Live-cell imaging demonstrates extracellular matrix degradation in association with active cathepsin B in caveolae of endothelial cells during tube formation.

Materials M199 medium, heparin, N-Octyl β-D-glucopyranoside, 2-[N-morpholino]ethanesulfonic acid (MES), methyl-β-cyclodextrin (MβCD), and all other chemicals unless otherwise stated were from Sigma (St. Louis, MO); fetal bovine serum (FBS), Lipofectin reagent, dye-quenched fluorescent (DQ)-gelatin and DQ-collagen IV were from Invitrogen (Carlsbad, CA); bovine endothelial cell growth factor (bECGF) was from Roche Applied Science (Indianapolis, IN); polyclonal anti-caveolin (610059), monoclonal anti-annexin II (mAb 5) and monoclonal anti-p11 (mAb 148) antibodies were purchased from BD Biosciences (Bedford, MA); polyclonal anti-cathepsin B [ 24 ] antibodies were produced, affinity-purified and characterized in our laboratory; polyclonal anti-β1 integrin antibodies were a kind gift from Dr. Kenneth Yamada (National Institutes of Health/National Institute of Dental and Craniofacial Research, Bethesda, MD); polyclonal anti-uPA (ab20046) and polyclonal anti-uPAR (ab27423) antibodies were from Abcam (Cambridge, MA); polyclonal anti-MMP-14 (AB815) antibodies were from Chemicon (Temecula, CA); horseradish peroxidase-labeled goat anti-rabbit and goat anti-mouse IgG were from Pierce (Rockford, IL); purified MMP-2 and MMP-9 enzymes were a kind gift from Dr. Rafael Fridman (Wayne State University, Detroit, MI); cysteine cathepsin activity-based probes, GB-111-FL and GB-123, were recently described in references [ 25 ] and [ 26 ], respectively; Cultrex was from Trevigen (Gaithersberg, MD); acrylamide and nitrocellulose membranes were from BioRad (Hercules, CA); protein markers and chemiluminescent western blotting detection kits were from Amersham Pharmacia Biotech (Piscataway, NJ); benzyloxycarbonyl-L-arginyl-L-arginine-4-methyl-7-coumarylamide (Z-Arg-Arg-NHMec) was from Bachem (Torrance, CA); and Amicon Ultra-4 10 K centrifugal filters were from Millipore (Bedford, CA).

Cell Culture

HUVEC from the American Type Culture Collection (ATCC) (Rockville, MD) were grown on 1.5% (w/v) gelatin-coated tissue culture lab-ware in endothelial cell M199 media supplemented with 40 μg/ml bECGF, 100 μg/ml heparin, antibiotics (penicillin/streptomycin) and 10% (v/v) FBS (as recommended by the ATCC) in 5% CO 2 /humidified atmosphere at 37°C unless otherwise stated. Transient transfection of HUVEC with cav-1-mRFP expression vector (a kind gift from Drs. Radu V. Stan and Steve F. Dowdy, University of California at San Diego, San Diego, CA) was performed using Lipofectin reagent according to the manufacturer’s instructions. In vitro Tube Formation Assay In vitro tube formation assays were performed in 35 mm tissue culture dishes coated with 200 μl of Cultrex [i.e., reconstituted basement membrane (rBM)] that was allowed to gel at 37 °C for 10 min. Thereafter, 2 × 10 5 cells were seeded onto the Cultrex in M199 media containing 2% (v/v) FBS and incubated at 37 °C for 16–18 h to allow formation of tubular structures [ 27 , 28 ].

