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From the Department of Cell Biology, Harvard Medical School, Boston, Massachusetts
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Abstract |
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Segments of thoracic aorta, 1 to 2 cm in length, were excised in a sterile manner from 4- to 7-month-old Col18a1-null mice, as well as from wt littermates. The specimens were dissected carefully to remove surrounding fibroadipose tissue, rinsed extensively with phosphate-buffered saline (PBS), opened and sectioned into 
1-mm rectangular pieces, and embedded in type I collagen gel. The gel was made by adding 1 ml of chilled collagen solution into prechilled culture inserts with 0.45-µm pore-size polyethylene terephthalate membrane of six-well plates (Becton Dickinson, Bedford, MA) and gelled at 37°C for 30 minutes. The final collagen solution was obtained by mixing 7 vol of 4.15 mg/ml rat tail type I collagen (Becton Dickinson) with 2 vol of 1.17% NaHCO3 and 1 vol of 10x minimal essential medium (Life Technologies Inc., Rockville, MD). After gelation, 2 ml of Endothelial-SFM medium (Life Technologies Inc.) was added into inserts and 2.5 ml of the same medium to the lower wells. The medium was changed every 2 days. The aortic explants, four to five explants in each well, were cultured at 37°C, 5% CO2 for a period of 15 to 21 days. Explants were treated with soluble recombinant ES at concentrations of 0.1, 0.25, 0.5, or 1 µg/ml. We used both mouse and human ES produced in 293EBNA cells and purified as described2
or human ES produced in Pichia pastoris (a generous gift from Dr. K. L. Sim, EntreMed, Inc., Rockville, MD). No differences in activities between mouse and human ESs were observed in the assays reported here. Untreated cultures were used as controls. Angiogenesis was quantitated in two different ways. One method consisted of counting the number of free tips of microvessels growing out from the two long edges of the rectangular explants. In this method we did not attempt to adjust the counts for variations in the size of the explant rectangles, but simply tried to select explants from different experiments and different animals that were similar in their lengths and widths for counting. The second method was designed to assess the number of long microvessels growing out from the long edges of the rectangular explants. In this method, we counted the number of microvessels extending to or beyond a distance of 414 µm from the long edges of the explanted tissue as illustrated in Figure 1
. To control for variations in size of the explants, the number of long microvessels was expressed as number of long microvessels per µm of long explant edge (microvessel density). This method was used to combine data from three experiments, with different experiments using aortic tissue from different animals. In experiments with wt tissue, one mouse was used for each experiment; in experiments with ko tissue, two mice (littermates) were used for each experiment. In each experiment four to five explants were analyzed in each group of wt and ko explants. The Student’s t-test was used for statistical analysis.
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Immunostaining was performed on whole mounts of aortic explant cultures. Gels containing aortic explants were fixed with 4% paraformaldehyde in PBS for 4 hours at 4°C, washed briefly in PBS, treated with 0.25% Triton X-100 in PBS for 1 hour, and blocked with 1.5% blocking reagent (BioGenex, San Ramon, CA) or 1% bovine serum albumin (BSA)/PBS (Sigma, St. Louis, MO) for 2 hours at room temperature. Anti-mouse CD31 (PECAM, 1:800; BD PharMingen, San Diego, CA), anti-
-smooth muscle actin (-SMA, 1:500; Sigma), anti-collagen IV (1:1000, Chemicon Int., Inc., Temecula, CA) and anti-FN monoclonal antibody (1: 400, Sigma) were used as primary antibodies and gels were incubated at 4°C overnight with gentle shaking. The gels were washed for 2 hours with three changes of PBS. For immunohistochemical staining, cultures were incubated with biotinylated secondary antibodies for 3 hours at room temperature with gentle shaking. After 2 hours of washing, the gels were incubated with ABC reagent for 1 hour, and the bound antibodies were detected by Vector VIP or diaminobenzidine substrate (Vector Laboratories, Inc., Burlingame, CA). For immunofluorescent staining, gels were incubated with fluorescein isothiocyanate (FITC)-conjugated secondary antibodies (Vector Laboratories, Inc.) for 3 hours. Images were analyzed using Nikon E800 upright microscope (Nikon, Melville, NY). Controls for immunostaining included incubations with species-matched immunoglobulin and incubations in which the primary antibody was omitted.
