Isolation and culture conditions of endothelial progenitor cells (EPC) from rat bone marrow
EPC were collected from the femurs of 6 to 8 weeks old male Sprague-Dawley rats (220–280 g). Aspirated bone marrow was mixed with 1000 U/ml heparin (Immuno, Vienna, Austria), deoxyribonuclease I 1000 U/ml (Sigma, St. Louis, MO) in Dulbecco's PBS (PAA Laboratories, Austria) as described . The mononuclear cell fraction was obtained from a Lymphoprep density gradient (Nycomed, Norway) after centrifugation for 30 min at 1700 rpm (centrifuge GPR, Beckman, Hettich, Germany). The mononuclear cell fraction was carded, washed and centrifuged at 800 rpm for 10 min. The cell pellet was then suspended in EBM-2 medium (Clonetics, San Diego, California) supplemented with 20% fetal calf serum (FCS, PAA Laboratories, Austria) and plated on rat-derived fibronectin-coated (10 μg/ml, Sigma, F0635, St. Louis, MO) 12-well plates (Costar, Corning, The Netherlands). After 24 h the non-adherent cell population was aspirated and transferred to a new fibronectin-coated plate. After another 24 h this procedure was repeated to remove rapidly adherent hematopoietic cells or mature EC being possibly present in the aspirate. Only the non-adherent cell population harvested after 48 h was evaluated further in all experiments. This fraction was cultured in EBM-2 medium containing vascular endothelial growth factor (VEGF), human fibroblast growth factor-B (hFGF-B), R3-insulin like growth factor (R3-IGF-1), human epidermal growth factor (hEGF), ascorbic acid, hydrocotisone, gentamycin, amphotericin B (MV-Kit, Clonetics, San Diego, California) and stem cell growth factor (SCGF, PreproTech EC Ltd., USA). After 2–3 days a kind of angioblast-like cells were observed and spindle-shaped cell outgrowth documented. After 7 to 10 days confluence of the outgrowing cell population was reached and cells were divided by collagenase (Type CLS-CI-22, Biochrom AG, Berlin, Germany).
Characterization of EPC from rat bone marrow
Cells were primarily characterized by phase contrast microscopy evaluating cobblestone morphology which is typical for confluent EC. EPC were further imaged for their incorporation of acetylated low density lipoprotein (aLDL) labeled with fluorescent Dil dye (Dil-acLDL; Biomedical Technologies, Stoughton, Massachusetts). Indirect immunofluorescence for detection of CD31 (PharMingen, USA), was performed using rabbit anti-rat PECAM-1 antibody by a standard protocol as given by the manufacturer. Secondary FITC-labeled antibodies (swine anti-rabbit Ig) were purchased from DAKO (Carpenteria, California). Von Willebrand Factor (vWF) was detected by direct immunofluorescence using a FITC-marked anti-vWF antibody (DAKO, Carpenteria, California). Direct and indirect immunofluorescence microscopy was done using a Olympus BH-2 RFCA fluorescence microscope and KAPPAImage software (Kappa Optoelectronics, Germany).
Additionally, flow cytometry (FACS) analyses were performed for further characterization of EPC. EPC were checked for the presence of CD146-PE (P1H12) (Chemicon, Temecula, USA), CD133-PE (Milteny-Biotec, Bergisch-Gladbach, Germany), VEGF receptor-2 (KDR; R&D, Wiesbaden, Germany) and CD106 (clone 1.G11B1, Serotec, Oxford, UK). Expression of cell surface markers were measured in a LSR flow cytometer (Becton Dickinson, USA) using the Cell Quest software (Becton Dickinson, USA).
Isolation and culture conditions of arterial endothelial cells from rat thoracic aorta (rAEC)
Female Sprague-Dawley rats weighing 230–280 g were housed in a light-, temperature-, and humidity-controlled environment and provided with food and water ad libitum. Before killing by decapitation, rats were anesthetized with dietylether and thoracic aortas prepared immediately after removal. Aortas were cut into consecutive 2 mm segmental rings, mounted on the plastic surface of 24-well tissue culture plates coated with a distinct mixture of collagen type I (0.1 mg/ml; Collaborative Biomedical Products, Bedford, MA), fibronectin (10 μg/ml; Collaborative Biomedical Products) and porcine gelatin (0.2%; Sigma, St. Louis, MO). Cells were cultured in M199 with 10% FCS, 100 U/ml penicillin, 100 mg/ml streptomycin and 100 mg/ml endothelial cell growth factor supplement (Sigma, St. Louis, MO) and kept in a humidified incubator at 37°C in 5% CO2. Rat aortic endothelial cells (rAEC) were used between passages three and five for all experiments.
