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Increased Angiogenic Response in Aortic Explants of Collagen XVIII/Endostatin-Null Mice

2004; Elsevier BV; Volume: 165; Issue: 2 Linguagem: Inglês

10.1016/s0002-9440(10)63307-x

1525-2191

Qing Li, Bjørn R. Olsen,

Platelet Disorders and Treatments

Endostatin, a proteolytic fragment of basement membrane-associated collagen XVIII, has been shown to be a potent angiogenesis inhibitor both in vivo and in vitro when given at high concentrations. The precise molecular mechanisms by which it functions and whether or not it plays a role in physiological regulation of angiogenesis are not clear. In mice with targeted null alleles of Col18a1, there appears to be no major abnormality in vascular patterns or capillary density in most organs. Furthermore, the growth of experimental tumors is not increased. However, a detailed analysis of induced angiogenesis in these mice has not been performed. Therefore, we compared the angiogenic responses induced by in vitro culture of aortic explants from collagen XVIII/endostatin-null mice (ko) to wild-type (wt) littermates. We found a twofold increase in microvessel outgrowth in explants from ko mice, relative to wt explants. This increased angiogenesis was reduced to the wt level by the addition of low levels (0.1 μg/ml) of recombinant mouse or human endostatin during the culture period. To address cellular/molecular mechanisms underlying this difference in angiogenic response between ko and wt mice, we isolated endothelial cells from both strains and compared their biological behavior. Proliferation assays showed no difference between the two types of endothelial cells. In contrast, adhesion assays showed a striking difference in their ability to adhere to fibronectin suggesting that collagen XVIII/endostatin may regulate interactions between endothelial cells and underlying basement membrane-associated components, including fibronectin, such that in the absence of collagen XVIII/endostatin, endothelial cells are more adhesive to fibronectin. In the aortic explant assay, characterized by dynamic processes of microvessel elongation and regression, this may result in stabilization of newly formed vessels, reduced regression, and a net increase in microvessel outgrowth in explants from ko mice compared to the wt littermates. Endostatin, a proteolytic fragment of basement membrane-associated collagen XVIII, has been shown to be a potent angiogenesis inhibitor both in vivo and in vitro when given at high concentrations. The precise molecular mechanisms by which it functions and whether or not it plays a role in physiological regulation of angiogenesis are not clear. In mice with targeted null alleles of Col18a1, there appears to be no major abnormality in vascular patterns or capillary density in most organs. Furthermore, the growth of experimental tumors is not increased. However, a detailed analysis of induced angiogenesis in these mice has not been performed. Therefore, we compared the angiogenic responses induced by in vitro culture of aortic explants from collagen XVIII/endostatin-null mice (ko) to wild-type (wt) littermates. We found a twofold increase in microvessel outgrowth in explants from ko mice, relative to wt explants. This increased angiogenesis was reduced to the wt level by the addition of low levels (0.1 μg/ml) of recombinant mouse or human endostatin during the culture period. To address cellular/molecular mechanisms underlying this difference in angiogenic response between ko and wt mice, we isolated endothelial cells from both strains and compared their biological behavior. Proliferation assays showed no difference between the two types of endothelial cells. In contrast, adhesion assays showed a striking difference in their ability to adhere to fibronectin suggesting that collagen XVIII/endostatin may regulate interactions between endothelial cells and underlying basement membrane-associated components, including fibronectin, such that in the absence of collagen XVIII/endostatin, endothelial cells are more adhesive to fibronectin. In the aortic explant assay, characterized by dynamic processes of microvessel elongation and regression, this may result in stabilization of newly formed vessels, reduced regression, and a net increase in microvessel outgrowth in explants from ko mice compared to the wt littermates. Collagen XVIII is a heparan sulfate-containing collagen and proteoglycan that is located in the basement membranes (BMs) of epithelia and vascular endothelium. Endostatin (ES), a proteolytic fragment of the C-terminal nontriple-helical (NC1) domain of collagen XVIII, has been identified as a potent angiogenesis inhibitor.1O'Reilly MS Boehm T Shing Y Fukai N Vasios G Lane WS Flynn E Birkhead JR Olsen BR Folkman J Endostatin: an endogenous inhibitor of angiogenesis and tumor growth.Cell. 1997; 88: 277-285Abstract Full Text Full Text PDF PubMed Scopus (4212) Google Scholar Recombinant ES has been reported to inhibit endothelial cell proliferation1O'Reilly MS Boehm T Shing Y Fukai N Vasios G Lane WS Flynn E Birkhead JR Olsen BR Folkman J Endostatin: an endogenous inhibitor of angiogenesis and tumor growth.Cell. 1997; 88: 277-285Abstract Full Text Full Text PDF PubMed Scopus (4212) Google Scholar and migration2Yamaguchi N Anand-Apte B Lee M Sasaki T Fukai N Shapiro R Que I Lowik C Timpl R Olsen BR Endostatin inhibits VEGF-induced endothelial cell migration and tumor growth independently of zinc binding.EMBO J. 1999; 18: 4414-4423Crossref PubMed Scopus (424) Google Scholar and to induce endothelial cell apoptosis,3Dhanabal M Ramchandran R Waterman MJ Lu H Knebelmann B Segal M Sukhatme VP Endostatin induces endothelial cell apoptosis.J Biol Chem. 1999; 274: 11721-11726Crossref PubMed Scopus (586) Google Scholar, 4Dixelius J Larsson H Sasaki T Holmqvist K Lu L Engstrom A Timpl R Welsh M Claesson-Welsh L Endostatin-induced tyrosine kinase signaling through the Shb adaptor protein regulates endothelial cell apoptosis.Blood. 