Electron Microscopy
Aortic explants embedded in collagen gels were fixed at room temperature for 1 hour with 4% glutaraldehyde and 4% paraformaldehyde in 0.1 mol/L cacodylate buffer, pH 7.4, rinsed and postfixed in cold 1% osmium tetroxide in the same buffer for 1 hour, and stained en bloc for 30 minutes with saturated uranyl acetate in distilled water. They were dehydrated through graded ethanols, cleared in propylene oxide, and embedded in Epon/Araldite. Thin sections were obtained and examined by transmission electron microscopy.
Isolation of Mouse Lung Endothelial Cells
The lungs of 2- to 3-month-old ko and wt mice (more than 10 mice in each group) were perfused with PBS-heparin (1 U/ml, Sigma) and collected into 40 ml of Dulbecco’s modified Eagle’s medium/F12. The tissue was minced into small pieces, and digested in 20 ml of 0.2% collagenase I (Sigma) at 37°C for 1 hour with occasional shaking. The solution was passed through a nylon gauze filter (two layers, 200 mesh) and centrifuged. The pellet was collected and resuspended in 1% BSA/PBS. Dynabeads M-450 with sheep anti-rat IgG (Dynal Biotech Inc., Lake Success, NY) were conjugated with rat anti-mouse CD31 (MEC13.3 no azide/low endotoxin, PharMingen) and added into the cell suspension. After incubating for 30 minutes with rotation, the cells bound to the beads were isolated by placing the tubes on a magnetic device (MPC, Dynal Biotech Inc.). The beads were removed by incubation with 0.25% trypsin/ethylenediaminetetraacetic acid (Irvine Scientific, Santa Ana, CA) for 5 to 10 minutes, and the isolated mouse lung endothelial cells (mLECs) were plated on a gelatin-coated (Cascade Biologics, Inc., Portland, OR) 10-cm-diameter culture dish and cultured in Dulbecco’s modified Eagle’s medium/F12 medium (Mediatech, Inc., Herndon, VA) supplemented with 10% fetal bovine serum (Paragon Biotech, Inc., Baltimore, MD), 20 mmol/L HEPES (Sigma), 100 µg/ml streptomycin, 100 U/ml penicillin (Irvine Scientific, Santa Ana, CA), 2 mmol/L sodium pyruvate, 20 U/ml heparin (Sigma), 100 µg/ml endothelial cell growth supplement (Becton Dickinson). The medium was changed every 2 days, and cells from passage 3 to 10 were used for the experiments.
Indirect Immunofluorescence of Endothelial Cells
ECs were cultured on gelatin-coated glass coverslips and fixed in acetone/methanol at 1:1 ratio for 15 minutes at –20°C. The cells were blocked in 1% BSA/PBS for 1 hour and probed with primary antibodies against mouse CD31 (1:400), Tie2 (1:200), mouse recombinant ES (1:400; Medical & Biological Laboratories Co., Ima-City, Japan), Von Willebrand Factor (vWF, 1:400; DAKO Corp., Carpinteria, CA) or -SMA (1:400). Secondary antibodies, conjugated with FITC, were used for detection by conventional fluorescence microscopy.
Proliferation Assay
Trypsinized ECs suspended in 5% fetal bovine serum-containing medium were seeded into gelatin-coated 96-well plates at a density of 2000 cells/well with or without human ES (0.5 µg/ml). At each time point of 0, 12, 24, 48, 72, 96, and 120 hours, CellTiter nonradioactive cell proliferation assay (Promega Corp., Madison, WI) was performed according to the manufacturer’s instructions. The amount of 490-nm absorbance, proportional to the number of living cells, was measured using an enzyme-linked immunosorbent assay plate reader (Molecular Devices, Sunnyvale, CA).