Preparation of caveolae-enriched fractions

Caveolae-enriched fractions were isolated by both non-detergent and detergent based methods. The non-detergent method was performed as previously described [ 18 , 29 ]. Briefly, cells from four 100 mm dishes were washed twice with PBS at 4°C and lysed in 2 ml 500 mM sodium carbonate buffer, pH 11.0. Cell lysates were homogenized on ice by 10 strokes in a loose-fitting Dounce homogenizer and then sonicated on ice in a 50 W ultrasonicator three times for 20 sec each. Cell homogenates were adjusted to 45% (w/v) sucrose by mixing them with 2 ml of 90% (w/v) sucrose prepared in MES-buffered saline (25 mM MES, pH 6.5, 0.15 m NaCl) and then placed on the bottom of a 12 ml ultracentrifuge tube. The homogenate was then overlaid with a 5–-35% sucrose gradient comprised of 4 ml of 35% (w/v) sucrose (in MES-buffered saline containing 250 mM sodium carbonate) and 4 ml of 5% (w/v) sucrose (in MES-buffered saline containing 250 mM sodium carbonate). Gradients were subjected to ultracentrifugation at 185,000 g in a SW41 rotor (Beckman Instruments) for 18–20 h at 4 °C. One ml fractions were collected from the top of the gradient and equal volume aliquots were analyzed by SDS-PAGE and immunoblotting. Where indicated, cells were treated overnight with 1 μM GB111-FL, a quenched activity-based probe for cysteine cathepsins [ 25 ], prior to caveolae isolation. SDS-PAGE gels were imaged on a Typhoon Imaging Scanner (Amersham Biosciences, Pittsburgh, PA) to detect bound probe. Caveolae-enriched fractions could not be isolated from HUVEC grown on rBM for in vitro tube-formation assays using the non-detergent based method described above because the amount of rBM (131.2 mg in 8 ml) required for sufficient cells, i.e., four 100 mm dishes, prevented proper preparation of sucrose gradients. Therefore, we employed a successive detergent based method that separates Triton X-100-soluble from -insoluble membrane components [ 30 ]. Cells were incubated in lysis buffer containing 1% Triton X-100 in the absence of octylglucoside for 30 min on ice, collected and centrifuged at 14,000 × g for 15 min at 4 °C. The supernatant, hereafter termed the Triton-soluble fraction, was recovered. The insoluble pellet was resuspended in an equal volume of lysis buffer containing both 1% Triton X-100 and 60 mM octylglucoside, incubated on ice for 30 min, passed 10 times through a syringe with a 20-gauge needle and centrifuged for 10 min at 14,000 × g at 4 °C. The supernatant, hereafter termed the Triton-insoluble fraction, was recovered and the final volume adjusted to that of the Triton-soluble fraction. Caveolae were enriched in the Triton-insoluble fraction as was verified by treating cells with 10 mM MβCD, a cholesterol sequestering drug that disrupts caveolae, at 37 °C for 1 h prior to successive detergent extraction of membranes.

Preparation of Conditioned Media

Equal numbers of cells were serum-starved overnight, media were collected, centrifuged at 800 × g to remove whole cells, re-centrifuged at 2,000 × g to remove cell debris, and concentrated to equal final volumes.

SDS-PAGE and Immunoblot Analysis

Samples were equally loaded and separated by SDS-PAGE (10%, 12% or 15% acrylamide) and transferred onto nitrocellulose membranes under either reducing or non-reducing conditions. Immunoblotting was performed with primary antibodies against cathepsin B (1:4000), caveolin (1:5000), annexin II (1:5000), p11 (1:5000), uPA (5 μg/ml), uPAR (5 μg/ml), β1-integrin (1:3000) and MMP-14 (1:1000) and secondary antibodies conjugated with horseradish peroxidase (1:10,000) in Tris-buffered saline wash buffer (20 mM Tris, pH 7.5, 0.5 M NaCl) containing 0.5% Tween 20 and 5% (w/v) non-fat dry milk. After washing, bound antibodies were detected by enhanced chemiluminescence according to the manufacturer’s instructions. Gelatin Zymography Subcellular fractions and conditioned media samples were equally loaded and applied without heating or reduction to a 10% SDS-polyacrylamide gel containing 1 mg/ml of gelatin [ 31 ]. After electrophoresis, the gel was washed twice for 15 min each time with 2.5% Triton X-100, followed by a 20 min wash with distilled water, and incubated overnight at 37 °C in 50 mM Tris buffer, pH 7.8, containing 5 mM CaCl 2 and 0.05% Brij35. The gel was stained with a solution of 0.5% Coomassie brilliant blue R-250 in 50% methanol and 10% acetic acid and then de-stained in a 50% methanol and 10% acetic acid solution. Clear bands represent areas of proteolytic activity. Purified MMP-2 and MMP-9 were loaded separately as positive controls. Cathepsin B Activity Assay Cathepsin B activity was measured in subcellular fractions and conditioned media as previously described [ 32 ]. Briefly, to measure intracellular cathepsin B activity 50 μl of sample was incubated with 300 μl of activator buffer (5 mM EDTA, 10 mM DTT, pH 5.2) for 15 min at 37 °C. In a 96 well flat-bottom black microtiter plate, 100 μl of cell lysate/activator buffer mixture was added to 200 μl of assay buffer [Hank’s balanced-salt solution lacking sodium bicarbonate and phenol red and containing 0.6 mM CaCl 2 , 0.6 mM MgCl 2 and 25 mM piperazin-N-N′-bis[2-ethanesμlfonic acid] (disodium salt), pH 7.3] containing 150 μM Z-Arg-Arg-NHMec substrate. The enzymatic assay was carried out at pH 6.0. Fluorescence was measured in triplicate at one-minute intervals for 30 minutes using a Tecan SpectraFluor Plus plate-reader at an excitation of 360 nm and an emission of 465 nm. To measure latent or procathepsin B activity in media samples, autoactivation of procathepsin B was accelerated in the presence of dextran sulfate [ 33 ]. Briefly, 50 μl of conditioned media was mixed with 300 μl of citrate buffer (20 mM citrate buffer, pH 4.6, 10 mM DTT and 25 μg/ml dextran sulfate) and incubated for 45 min at 37 °C. Then 100 μl of media-citrate buffer solution was mixed in a 96 well flat-bottom black microtiter plate with 200 μl of 150 μM Z-Arg-Arg-NHMec substrate prepared in 200 mM sodium phosphate buffer, pH 6.8. Fluorescence was read in triplicate at one-minute intervals over a 30 min period at an excitation of 360 nm and an emission of 465 nm. Cathepsin B activity was recorded as pmoles/min/cell number. Cells were counted using a hemocytometer prior to setting up overnight experiments. Statistical significance was determined by a two-tailed t-test with assumed equal variance. In the figures, * represents a p value less than 0.05 and ** represents a p value less than 0.01.