Isolation and culture conditions of arterial endothelial cells from rat pulmonary arteries (rPAEC)
As given above two female Sprague-Dawley rats weighing 230–280 g were killed by decapitation: rats were anesthetized with dietylether and main pulmonary arteries prepared immediately after removal. Pulmonary arteries were cut into consecutive 2 mm segmental rings, mounted on the plastic surface of 24-well tissue culture plates coated with rat 10 μg/ml fibronectin. Rat pulmonary artery endothelial cells (rPAEC) were cultured in endothelial culture medium (Promo Cell, Heidelberg, Germany) containing 10% FCS and 2% endothelial cell growth supplement (Promo Cell, Heidelberg, Germany), 1% penicillin/streptomycin solution (Sigma, St. Louis, MO) and kept in a humidified incubator at 37°C in 5% CO2. rPAEC were used between passages three and five for all experiments.
Culture conditions of human lung microvascular endothelial cells (hL-MVEC)
Primary human lung microvascular endothelial cells (hL-MVEC; Clonetics, San Diego, CA, USA) were cultured according to the manufacturer's protocol in EBM-2 medium containing vascular endothelial growth factor (VEGF), human fibroblast growth factor-B (hFGF-B), R3-insulin like growth factor (R3-IGF-1), human epidermal growth factor (hEGF), ascorbic acid, hydrocotisone, gentamycin, amphotericin B (MV-Kit, Clonetics, San Diego, California).
After incubation at 37°C for various time periods cellular proliferation was measured using a colorimetric assay for cell growth and chemosensitivity. This colorimetric assay based on the tetrazolium salt MTT ((3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyl tetrazolium bromide; Sigma, St. Louis, MO) detects living but not dead cells, and the signal generated is directly proportional to the number of cells . After 6 h of incubation, medium was aspirated from adherent cells without disturbing formazan crystals formed within the cells. Subsequently, dimethylsulfoxide (Merck, Darmstadt, Germany) was added to each well, the plates were agitated on a plate shaker, and the optical density was read with an enzyme-linked immunoabsorbent assay reader at 570 nm (MR 700; Dynatech Labs, Guernsey, United Kingdom).
In vitro capillary tube formation assay in Matrigel
For analysis of capillary tube formation, 150 μl Matrigel (Becton Dickinson, Heidelberg, Germany), an extracellular mouse sarcoma matrix (Engelbreth-Holm-Swarm tumor) known to be in vivo and in vitro a pro-angiogenic stimulus, was laid into the wells of a 48-well plate (Falcon, Heidelberg, Germany) and incubated at 37°C for 60 minutes. EPC or hL-MVEC were harvested and 3 × 104 cells resuspended in 200 μl EBM-2/MV medium and plated. Conditions with EBM-2/MV with 10% FCS or supplemented with 50 ng/ml VEGF were studied. Capillary tube formation on Matrigel was observed under an inverted Zeiss Axiovert microscope after 5 or 18 h of incubation.
Application of subcutanous Matrigel
200 μl of Matrigel (Becton Dickinson, Heidelberg, Germany) was subcutanously administered into the left-sided flank subcutis of lung transplant recipients with EPC injection eight days before transplantation in order to assess angiogenesis as shown in figure five.
Ex vivo cell tracer labeling of EPC
EPC were kept on fibronectin-coated culture flasks within EBM-2/MV medium as given above without further complementation prior to in vivo coloration. After a washing procedure in buffer solution EPC were stained with the anionic sulfophenyl cell tracer SP-DiIC18(3) (Molecular Probes, Leyden, The Netherlands), a formaldehyde and acetone resistant Dil dye at a concentration of 2 μg/ml solution in standard PBS. Staining was performed on adherent EPC at 37°C for 10 min followed by a further incubation period of 35 min at 4°C. After staining, cells were washed in EBM-2 supplemented with 10% FCS. Efficiacy of coloration and cell morphology was checked by fluorescence microscopy twice before transplantation. Furthermore, growth, morphology and fluorescence intensity of SP-DiIC18(3)-in vivo staining was checked at the end of each experiment. No differences in biological functions of SP-DiIC18(3)-stained EPC tested have been observed (data not shown). SP-DiIC18(3) staining was detectable up to 14 days in in vitro cultured EPC (data not shown).