Adhesion Assay
The cell adhesion assay was performed as previously described9 with slight modifications. Briefly, 96-well plates were coated overnight at 4°C with collagen I (50 µg/ml, Becton Dickinson), laminin (LN, 20 µg/ml), collagen IV (10 µg/ml), or FN (10 µg/ml) diluted in PBS. PBS alone was used as control. All of the above reagents were purchased from Sigma unless otherwise indicated. Wells were washed with PBS and blocked with 0.1% BSA/PBS for 1 hour at room temperature. mLECs were harvested by trypsin treatment, labeled with 5 µmol/L Calcein AM (Molecular Probes, Inc., Eugene, OR), collected in Dulbecco’s modified Eagle’s medium/F12 containing 0.1% BSA, and washed three times with the same medium. Subsequently, 2 x 104 cells in 100 µl of medium/BSA were added to each well and incubated for 45 minutes at 37°C in 5% CO2. Nonadherent cells were removed with four vigorous PBS washes. The relative number of adherent cells was determined by fluorescent intensity measured by a fluorescence microplate reader (LJL Biosystem, Analyst).
To study the effect of exogenous ES on cell adhesion to FN, mLECs were either preincubated with 0.5 µg/ml human ES for 30 minutes at 37°C and washed two times in medium/BSA before seeding or ES of indicated concentrations was added to the cell suspensions immediately before seeding. The absence of ES was used as control.
For heparin treatment studies, 96-well plates were coated with FN overnight at 4°C, and 9 µg/ml of heparin either alone or together with 0.5 µg/ml of ES were added to cell suspensions. The omission of both heparin and ES was used as control.
For studies of the effects of RGD-containing peptides, 96-well plates were coated with FN overnight at 4°C, and the peptide GRGDS (Sigma) added at concentrations of 0.01, 0.05, 0.1, or 0.2 mmol/L to the cell suspensions, followed by a 20-minute incubation at 37°C before the cells were added to the FN-coated wells. The absence of GRGDS or the addition of SDGRG peptide (Sigma) at a concentration of 0.1 mmol/L were used as controls.
To block synthesis of endogenous collagen XVIII/ES, confluent cells of both ko (control) and wt were incubated with 2.5 µg/ml cycloheximide (Sigma) for 12 hours at 37°C, 5% CO2. To completely eliminate collagen XVIII-associated ES from cell surfaces, cells were first treated with cycloheximide for 12 hours and then treated with 0.5 µg/ml Cathepsin L (Cat L, Calbiochem Corp., La Jolla, CA) in 50 mmol/L Tris-acetate, pH 5.5, containing 5 mmol/L dithiothreitol at 37°C for 8 minutes. The treatment with Cat L was based on previous studies of proteolytic release of ES10 and on experiments in which a brief Cat L treatment was shown to release ES from mLECs (see below). The subsequent adhesion assays to FN were performed as described above. For all adhesion assays, the results are presented as mean values from three individual experiments. In each experiment eight wells were analyzed for each treatment group of cells. Student’s t-test was used for statistical analysis.
Western Blotting
Confluent wt mLECs were washed two times with PBS and incubated with 0.5 µg/ml Cat L in 50 mmol/L Tris-acetate, pH 5.5, containing 5 mmol/L dithiothreitol at 37°C for 5, 10, and 30 minutes. Cathepsin inhibitor E64 (2 µmol/L) (Sigma) was added at the end of each incubation. Supernatants were collected and concentrated approximately sixfold using Centricon (Millipore Corp., Bedford, MA). The treated cells, as well as control wt cells without Cat L treatment and untreated ko mLECs were solubilized for 30 minutes at 4°C with rotation in a lysis buffer, containing 10 mmol/L Tris-HCl, pH 7.5, 1% Nonidet P-40, 0.25% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 0.15 mol/L NaCl, 6 mmol/L ethylenediaminetetraacetic acid, a cocktail of protease inhibitors (Sigma) and 2 µmol/L E64. Lysates were centrifuged at 14,000 rpm for 10 minutes and supernatants were concentrated as described above. Samples were separated on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing condition (4% ß-mercaptoethanol) and proteins were transferred onto nitrocellulose membranes. Membranes were probed for 1 hour with anti-mouse ES polyclonal antibody and 30 minutes with horseradish peroxidase anti-rabbit IgG (1:5000; Santa Cruz Biotechnology, Santa Cruz, CA). The blots were visualized with chemiluminescent substrate (Pierce, Rockford, IL).
