas represented large choroidal neovascular membranes from large neighboring lesions that became confluent. Such confluent hyperfluorescent lesions were consistently observed in eyes of Col18a1–/– mice (60%) but not in the eyes of control mice (0%).
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Ultrastructural analysis of CNV lesions in mutant and control mice showed increased neoangiogenesis in mutant mice. In control mice, the retina is displaced into the choroid at the laser scar. CNV lesions in Col18a1–/– mice showed large networks of blood vessels with intraluminal erythrocytes surrounded by elongated cells (Fig. 4)
.
Lack of collagen XVIII/endostatin results in increased CNV lesion size
FITC-dextran perfusion of 2-month-old age- and gender-matched mice was performed before preparation of choroidal flat mounts in order to evaluate the size of CNV lesions 2 wk after laser photocoagulation. The angiographic findings were consistent with observations made with the choroidal flat mounts, which showed large neovascular choroidal outgrowths in Col18a1–/– mice (Fig. 6
A–E) and only small circular areas of CNV in control mice (Fig. 6K-O
). Total CNV area per eye was significantly increased in Col18a1–/– mice (mean±SD: 482x103±224x103 µm2, n=10 eyes) compared with the control animals (almost 3-fold as much) (181x103±66x103 µm2, n=9 eyes; P=0.0019) (Fig. 7
).
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Increased CNV lesion size and fluorescein leakage in Col18a1–/– mutant mice independent of age
Whereas young (2-month-old) mutant mice do not show major functional defects of RPE cells or accumulation of sub-RPE deposits, aged mutant mice (10-month-old) show significant RPE and retinal defects (14)
. If the differences in CNV lesion sizes observed between young Col18a1–/– mice and control mice were due in part to functional abnormalities of the RPE/choroid complex in mutant mice not readily observable at that age, this difference would be expected to become more significant in aged mice.
To investigate whether the age-dependent RPE defects in mutant mice contribute to the increase of CNV lesion size formation in these mice, we induced CNV lesions in young (2-month-old) and aged (10- to 12-month-old) mutant and control mice. In both age groups, Col18a1–/– mice (Figs. 8
and 9
) had large CNV lesions that often became confluent, whereas control mice had well-defined smaller CNV lesions. The relative difference in CNV lesion size between mutant and control mice was maintained in the young and aged groups (Fig. 8)
, with a well-documented increase of CNV lesion size with age (25)
. Total CNV area per eye was significantly increased in aged Col18a1–/– mice (mean±SD: 872x103±347x103 µm2, n=6 eyes) compared with age-matched control animals (almost 3-fold as much) (328x103±71x103 µm2, n=7 eyes; P=0.0130) (Fig. 8)
. Similarly, vascular leakage was increased in aged mutant mice (n=6) compared with control mice (n=4). Whereas 100% of CNV lesions in mutant mice were graded as 2B lesions, only 75% of CNV lesions in age-matched control mice were graded as 2B. Also, fused CNV lesions were found almost exclusively in the eyes of mutant mice (KO vs. WT: 83% vs. 25%).
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These data suggest that the increase in CNV lesion size and vascular leakage observed in mutant mice is not a consequence of RPE defects in these mice, but imply that collagen XVIII/endostatin has a direct antiangiogenic activity that inhibits CNV lesion size and vascular leakage.
Endostatin inhibits CNV formation and fluorescein leakage
To determine whether the increased CNV lesions observed in mutant mice are the result of the lack of collagen XVIII as a structural basement membrane component or instead due to the lack of the antiangiogenic activity of its endostatin domain, we administered recombinant endostatin to mutant mice to achieve serum concentrations approximating physiological serum levels of endostatin. In this rescue experiment, the total CNV area per eye in Col18a1–/– mice that received endostatin was significantly smaller than in mutant mice, which received no endostatin (PBS) (KO: mean±SD: 482x103±224x103 µm2, n=10 eyes; KO+ES: 204x103±92x103 µm2, n=12 eyes; P=0.0031); the total CNV area per eye was almost the same size as seen in WT mice, with no detectable significant statistical difference between the two groups (KO+ES: mean±SD: 203x103±92x103 µm2, n=12 eyes; WT: 181x103±66x103 µm2, n=9 eyes; P=0.5231) (Fig. 7)
. Administration of recombinant endostatin to WT mice potently inhibited CNV lesion formation (WT: mean±SD: 181x103±66x103 µm2, n=10 eyes; WT+ES: 53x103±38x103 µm2, n=12 eyes; P=0.0002) (Fig. 7)
. Thus, endostatin administration rescues the CNV phenotype in mutant mice. The data suggest that the antiangiogenic activity of the endostatin domain inhibits CNV lesion size.