Flow cytometry (FACS) of EPC in rat blood samples
100 μl of EDTA blood was withdrawn from an EPC-injected lung transplant recipient 12 h post reperfusion from the jugular vein. Whole blood was stained with 10 μl anti rat CD42d-FITC (Becton Dickinson, Heidelberg, Germany), 10 μl anti rat CD45-FITC (Becton Dickinson), and 10 μl anti human CD146-PE (clone P1H12, Chemicon, Hofheim, Germany) for 30 min at room temperature. Red blood cells were lysed with 1 ml of BD Lysing Solution (Becton Dickinson) for 10 min at room temperature. After washing twice with 3 ml PBS, cells were measured in a BD LSR flow cytometer (Becton Dickinson) using Cell Quest software (Becton Dickinson). To quantify the amount of circulating EC in the blood samples a standardized amount of 6 μm latex microspheres (Polyscienes, Eppelheim, Germany) was added to each blood sample. With this internal standard it was possible to calculate the amount of circulating EC per ml of blood.
In vivo experimental protocol including the intravenous injection of EPC
All experiments were performed according to the Helsinki convention for the use and care of animals and were approved by the local review boards for animal care. Briefly, weight matched female Sprague-Dawley rats of 220 – 270 g received orthotopic single left lung allografts under general anesthesia with 2% halothane from female Sprague-Dawley rats after a total graft ischemia of 20 h. A standard cuff technique for the vessel anastomoses and a running suture for the bronchial anastomosis were applied, as well as for the donor procedure and transplantation . Immediately before injection of EPC into the host rat, SP-DiIC18(3)-labelled cells were harvested, washed and resuspended in EBM-2 medium at a concentration of 1 × 106/ml. Injection of EPC was done under general anesthesia with 2% halothane into the saphenous vein of the right hind leg under microscopic vision to ascertain the successful and complete venous administration into each host animal. Intravenous application of EPC was performed 50 to 120 min after reperfusion of the transplanted left lung (n = 9). Two further control animals were not lung transplanted but received labelled EPC as given above.
In vivo experimental protocol
Weight matched female Sprague-Dawley rats of 220 – 270 g received orthotopic single left lung allografts from female Sprague-Dawley rats after a total graft ischemia of 20 h. A cuff technique for the vessel anastomoses and a running suture for the bronchial anastomosis were applied. The experiments were performed according to the Helsinki convention for the use and care of animals and were approved by the local review boards for animal care.
Animals were anaesthetized by intraperitoneally administered pentobarbital (50 mg/kg) and heparinized (500 I.U./kg). After tracheotomy the animals were ventilated through a 14 gauge cannula (FiO2 = 1.0) by a Unno rodent ventilator (Hugo Sachs Harvard Apparatus, March-Hugstetten, Germany) at a tidal volume of 8 ml/kg at 100/min. After division of the inferior vena cava and resection of the left appendix of the heart, a small silicon tube was inserted into the main pulmonary artery. Both lungs were flushed with 20 ml of Low Potassium Dextrane (LPD) solution (Perfadex, kindly provided from Xvivo, Göteborg, Sweden) at a pressure of 20 cm H2O. The trachea was tied in end-inspiration, the heart-lung block removed and 16 gauge cuffs (Abbocath-T, Abbott, Sligo, Ireland) were placed around the pulmonary artery and vein. The vessels were inverted and tied onto the cuff with an 8-0 monomeric filament. The lung was stored in LPD solution at 1.5°C until implantation.