Tube Formation
The assay for endothelial cell tube formation was performed as previously described11 with few modifications. Briefly, 45 µl of reduced Matrigel (Becton Dickinson) was added to prechilled 96-well plates and allowed to gel at 37°C for 30 minutes. mLECs from collagen XVIII ko and wt mice (15,000 cells/well) were seeded with or without exogenous human ES (0.5 µg/ml). Cells were incubated for 8 hours at 37°C, 5% CO2. The formation of capillary-like tubes was studied by phase contrast microscopy.
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Results |
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Aortic explants embedded in collagen gels and cultured in serum-free medium gave rise to microvessels that resemble capillary structures. The cells at the core of the sprouts were immunoreactive with the endothelial cell-specific anti-CD31 antibody as shown in Figure 2, A to C
(arrows), whereas peri-endothelial cells were immunonegative. The sprouts showed positive staining with anti--SMA antibody in the outer layer of sprouts (Figure 2; D to F
, arrows). We also demonstrated positive staining for other endothelial cell markers, such as Tie2, Flk1, and endoglin (data not shown). The microvessel sprouts showed immunoreactivity with anti-BM component antibodies, as shown in Figure 2G
for collagen IV and Figure 2H
for FN. Electron microscopy revealed the presence of a lumen (Figure 2I
, arrow) in the sprouting vessels and the presence of typical endothelial cell-cell junctions (Figure 2I
, arrowhead). The growth of microvessels in this culture system is a self-regulated, highly dynamic process, consisting of capillary growth and regression that recapitulates vascular remodeling during angiogenesis. In some regions of the cultures, regression resembling in vivo vascular remodeling could be observed (Figure 2, C and F
; arrowheads). These cellular processes continued during a 2- to 3-week or longer period of culture.
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Previous studies have shown that collagen XVIII/ES is present at relatively high levels in the aortic wall.12
We used the aortic explant assay to test whether these levels are functionally sufficient for local regulation of induced angiogenesis. We cultured aortic explants from homozygous mutant and wt mice and examined the outgrowth of microvessels. We used serum-free media without adding exogenous angiogenic factors. The results showed that explants from Col18a1-null mice had more than a twofold increase in the number of microvessel ends, as compared to explants from wt littermates (Figure 3, A and B)
. The number of sprout ends in Figure 3C
represent the average number of tips of microvessels growing out from the long edges of explants derived from ko and wt mice. The numbers in Figure 3D
represent the average value of long vessel number per unit length of explant edge (see Materials and Methods). The addition of recombinant ES to the cultures at a concentration as low as 0.1 µg/ml reduced vascular outgrowth in the mutant cultures to the wt control level (Figure 3, C and D)
, but had no effects on the wt cultures. Further inhibition of outgrowth in both wt and ko explants required a much higher concentration (>2 µg/ml, data not shown).
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To address the question of what mechanism(s) contribute to the increased in vitro angiogenic response in aortic explants from Col18a1-null mice, we isolated endothelial cells (mLECs) from mouse lungs of both ko and wt mice. The endothelial cells were characterized by staining for endothelial-specific markers. Both mutant and wt mLECs were immunoreactive for vWF (Figure 4A)
, CD31 (Figure 4B)
, and Tie 2 (Figure 4C)
. There were few -SMA-positive cells (Figure 4D)
. First we tested whether there was a difference in proliferative activity between mutant and wt mLECs; however, the results showed no significant differences in the growth rates (Figure 5)
. There was no inhibitory effect of exogenous ES (0.5 µg/ml) on proliferation of both cell types. In addition, mutant and wt cells showed similar stimulatory responses to vascular endothelial growth factor and basic fibroblast growth factor and inhibitory responses to transforming growth factor-ß1 (data not shown).