2000; 95: 3403-3411Crossref PubMed Google Scholar but it is unclear whether ES functions as a physiological regulator of angiogenesis. To gain insights into the potential physiological role of collagen XVIII/ES as a local regulator of angiogenesis, we generated Col18a1-null mice by gene targeting. Surprisingly, the mice were fertile and had a normal life span. Histological studies of embryos and adult mice have shown ocular abnormalities, including 1) delayed hyaloid vessel regression and abnormal outgrowth of the retinal vasculature;5Fukai N Eklund L Marneros AG Oh SP Keene DR Tamarkin L Niemela M Ilves M Li E Pihlajaniemi T Olsen BR Lack of collagen XVIII/endostatin results in eye abnormalities.EMBO J. 2002; 21: 1535-1544Crossref PubMed Scopus (290) Google Scholar 2) developmental defects in the iris characterized by rupture of the posterior iris pigment epithelium and flattening of the ciliary epithelium;6Marneros AG Olsen BR Age-dependent iris abnormalities in collagen XVIII/endostatin deficient mice with similarities to human pigment dispersion syndrome.Invest Ophthalmol Vis Sci. 2003; 44: 2367-2372Crossref PubMed Scopus (66) Google Scholar, 7Ylikarppa R Eklund L Sormunen R Kontiola AI Utriainen A Maatta M Fukai N Olsen BR Pihlajaniemi T Lack of type XVIII collagen results in anterior ocular defects.EMBO J. 2003; 17: 2257-2259Google Scholar 3) age-dependent thickening of the anterior iris BM and abnormal migration of pigmented macrophage-like cells from the iris along the retina;6Marneros AG Olsen BR Age-dependent iris abnormalities in collagen XVIII/endostatin deficient mice with similarities to human pigment dispersion syndrome.Invest Ophthalmol Vis Sci. 2003; 44: 2367-2372Crossref PubMed Scopus (66) Google Scholar and 4) age-dependent formation of abnormal deposits between the basal infoldings of the retinal pigment epithelium, resulting in deterioration of retinal pigment epithelium function and attenuation of visual function with pathological electroretinograms.8Marneros AG Keene DR Hansen U Fukai N Moulton K Goeltz PL Moiseyev G Pawlyk BS Halfter W Dong S Shibata M Li T Crouch RK Bruckner P Olsen BR Collagen XVIII/endostatin is essential for vision and retinal pigment epithelial function.EMBO J. 2004; 23: 89-99Crossref PubMed Scopus (106) Google Scholar No microscopic defects were observed in the extraocular vascular system, indicating that collagen XVIII/ES is not required for normal vasculogenesis and angiogenesis in most tissues/organs. However, this does not rule out the possibility that collagen XVIII/ES plays some role as a local, BM-associated regulator of angiogenesis in some physiological/pathological contexts. To address this possibility we used an aortic explant assay to compare in vitro angiogenesis with wild-type (wt) and Col18a1-null tissues. The aortic explants from Col18a1-null mice showed a significantly higher number of long microvessel sprouts than explants from wt littermates. This increased outgrowth of microvessels could be reduced to wt levels by addition of recombinant ES to the explant cultures. To further study the possible mechanism(s) involved in the increased aortic explant angiogenesis in ko mice, we compared the biological behavior of endothelial cells derived from lung tissues of wt and ko mice. We conclude that collagen XVIII/ES can negatively modulate angiogenic processes by mediating interactions between endothelial cells and underlying extracellular matrix components, such as fibronectin (FN).Materials and MethodsAortic Explant AssaySegments 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 10× 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 described2Yamaguchi N Anand-Apte B Lee M Sasaki T Fukai N Shapiro R Que I Lowik C Timpl R Olsen BR Endostatin inhibits VEGF-induced endothelial cell migration and tumor growth independently of zinc binding.EMBO J. 1999; 18: 4414-4423Crossref PubMed Scopus (424) Google Scholar 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.Immunohistochemical and Immunofluorescent AnalysisImmunostaining 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-α-smooth muscle actin (α-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 MicroscopyAortic 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 CellsThe 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 CellsECs 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 α-SMA (1:400). Secondary antibodies, conjugated with FITC, were used for detection by conventional