Transplantation was performed after 20 h of cold ischemia at 1.5°C. The recipient rat was anesthetized by breathing 4% halothane in a glass chamber followed by intubation. Anesthesia was maintained throughout the operative procedure with 2% halothane. A left lateral thoracotomy was performed in the 4th intercostal space. The left hilum was dissected and after clamping of the left pulmonary artery and vein with removable microvascular clips, the pulmonary vein was opened, flushed with heparinized saline solution, and the cuff was inserted and fixed with 6-0 Silk. With the same technique, the pulmonary artery was anastomosed. The native left lung was removed and the bronchial anastomosis performed with a running over-and-over suture with 9-0 Monosof (Tyco Healthcare, Wollerau, Switzerland). The lung was first reventilated and then reperfused. A chest tube was inserted and the thoracotomy closed. The chest tube was removed after restoration of spontaneous breathing when the animal was extubated.
Intravenous injection of EPC
Immediately before injection of EPC into the host rat, SP-DiIC18(3)-labelled cells were harvested, washed and resuspended in EBM-2 medium at a concentration of 1 × 106/ml. Injection of EPC (1 × 106 cells) was done under general anesthesia with 2% halothane into the saphenous vein of the right hind leg under microscopic vision to ascertain the successful and complete venous administration into each host animal. In preliminary experiments tolerability of intraveinous application of EBM-2 (1 ml) alone turned out to be safe. Intravenous application of EPC was performed 50 to 120 min after reperfusion of the transplanted left lung.
Assessment of transplanted EPC in the host animal
To evaluate the incorporation of EPC into rat organs, animals were anesthetized by intraperitoneal pentobarbital (50 mg/kg) and ventilated after tracheotomy with an FiO2 of 1.0 at 100/min, a tidal volume of 8 ml/kg, and a positive end-expiratory pressure (PEEP) of 5 cm H2O. Lung transplanted animals were sacrificed after one day (n = 7), 3 days (n = 1), or 9 days (n = 1) post transplantation. Controls were killed at day one after peripheral EPC injection. Animals were sacrificed after median thoracotomy and intracardiac heparinization with 500 U/kg, when lungs were flushed with 20 ml saline solution through the pulmonary artery. The heart-lung block was excised and the lungs separated: Each lung was divided and one part put into 10% PBS-buffered formalin solution, and the remainder part was deep-frozen in liquid nitrogen and stored at -70°C.
Further organs of the host rats (spleen, liver, kidney and adrenals, stomach, small intestine, colon, bone) were preserved in 10% PBS-buffered formalin solution as well as deep-frozen in liquid nitrogen and stored at -70°C.
Immunofluorescence staining of tissue specimens
The formalin-fixed tissue was paraffin-embedded and cut at 4 μm to 10 μm (as given in detail in some experiments). Slides were heated in an incubator at 70°C for 30 min before they were deparaffinized in xylene and hydrated in graded ethanol. Slides were incubated with FITC-labelled lectin from Bandeiraea simplicifolia (Griffonia simplicifolia) BS-I (Sigma, St. Louis, MO) and 3', 6'-diamidino-2-phenylindole, dihydrochloride (DAPI; Molecular Probes, Leyden, The Netherlands) according to the manufacturers' protocol. Bandeiraea simplicifolia lectin was chosen due to its affinity to EC, and DAPI staining was used to stain nuclei specifically with blue fluorescence. Lectin was diluted at 1:100 and DAPI at 1:1000 in PBS containing 1% bovine serum albumin (BSA). Analysis was performed by three of the authors (H. N., J.H., C.M.K.) using a Zeiss Axioskop 2 light and fluorescence microscope (Zeiss, Göttingen, Germany). For additional confocal microscopic analysis, histological sections with a thickness up to 10 μm (left-sided injured lung, right lung and the other organs investigated) were examined with an Inverse Axiovert 100 M BP (Base Port) confocal microscope LSM 510 (Zeiss, Göttingen, Germany) using the following laser emissions: DAPI: excitation 364 nm, emission BP 385–470 nm; FITC: excitation 488 nm, emission BP 405–430 nm; SP-DiIC18(3): excitation 543 nm: emission LP 585 nm. Fluorescent signals from DAPI, FITC-lectin and SP-DiIC18(3) were viewed simultaneously in separate detector channels. True color overlays of single and serial sections were generated with Zeiss confocal software 2.8 SP1.
Values are presented as mean ± S.E.M. The values were compared by Mann-Whitney U test as given in the text. Differences were considered statistically significant at p ≤ 0.05.