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Because collagen XVIII is a heparan sulfate proteoglycan and component of BMs,13
it is possible that it may regulate angiogenesis by modulating interactions between endothelial cells and the underlying matrix. To examine this possibility, we evaluated the ability of mLECs to adhere to different components of BM. As shown in Figure 6A
, mutant cells had strikingly higher adherence to FN compared to wt mLECs. In contrast, there were no significant differences in the adhesion to LN, collagen I, or collagen IV between the two cell types. Exogenous ES added during the assay inhibited adhesion of mutant mLECs to FN, but had no effect on wt cells as seen in Figure 6B
. Furthermore, the addition of heparin at 9 µg/ml, either alone or together with ES, inhibited the increased adhesion of mutant mLECs to FN in a similar manner (Figure 6C)
. We did not observe significant inhibitory effect of ES when mutant cells were preincubated with ES for 30 minutes and washed before seeding (Figure 6B)
. Addition of RGD peptide reduced the adhesion of both ko and wt cells to FN proportionally; even at a peptide concentration of 0.2 mmol/L the mutant cells adhered better to FN than wt cells (Figure 6D)
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. Mutant cells were immunonegative. Cathepsin L is responsible for the generation of ES in hemangioendothelioma cell culture and can generate ES from recombinant NC1 protein.10
To evaluate the role of endogenous collagen XVIII/ES in the low degree of wt endothelial cell adhesion to FN (compared to ko cells), we first tested whether collagen XVIII on the cell surface could be cleaved by Cat L. As shown in Figure 7B
, ES was released into cell-free supernatants after treatment of wt mLECs with Cat L for 10 minutes. Treatment for 30 minutes resulted in further degradation of the released ES, consistent with previous studies.10
In an attempt to eliminate endogenous ES from the cell surface of mLECs, we therefore treated cells with Cat L for 8 minutes after incubation for 12 hours with cycloheximide to block protein synthesis. When such wt and ko mLECs, treated with cycloheximide and Cat L, were used in adhesion assays, they both showed reduced adhesion and there was no difference in their adhesion to FN (Figure 7C)
.
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To compare the ability of mutant and wt mLECs to form capillary-like tubes in vitro, mLECs were plated on reduced Matrigel. This assay reflects cell-extracellular matrix interactions. As shown in Figure 8
, under the same conditions, mutant mLECs formed more continuous capillary-like structures than wt cells. The addition of exogenous ES inhibited tube formation in the ko mLEC cultures so that the extent of tube formation appeared similar with mutant and wt cells.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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. The assay allowed the function of collagen XVIII/ES in induced angiogenesis to be examined independently of or in the presence of exogenous ES without interference by other unknown serum factors and without the complication of endothelial cell differentiation from circulating progenitor cells. Microvessel sprouting continues at a high level during the 2- to 3-week culture period. This suggests that continuous synthesis and secretion of local soluble autocrine and paracrine factors from aortic tissue or newly formed microvessels, sustains or amplifies the overall angiogenic sprouting. This behavior differs from the rat aortic ring assay in which vessel sprouting reaches a peak after 1 week of culture and begins to regress from the second week in the absence of exogenous vascular endothelial growth factor or basic fibroblast growth factor. Using the aortic explant assay, we observed a twofold increase in the number and length of microvessels in explants from Col18a1-null mice. This suggests that aortic tissue is more angiogenic in the absence of endogenous collagen XVIII/ES. The underlying mechanism could be increased proliferation or migration of endothelial cells in the mutant explants or increased vascular stabilization by altered cell-matrix interactions during remodeling. We measured the length of the outgrowing vessels on a daily basis and did not observe any differences in the rate of sprout elongation between mutant and wt explants (data not shown). Hence, a difference in endothelial cell migration seems to be an unlikely explanation for the increased vessel density in the mutant explants. Furthermore, there was no significant difference in the proliferative activity of mLECs isolated from ko and wt mice and addition of exogenous ES did not significantly inhibit cell proliferation in both cell types. In addition, the cells also showed similar responses to growth factors; both were stimulated by basic fibroblast growth factor and to a lesser extent by vascular endothelial growth factor and inhibited by transforming growth factor-ß1 (data now shown).
