MethodsMolMed_135_305

Assessment of Endothelial Cell Activation 305 305 From: Methods in Molecular Medicine, Vol. 135: Arthritis Research, Volume 1 Edited by: A. P. Cope © Humana Press Inc., Totowa, NJ 19 Isolation and Analysis of Large and Small Vessel Endothelial Cells Justin C. Mason, Elaine A. Lidington, and Helen Yarwood Summary The in vitro isolation, propagation and study of endothelial cells (EC) is an invalu- able means by which the function of the vascular endothelium in physiology and patho- physiology can be explored. In recent years heterogeneity between large and small vessel EC, between arteries and veins, and between microvascular EC derived from different organs has become increasingly apparent. This has led to the development of protocols for the isolation of these different EC. In addition, the data emerging on vascular EC function in gene-targeted mice has highlighted the need for reliable methods of isolation of murine EC. This chapter describes methods for the isolation, characterization and culture of macro- and microvascular EC from a variety of species and introduces simple approaches to investigating their surface antigen expression. Key Words: Human umbilical vein endothelial cells; dermal microvascular endothe- lial cells; murine endothelial cells; porcine endothelial cells; flow-cytometry; enzyme- linked immunosorbent assay. 1. Introduction The in vitro isolation, propagation and study of endothelial cells (EC) remains an invaluable method by which the role of the vascular endothelium in a variety of physiological and pathophysiological processes can be studied. This approach has resulted in a dramatic increase in our understanding of endo- thelial function. The culture of EC in vitro has been in routine laboratory use for over 30 yr (1), and during this time methodology for the isolation of EC from specific vascular beds has constantly evolved. In particular, methods for the isolation of EC from microvascular beds have been developed (2–9). In addition, the development of genetically modified mice provides the opportunity to study the role of individual endothelial genes not only in vivo, but also in vitro through isolation of endothelial cells from different murine 306 Mason, Lidington, and Yarwood organs (10–14). This approach is likely to be of specific importance in view of previous studies, in both human and murine models, in which the presence of functional heterogeneity between endothelial cells derived from different vascular beds has been demonstrated (14–18). The aim of this chapter is to describe methods for the isolation, culture and study of endothelial cells from large and small vessels in a variety of species. 2. Materials 2.1. Isolation of Endothelial Cells 1. Penicillin/streptomycin (Sigma; cat no. P0781). Store in aliquots at –20°C. 2. Hanks balanced salt solution (HBSS) (Sigma; cat no. H9269). 3. Hanks balanced salt solution without calcium and magnesium (HBSS w/o Ca/Mg) (Sigma; cat no. H9394). 4. Sodium pyruvate (Invitrogen; cat no. 11360-039). Store at 4°C. 5. Gentamicin (Invitrogen; cat no. 15710-049). Store at room temperature. 6. Amphotericin B (Fungizone) (Invitrogen; cat no. 15290-018). Aliquot and store at –20°C. 7. Dispase (Boehringer Mannheim). Store at –20°C and make up fresh on each occasion. 8. Medium 199 (MP Biomedicals; cat. No. 1220254). Store at 4°C. 9. Heat-inactivated human AB positive serum (Sigma; cat. no. S7418). To heat in- activate, incubate in a water bath at 56°C for 45 min. Store in aliquots at –20°C. 10. L-glutamine (Sigma; cat no. G7513). Store in aliquots at –20°C. 11. Preservative free heparin 1000 iu/mL (CP Pharmaceuticals, Wrexham UK). Store at 4°C. 12. Endothelial cell growth factor (ECGF) (Sigma; cat no. E2759). Reconstitute each vial with 5 mL of M199 and pass through a 0.2 µM filter and collect into a sterile universal container. Divide into 1-mL aliquots and freeze at –20°C. Reconsti- tuted ECGF is stable at 4°C for 2 wk. 13. Bovine fibronectin (Sigma; cat no. F-1141). Reconstitute in sterile water, aliquot and store at –20°C. 14. Trypsin/ethylene diamine tetraacetic acid (EDTA) (ICN; cat no. 16-891-49). Aliquot and store at –20°C. 15. MiniMacs beads (cat no. 130-048-502 or 130-047-202), preseparation filters (cat no. 130-041-407), and columns (cat no. 130-042-401 or 130-042-201) (Miltenyi Biotec). 