fluorescence microscopy.Proliferation AssayTrypsinized 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 AssayThe cell adhesion assay was performed as previously described9Martens CL Cwirla SE Lee RY Whitehorn E Chen EY Bakker A Martin EL Wagstrom C Gopalan P Smith CW Tate E Koller KJ Schatz PJ Dower WJ Barrett RW Peptides which bind to E-selectin and block neutrophil adhesion.J Biol Chem. 1995; 270: 21129-21136Crossref PubMed Scopus (144) Google Scholar 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 × 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 ES10Felbor U Dreier L Bryant RA Ploegh HL Olsen BR Mothes W Secreted cathepsin L generates endostatin from collagen XVIII.EMBO J. 2000; 19: 1187-1194Crossref PubMed Scopus (400) Google Scholar 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 BlottingConfluent 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% β-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 FormationThe assay for endothelial cell tube formation was performed as previously described11Malinda KM Nomizu M Chung M Delgado M Kuratomi Y Yamada Y Kleinman HK Ponce ML Identification of laminin alpha1 and beta1 chain peptides active for endothelial cell adhesion, tube formation, and aortic sprouting.EMBO J. 1999; 13: 53-62Google Scholar 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.ResultsMicrovessel Formation in Mouse Aortic ExplantsAortic 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-α-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.Figure 2Whole mount immunostaining of microvessel sprouts from mouse aortic explants embedded in collagen gels and cultured for 15 to 21 days. A to C: Staining for endothelial cell-specific marker, CD31. A: CD31 staining is visualized by FITC-conjugated secondary antibody. B and C: Staining pattern (diaminobenzidine substrate) shows specific immunoreactivity in the core of sprouts, as indicated by arrows. D to F: Staining for smooth muscle cell marker, α-SMA. D: Anti-α-SMA antibody detected by FITC-conjugated secondary antibody. Note that the diameter of the sprout is much bigger than when the sprouts are stained for CD31 (A). A lumen can be seen in the middle of sprouts, as indicated by arrows. E and F: Outer cell layer of sprouts is α-SMA-positive (VIP substrate for E and diaminobenzidine substrate for F), arrows. C and F: Images of vessel regression (arrowheads) during outgrowth demonstrate cell debris and silhouettes of previously existing vessels. G: Sprouts show positive staining (VIP substrate) for collagen IV. H: Fluorescent staining for FN. Secondary antibody conjugated with FITC. Nuclei labeled with DAPI. I: Sprouts, examined by EM, reveal lumen (arrow) and cell-cell junction (arrowhead). Original magnifications: ×8 (A, D); ×20 (B, C, E, F); ×5 (G); ×40 (H); ×10,000 (I).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Quantitative Analysis of Angiogenesis in Aortic Explant AssayPrevious studies have shown that collagen XVIII/ES is present at relatively high levels in the aortic wall.12Miosge N Sasaki T Timpl R Angiogenesis inhibitor endostatin is a distinct component of elastic fibers in vessel walls.EMBO J. 1999; 13: 1743-1750Google Scholar 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).Figure 3Microvessel outgrowth in aortic explants from Col18 a1-null mice and wt littermates. A and B: Cultures (incubated for 17 days) stained with anti-CD31 antibody (VIP substrate) (A, ko; B, wt). C: Quantitation of microvessel outgrowth in ko and wt explants by counting the number of ends of vessel sprouts that had grown out from the two long edges of the rectangular explants. The cultures were incubated in the absence or presence of various concentrations of recombinant ES. The heights of the columns in the histogram show the arithmetic mean number of vessel ends in the various groups; the standard deviations are indicated by the vertical error bars. Except for the groups of wt explants treated with 0.1 μg/ml or 0.5 μg/ml of ES in which three explants were used for counting, all other groups show the results of counting vessel ends in four to seven explants. The difference in number of vessel ends between ko and wt explants in the absence of ES was highly significant (*, P < 0.005). D: Quantitation of long microvessel sprouts in aortic explants shows that the arithmetic mean number of long vessels growing from the two long edges of explants (expressed as vessel density, see Materials and Methods) was significantly higher (*, P < 0.005) in explants from ko mice than from wt mice. The results shown are derived from several different explants, experiments, and mice as described in Materials and Methods. The increased outgrowth in ko explants was inhibited by the addition of exogenous ES at a concentration as low as 0.1 μg/ml (*, P < 0.005 when comparing ko sample without ES and ko samples with ES). Standard deviations are indicated by the vertical error bars. Original magnifications, ×4 (A, B).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Proliferative Activities of mLECsTo 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 α-SMA-positive cells (Figure 4D). First we tested whether there was a difference in proliferative activity between mutant and wt mLECs;