The most striking difference in biological behavior between wt and mutant ECs was in their ability to adhere to FN. Exogenous ES (at a concentration of 100 ng/ml) suppressed the elevated adhesion of mutant ECs to the wt level. The molecular basis for the adhesion of endothelial cells to extracellular matrix components involves two major families of cell-surface molecules, integrins, and heparan sulfate proteoglycans. FN contains several discrete domains that are involved in the binding to cell surfaces, including the C-terminal Hep II domain,17,18
the alternatively spliced type III connecting segment,19
and the central cell-binding domain.20
EC adhesion to FN is primarily mediated by 5ß1,21
but also by proteoglycans on the cell surface. A chondroitin sulfate proteoglycan with weak affinity for FN reduced binding of FN to both cells and the purified FN receptor (5ß1), probably by steric hindrance.22
Because the adhesion of both mutant and wt cells to FN was inhibited proportionally by the addition of RGD peptide (the number of adhered cells was reduced to 35% in both cell types with 0.1 mmol/L RGD peptide added to the assay), we believe that differential binding to 5ß1 integrin is unlikely to explain the difference in adhesion to FN of ko and wt mLECs.
Several lines of evidence point to an important role for intercalated heparan sulfate proteoglycan on cell surfaces in interactions with FN via its heparin binding sites.23,24 The ES domain of intact collagen XVIII, as well as proteolytically cleaved ES, can bind to heparan sulfate, and the affinity for binding to heparan sulfate is higher when ES is a trimeric domain within intact collagen XVIII than when it is proteolytically cleaved and released as a monomer.25,26 Thus, it is possible that endogenous collagen XVIII/ES affects cell-matrix interactions by occupying cell surface heparan sulfate sites, resulting in fewer binding sites for FN in wt cells than in collagen Col18a1-null cells. In support of this hypothesis is the finding that when heparin was added to the mutant and wt cell suspensions, either alone or together with ES before seeding, the cells exhibited similar adhesion to FN. This observation suggests that ES and heparin inhibit cell adhesion to FN through the same pathway, and that the difference in adhesion to FN between ko and wt endothelial cells is because of a heparan sulfate-dependent mechanism. The hypothesis implies that collagenXVIII/ES produced by wt mLECs before and/or during the adhesion assay inhibits cell-FN interactions. Consistent with this idea, we found that treatment of the cells with cycloheximide to inhibit protein synthesis followed by treatment with cathepsin L to release ES from cell surface-bound collagen XVIII, eliminated the difference in adhesion of wt and ko cells to FN. It has been shown that ES inhibits basic fibroblast growth factor-induced angiogenesis in the chorio-allantoic membrane assay, and that this inhibitory activity is abolished when two arginine residues in the heparin-binding site are converted to alanine.25 This suggests that in situations in which heparin-binding of ES is required for its anti-angiogenic activity, for example during inhibition of tumor-induced angiogenesis by exogenous ES, the effect of ES may be based on a mechanism similar to the one described here for the aortic explant angiogenesis model.
Tube formation during angiogenesis involves complex cell-cell and cell-matrix interactions. The increased capillary-like tube formation in mutant mLECs and its inhibition by exogenous ES further support the hypothesis that collagen XVIII/ES modulates cell-matrix interactions. On the basis of the above data, we conclude that collagen XVIII/ES can act as a local negative regulator of angiogenesis by modulating endothelial cell-extracellular matrix interactions, such as those involving FN. This modulation can lead to destabilization of newly formed vessels and cause regression.
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Acknowledgements |
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Footnotes |
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Supported by the National Institutes of Health (grants AR 36820 and AR 048564).
Accepted for publication April 6, 2004.
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