Similarly, endostatin administration to mutant mice reduced vascular leakage from CNV lesions to the amount seen in control mice. Furthermore, endostatin acted as a potent inhibitor of CNV leakage even when administered to control mice. Whereas 93% of CNV lesions in KO mice where grade 2B lesions, WT mice that received endostatin had only 23% grade 2B lesions (Fig. 3)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We previously showed that collagen XVIII and its endostatin domain are localized in Bruch’s membrane and in the choroidal subendothelial basement membrane (14)
. It has been proposed that proteolytic degradation of the subendothelial basement membrane releases antiangiogenic fragments of basement membrane components, such as endostatin, in areas of induced angiogenesis that would oppose the induced vascular outgrowth via a negative effect on vessel growth. This hypothesis is supported by evidence showing proteolytic cleavage of endostatin and endostatin-like protein fragments from the C-terminal region of collagen XVIII through proteolytic enzymes that initiate sprouting angiogenesis by degrading the basement membrane (26
, 27)
. CNV is such a process of induced neoangiogenesis, requiring degradation of the basement membrane as an initial pathogenetic step. In fact, studies of various mouse models have suggested that invasive CNV requires a disruption of Bruch’s membrane in addition to increased levels of vascular growth factors (such as increased VEGF expression in the RPE or inflammatory cells) (28
29
30
31)
. Thus, in the absence of the antiangiogenic collagen XVIII/endostatin, the balance between proangiogenic and antiangiogenic factors may be shifted toward proangiogenic factors. This could explain the increased size of CNV lesions in Col18a1–/– mice. Decreased endostatin levels in human CNV lesions support this hypothesis as well (32)
. Conversely, i.v. injection of adenoviral vectors expressing secretable endostatin resulted in decreased experimental CNV lesion size in mice (33)
. This finding is not surprising, since injection of such adenoviral vectors resulted in endostatin serum concentrations of up to 10 µg/ml, 100-fold greater than reported physiological serum levels of endostatin in mice (50–100 ng/ml) (15
, 34)
. The antiangiogenic activity of such high doses of endostatin and other C-terminal proteolytic fragments of collagen XVIII is well documented (20
, 21
, 35)
.
In contrast, our data are the first to demonstrate a regulatory effect of physiological levels of collagen XVIII/endostatin on neoangiogenesis in vivo. Physiological levels of endogenous endostatin regulate the size of CNV lesions and leakage from these lesions in the experimental CNV model, and thus it is likely that reduced endostatin levels, as reported for human CNV lesions (32)
, may predispose to the development of CNV in AMD. Notably, we did not observe CNV in aged Col18a1–/– mice unless it was experimentally induced. Thus, the absence of collagen XVIII/endostatin alone is not sufficient to cause CNV; additional factors, such as injury to Bruch’s membrane, are required.
In addition to the increased growth of CNV lesions in mutant mice, we also observed increased vascular leakage from these lesions in Col18a1–/– mice, which was inhibited by administration of recombinant endostatin. This finding suggests that endostatin inhibits vascular leakage directly and is consistent with previous observations showing that endostatin inhibits VEGF-induced retinal vascular permeability, possibly through an up-regulation of tight junction proteins (36
, 37)
.
In vitro aortic explant cultures from Col18a1–/– and control mice showed an increased vascular outgrowth in mutant mice, resembling in vitro the increased angiogenesis and increased endothelial cells in the in vivo CNV model (22)
. This difference was not observed after the addition of low levels of endostatin to the cultures. While endothelial cells isolated from Col18a1–/– mutant mice showed no difference in their proliferation or growth factor-induced migration in vitro, they showed an increased adhesion to fibronectin, a component of the extracellular matrix that binds to endothelial cells via integrins and cell surface proteoglycans, which is highly expressed in CNV lesions as well (22
, 38
39
40)
. Endostatin can bind to cell surface proteoglycans and therefore may reduce fibronectin binding sites on endothelial cells (22
, 34
, 35)
. The antiangiogenic activity of endostatin thus may result from a localized inhibition of endothelial cell/extracellular matrix interactions during vascular tube formation at areas of neoangiogenesis, with subsequent destabilization and regression of vessels. Lack of endostatin, in contrast, would result in an increased angiogenic response through increased endothelial cell-extracellular matrix interactions, consistent with the increased angiogenesis seen in vitro in the aortic explant assay and in vivo in the CNV model in Col18a1–/– mice.
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ACKNOWLEDGMENTS |
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Received for publication April 14, 2007. Accepted for publication May 17, 2007.
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