16. 2% Gelatin (Sigma; cat no. G1393). Prewarm at 37°C for 30 min and dilute to 1% in sterile water or phosphate buffered saline (PBS). Aliquot and store at 4°C. 17. Collagenase A (Roche Diagnostics). Store at –20°C and make up fresh on each occasion in HBSS. 18. Three-way tap (Becton Dickinson; cat no. 394601). 19. RPMI 1640 (Sigma; cat no. R0883). 20. 70 µm filters (Becton Dickinson; cat no. 352350). Assessment of Endothelial Cell Activation 307 21. Rat anti-mouse endoglin antibody (MJ7/18) (BD Pharmingen or hybridoma from developmental studies hybridoma bank, University of Iowa). 22. DMEM (Invitrogen; cat no. 41965-039). 23. Fetal calf serum (FCS) batch tested for EC growth properties and heat inacti- vated to 56°C for 30 min. 30% Bovine serum albumin solution (BSA) (Sigma; cat no. A-3424). 2.2. Storage of Endothelial Cells in Liquid Nitrogen 1. Cryogenic vials (Corning Inc.; cat no. 430489). 2. Dimethyl sulfoxide (DMSO). Use tissue culture grade and store at room tem- perature. (Sigma; cat no. D2650). 2.3. Characterization of Cultured Endothelial Cells 1. Matrigel (Becton Dickinson; cat no. 35 40234). Store at –20°C. 2. Acetylated low-density lipoprotein (Intracel; cat no. RP-078). Store at 4°C. 3. FITC-anti-von Willebrand Factor polyclonal Ab (Serotec; cat no. AHPO62F). Store at 4°C. 4. Biotinylated Ulex europaeus agglutinin-I (UEA-I) (Vector Laboratories; cat no. B-1065). 5. Biotinylated Griffonia simplicifolia isolectin B4 (Vector Laboratories; cat no. B-1205). 2.4. Analysis of Endothelial Cell Activation 1. Paraformaldehyde (BDH Laboratory supplies; cat no. 294474L). 2. L-lysine (Sigma; cat no. L-5626) 3. Sodium m-periodate (Sigma; cat no. S-1147). 4. Biotinylated rabbit anti-mouse Ig F(ab')2 (DAKO; cat no. E0413). 5. FITC- rabbit anti-mouse Ig F(ab')2 (DAKO; cat no. E0313). 6. RPE- rabbit anti-mouse Ig F(ab')2 (DAKO; cat no. R0439) 7. Alexa Fluor 488-labeled anti-mouse Ig (Molecular Probes; cat no. A-11011 or A-11017). 8. Streptavidin-biotin-peroxidase (DAKO; cat no. P0347) 9. O-phenylenediamine (Sigma; cat no. P-5412). 10. Hydrogen peroxidase (Sigma; cat no. H-1009). 11. Crystal violet (Sigma; cat no. C-0775). 12. FACS tubes (Becton Dickinson; cat no. 62052). 13. V-bottomed 96-well plate (Nunc; cat no. 245128). 3. Methods 3.1. Isolation of Human Dermal Microvascular Endothelial Cells 1. Human skin should be collected into HBSS, supplemented with 100 IU/mL penicillin, 100 µg/mL streptomycin and 20 µg/mL amphotericin B (see Note 1). 308 Mason, Lidington, and Yarwood 2. In a tissue culture hood, the skin is cut into strips (2 × 0.5 cm), washed twice in HBSS and incubated overnight in 2 µg/mL Dispase at 4°C. Following enzymatic digestion the epidermis should be easily removed with the use of sterile fine for- ceps. The remaining tissue is cut into 0.5 cm2 pieces, washed twice in HBSS and transferred into Medium 199 (M199). 3. Skin segments are transferred individually into a single well of a 6-well tissue culture plate containing 1 mL M199. Microvascular segments are expressed from the edge of the skin, into the M199, by firm downward pressure with the curved edge of a sterile stitch cutter (19) (see Note 2). Once all the tissue segments have been used, carefully discard them (following local regulations for disposal of human tissue). Collect the M199 containing expressed microvascular fragments and pass through a sterile 100 µm filter to remove contaminating cell debris. 