Immunocytochemistry

Cells were grown on coverslips for 16–24 hours, permeabilized with saponin, fixed with cold methanol for 5 min and then blocked for 45 minutes by incubating with PBS containing 2 mg/ml BSA. Cells were then washed with PBS and incubated with primary antibody (rabbit anti-human caveolin or preimmune rabbit IgG) for 2 hours at room temperature according to our published procedures [ 34 ]. After washing with PBS, the cells were incubated for 1 hour with Texas red-conjugated affinity-purified donkey anti-rabbit IgG containing 5% normal donkey serum (Jackson ImmunoResearch). Cells were then washed, mounted upside-down with Slow Fade anti-fade reagent (Invitrogen Life Technologies) on glass slides and observed with a Zeiss 510 LSM confocal microscope.

Live Cell Proteolysis Assay

Proteolysis by live cells was assessed as previously described [ 35 , 36 ]. Briefly, glass coverslips were coated with either 50 μl of 1.5% (w/v) gelatin containing 25 μg/ml DQ-gelatin and incubated on ice for 15 min to solidify or 50 μl of 16.4 mg/ml Cultrex containing 25 μg/ml DQ-collagen IV and incubated for 10 min at 37 °C to solidify. Subsequent steps were carried out at 37 °C. Cells (2.5 × 10 4 ) were seeded onto coated coverslips and incubated for 60 min. Medium containing 2% FBS was added to the cells and incubated for 16–18 h. Proteolysis of DQ-gelatin and DQ-collagen IV (green fluorescence) was observed in live cells with a Zeiss LSM 510 META NLO microscope with 10x and 40x water immersion objectives. Time series analyses of DQ-collagen IV degradation were performed with a Zeiss LSM 510 META NLO microscope equipped with a controlled environmental chamber that maintains a 5% CO 2 /humidified atmosphere at 37 °C. Where indicated, assays were conducted in the presence of 1 μM GB-123, a non-quenched cysteine cathepsin activity-based probe, for 16–18 h. Cells were washed 3 times with PBS and incubated in complete growth medium in the absence of the probe immediately prior to imaging.

Materials M199 medium, heparin, N-Octyl β-D-glucopyranoside, 2-[N-morpholino]ethanesulfonic acid (MES), methyl-β-cyclodextrin (MβCD), and all other chemicals unless otherwise stated were from Sigma (St. Louis, MO); fetal bovine serum (FBS), Lipofectin reagent, dye-quenched fluorescent (DQ)-gelatin and DQ-collagen IV were from Invitrogen (Carlsbad, CA); bovine endothelial cell growth factor (bECGF) was from Roche Applied Science (Indianapolis, IN); polyclonal anti-caveolin (610059), monoclonal anti-annexin II (mAb 5) and monoclonal anti-p11 (mAb 148) antibodies were purchased from BD Biosciences (Bedford, MA); polyclonal anti-cathepsin B [ 24 ] antibodies were produced, affinity-purified and characterized in our laboratory; polyclonal anti-β1 integrin antibodies were a kind gift from Dr. Kenneth Yamada (National Institutes of Health/National Institute of Dental and Craniofacial Research, Bethesda, MD); polyclonal anti-uPA (ab20046) and polyclonal anti-uPAR (ab27423) antibodies were from Abcam (Cambridge, MA); polyclonal anti-MMP-14 (AB815) antibodies were from Chemicon (Temecula, CA); horseradish peroxidase-labeled goat anti-rabbit and goat anti-mouse IgG were from Pierce (Rockford, IL); purified MMP-2 and MMP-9 enzymes were a kind gift from Dr. Rafael Fridman (Wayne State University, Detroit, MI); cysteine cathepsin activity-based probes, GB-111-FL and GB-123, were recently described in references [ 25 ] and [ 26 ], respectively; Cultrex was from Trevigen (Gaithersberg, MD); acrylamide and nitrocellulose membranes were from BioRad (Hercules, CA); protein markers and chemiluminescent western blotting detection kits were from Amersham Pharmacia Biotech (Piscataway, NJ); benzyloxycarbonyl-L-arginyl-L-arginine-4-methyl-7-coumarylamide (Z-Arg-Arg-NHMec) was from Bachem (Torrance, CA); and Amicon Ultra-4 10 K centrifugal filters were from Millipore (Bedford, CA).