4. Following centrifugation of the cell suspension at 200g for 5 min, aspirate the M199, and gently resuspend the cell pellet in DMEC growth medium (M199 supplemented with 20% heat-inactivated human AB positive serum, 100 IU/mL penicillin, 100 µg/mL streptomycin and 5 µg/mL amphotericin B, 2mM L-glutamine, 10 U/mL preservative free heparin, and 50 µg/mL endothelial cell growth factor). 5. The initial isolate is plated out onto a single well of a 6-well tissue culture plate, precoated for 30 min at 37°C with human fibronectin (10 µg/mL) and cultured for 72 h. The cell monolayer is then washed twice in calcium and magnesium free HBSS and the cells detached by incubation for 1 to 2 min with 0.125% trypsin/ EDTA at 37°C. The harvested EC are transferred to a 25cm2 fibronectin-coated tissue culture flask containing prewarmed (37°C) and pregassed (5% CO2) DMEC medium and grown to confluence. 6. The initial isolate consists of approx 70% DMEC and therefore requires further purification by positive selection. EC are harvested with trypsin/EDTA, as described in Subheading 3.1.5., and resuspended in HBSS/5% FCS containing a mouse anti-human endoglin (CD105) MAb (final concentration 10 µg/mL) (see Note 3). Following incubation for 20 min at 4°C, the DMEC are centrifuged at 200g for 5 min, washed twice in HBSS/1% FCS and resuspended with anti-mouse IgG-coated MiniMacs beads and incubated for 20 mins at 4°C (see Note 4). 7. The DMEC suspension is centrifuged at 200g for 5 min, washed thrice in HBSS/ 1% FCS, and passed over a MS+ miniMac column suspended in a miniMac mag- net. Following washing in 3-column volumes of HBSS/1% FCS, the purified DMEC are eluted from the column following removal of the column from the magnetic field. 8. DMEC are collected by centrifugation at 200g for 5 min and resuspended in DMEC growth medium and transferred into a 25 cm2 fibronectin-coated tissue culture flask and grown to confluence prior to detachment with trypsin/EDTA and transfer to a 75 cm2 fibronectin-coated tissue culture flask. The DMEC can be sub-cultured up to passage 6 in 75 cm2 fibronectin-coated tissue culture flask (see Note 5). DMEC require feeding every 2 to 3 d by exchanging half the growth medium. Assessment of Endothelial Cell Activation 309 3.2. Isolation of Human Umbilical Vein Endothelial Cells 1. Collect cords into HBSS supplemented with 1 mM sodium pyruvate, 100 IU/mL penicillin, 100 µg/mL streptomycin, and 100 µg/mL gentamicin. Place umbilical cord onto a silver foil covered tray, sprayed with 70% ethanol and air dried in a tissue culture hood (1). 2. Wipe cord with tissues soaked in 70% ethanol to remove blood. 3. Prepare collagenase A solution (0.5 mg/mL) in HBSS (without calcium and magnesium) allowing 10 to 15 mL per umbilical cord. Filter collagenase through 0.2 µm filter and prewarm to 37°C. Add 2 mL of 1% gelatin to one 25 cm2 tissue culture flask for each umbilical cord and incubate for 30 min at 37°C. 4. Cut approx 1 cm off each end of the cord and identify the two umbilical arteries and one umbilical vein. Dissect 0.5 to 1 cm of the vein at each end of the cord and dilate with a pair of blunt forceps. Cannulate one end with a three-way tap, secure firmly with fine thread. 5. Attach a sterile 20-mL syringe to the three way tap and repeatedly flush the umbilical vein with HBSS (without calcium and magnesium) until all blood and blood clots are removed. Ensure that no holes exist in the vein and if found use a fine artery clamp or a crocodile clip to occlude them. Finally, push 15 mL of air through the umbilical vein to remove residual HBSS. 6. Cannulate the other end of the umbilical vein with a three-way tap, secure, and close the three-way tap. Attach an empty 20-mL syringe to this end and a second 20-mL syringe containing the prewarmed collagenase to the opposite end. 7. Place umbilical cord on saran wrap on a small tray. Distend the cord with collagenase. Wrap the cord in Saran™ wrap and place in an incubator at 37°C for 8 to 10 min (see Note 6). 8. Remove cord from incubator and relieve pressure by drawing 5 mL of collage- nase into the previously empty syringe. Massage the cord firmly along the whole length between thumb and forefinger for 1 min. Push any remaining collagenase from the original syringe through the cord into the collecting syringe and then push 15 mL of air through the umbilical vein to remove residual collagenase. 9. Remove syringe containing collagenase and empty contents into a 50-mL centri- fuge tube. Replace the syringe with one containing 20 mL HBSS. Pass 10 mL into the cord and repeat the massage step and collect into an empty syringe attached to the other end of the cord. Repeat this step 4 times adding each 10 mL of HBSS to the 50-mL centrifuge tube (see Note 7). Pellet human umbilical vein endothelial cells (HUVEC) by centrifugation at 200g for 5 min. 10. Carefully dispose of umbilical cord in a sealed container following local regula- tions for disposal of human tissue. 11. Aspirate gelatin from 25 cm2 tissue culture flask and add 4 mL of prewarmed and pregassed (37°C in 5% CO2 incubator) HUVEC growth medium (M199 supplemented with 20% heat-inactivated FCS [see Note 8], 100 IU/mL penicillin, 100 µg/mL streptomycin, 2 mM L-glutamine, 10 U/mL preservative free heparin, and 30 µg/mL endothelial cell growth factor). 310 Mason, Lidington, and Yarwood 12. Resuspend HUVEC pellet gently in 1 mL of growth medium and transfer to 25 cm2 tissue culture flask and leave EC to attach overnight. 13. The following day prewarm and pregas 5 mL of HUVEC growth medium for 30 min. Aspirate medium from tissue culture flask and wash HUVEC monolayer in HBSS 3 times to remove all residual blood and cell debris. Add prewarmed HUVEC growth medium. 14. HUVEC will reach confluence in 1 to 5 d and require feeding every 2 to 3 d by exchanging half the growth medium. At confluence HUVEC should be harvested and transferred to a gelatin-coated 75 cm2 containing 12 mL of HUVEC growth medium. For harvesting, wash the monolayer twice in calcium and magnesium free HBSS and detach cells by incubation for 1 to 2 min in 0.125% trypsin/EDTA at 37°C. HUVEC can be subcultured up to passage 6 in 75 cm2 gelatin-coated tissue culture flask (see Notes 9 and 10). 3.3. Isolation of Porcine Arterial Endothelial Cells 1. Collect porcine aortas into HBSS supplemented with 1 mM sodium pyruvate, 100 IU/mL penicillin, 100 µg/mL streptomycin, 100 µg/mL gentamicin, 20 µg/mL, and amphotericin B (see Note 11). 2. Place aorta onto a silver foil covered tray, sprayed with 70% ethanol and air dry in a tissue culture hood. Cut off excess fat and connective tissue and carefully wash vessel lumen with HBSS supplemented with 1 mM sodium pyruvate, 100 IU/mL penicillin, 100 µg/mL streptomycin, 100 µg/mL gentamicin, and 20 µg/mL amphotericin B. 3. Prepare collagenase A solution (0.25 mg/mL) in HBSS (without calcium and magnesium) allowing 20 to 25 mL per aorta. Filter collagenase through 0.2 µM filter and prewarm to 37°C. Add 2 mL of 1% gelatin to one 25 cm2 tissue culture flask for each aorta and incubate for 30 min at 37°C. 4. Any vessels arising from the aorta should be carefully clipped using fine artery clips and one end of the aorta closed with an arterial clamp. The aorta should then be filled with HBSS to check for leaks (see Note 12). 5. Remove HBSS and fill aorta with collagenase, clamp the open end, wrap aorta in Saran wrap, and incubate for 15 min at 37°C. 6. The porcine arterial endothelial cells (PAEC) are harvested by careful collection of the collagenase into a 50-mL centrifuge tube and repeated gentle massage of the aorta and washing of the lumen with 10 mL HBSS supplemented with antiobiotics as above. Pellet PAEC by centrifugation at 200g for 5 min. 7. Carefully dispose of each aorta in a sealed container following local regulations for disposal of animal tissue. 8. Aspirate gelatin from 25 cm2 tissue culture flask and add 4 mL of PAEC growth medium (RPMI 1640, supplemented with 20% heat-inactivated FCS, 100 IU/mL penicillin, 100 µg/mL streptomycin, 2 mM L-glutamine, 10 U/mL preservative free heparin, and 30 µg/mL endothelial cell growth factor). 9. Resuspend PAEC pellet gently in 1 mL of growth medium and transfer to the 25 cm2 tissue culture flask and leave EC to attach overnight. Assessment of Endothelial Cell Activation 311 10. The following day prewarm 5 mL of PAEC growth medium for 30 min. Aspirate medium from tissue culture flask and wash PAEC monolayer in HBSS 3 times to remove all residual blood and cell debris. Add prewarmed PAEC growth medium. 11. PAEC will reach confluence in 1 to 5 d and require feeding every 2 to 3 d by exchanging half the growth medium. At confluence PAEC should be harvested and transferred to a gelatin-coated 75 cm2 containing 12 mL of growth medium. For harvesting, wash the monolayer twice in calcium and magnesium free HBSS and detach cells by incubation for 1 to 2 min in 0.125% trypsin/EDTA at 37°C. PAEC can be subcultured up to passage 6 in 75 cm2 gelatin-coated tissue culture flask (see Notes 9 and 10). 3.4. Isolation of Murine Cardiac Endothelial Cells 1. Prepare collagenase A solution (1 mg/mL) in HBSS, allowing 5 mL per isolation. Filter collagenase through 0.2 µM filter and prewarm to 37°C. In addition, prewarm 5 mL 0.125% trypsin/EDTA to 37°C and one 25 cm2 tissue culture flask containing 2 mL of 1% gelatin per preparation (see Note 13 on preparation of control EC). 2. Use 6 mice per isolation, killed via an approved schedule 1 method. Immerse each mouse briefly in 70% ethanol and pin-out onto a silver foil covered polysty- rene board, in a tissue culture hood. 3. Using sterile scissors and fine forceps carefully remove skin from the chest, making incisions across the abdomen and up the right flank without piercing the peritoneum. Reflect the skin to the left and rinse loose hairs off with HBSS. Flame instruments and once cool, hold the sternum and make a horizontal incision just beneath the sternum and above the diaphragm followed by a vertical incision along the right border of the sternum and reflect the ribcage to expose the heart. Flame instruments again, remove the heart and transfer into 3 mL HBSS supple- mented with 100 IU/mL penicillin, 100 µg/mL streptomycin, and 100 µg/mL gentamicin in a 60-mm Petri-dish. Repeat for all six mice. EC can also be obtained from lung and brain tissue (see Note 14). 4. Using a sterile scalpel cut hearts in half, remove any connective tissue, blood clots, and the base of the aorta. Collect tissue into a fresh 60-mm Petri-dish con- taining HBSS (with antibiotics), dice-up tissue into pieces of about 1 to 2 mm3. Remove HBSS using a P1000 pipette and wash tissue in 3 ml HBSS removing the HBSS again using a P1000 pipette. 5. Place tissue into 5 mL of prewarmed collagenase A (0.5 mg/mL) in a Universal tube and incubate for 30 min at 37°C, shaking tube at regular intervals. After a final vigorous shake, allow the undigested tissue to fall to the bottom of the tube. Collect the cell suspension into a 50-mL Falcon tube. Wash the undigested tissue in 10 mL calcium and magnesium free HBSS, shake vigorously and once settled add suspension to the previously collected suspension. Repeat wash twice more and centrifuge total cell suspension at 700g for 8 min at 4°C. 6. Aspirate supernatant and wash pellet in 30 mL calcium and magnesium free HBSS, centrifuging at 700g for 8 min at 4°C. Aspirate the supernatant and incu- 312 Mason, Lidington, and Yarwood bate the pellet with 5 mL Trypsin/EDTA at 37°C for 10 min. Pipette up and down several times before passing suspension through a 70 µm filter placed over a 50-mL Falcon tube, wash through with 25 mL cold HBSS (Ca/Mg) 1% BSA. Centrifuge cell suspension at 700g for 8 min at 4°C. 7. Aspirate the supernatant and incubate the pellet with 5 µg irrelevant MAb (see Note 15) in 500 µL at 4°C for 20 min, to block Fc receptors on macrophages (optional wash in HBSS/1%BSA). Add 5 µg rat anti-murine endoglin MAb MJ7/ 18 (10 µg/mL) and incubate at 4°C for 20 min. Add 30 mL HBSS/1% FCS and centrifuge