Human vWF-Factor VIII free was obtained from American Diagnostica Inc. (Stamford, CT, USA). P38 MAPK and ERK-1,2 inhibitors, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4'-pyridyl)-1-H-imidasole (SB203580), 4-ethyl-2(p-methoxyphenyl)-5-(4'-pyridyl)-1-H-imidazole (SB202474), 2'-amino-3'-methoxyflavone (PD98059), 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U0126), were purchased from Calbiochem (Gibbstown, NJ, USA). Neutralizing antibodies against human P-selectin, E-selectin, ICAM1, VCAM1 and normal IgG (isotype-matching control) were purchased from R&D Systems (Minneapolis, MN, USA).
Human mesenchymal stem cells (hMSCs) and human umbilical vein endothelial cells (HUVECs) were purchased from Lonza Group Ltd. (Basel, Switzerland) and cultured in MSCGM BulletKit (Lonza) and EGM-2 BulletKit (Lonza), accordingly. Passages 2 to 5 were used. Cells were maintained at 37°C in a humidified atmosphere of 5% CO2.
HMSC adhesion assay
HMSC adhesion to HUVECs was conducted as previously described . HMSCs grown as a monolayer were dissociated with trypsin-EDTA solution (Lonza), washed with Hank's balanced salt solution (HBSS), and labeled with 4 μg/ml calcein AM (Molecular Probes, Invitrogen, Carlsbad, CA, USA) in HBSS for 45 minutes at 37°C and 5% CO2. After the labeling, hMSCs were washed with HBSS and resuspended in Dulbecco's modified Eagle's medium (DMEM; Sigma, St. Louis, MO, USA). HUVECs were prepared for the adhesion assay as follows. A confluent monolayer of HUVECs in a 96-well cell culture clear-bottom black plate (Corning Incorporated Life Sciences, Lowell, MA, USA) was washed twice with HBSS and treated with vWF (0 to 6 μg/ml) in HBSS for 0 to 9 hours at 37°C and 5% CO2.
Before the adhesion assay cells were washed with HBSS and left in 50 μl of HBSS. HMSC suspension (50 μl, 10,000 cells per well) was added to HUVECs and incubated for 30 minutes at 37°C and 5% CO2. The cell load was estimated by scanning the plate in a POLARstar OPTIMA microplate reader (BMG Labtech Inc., Cary, NC, USA) at excitation/emission wavelengths of 485/520 nm. Wells without hMSCs were used to assay the background fluorescence. Unbound hMSCs were aspirated and wells were washed with 200 μl of HBSS two times, 100 μl HBSS was added to each well and plates were scanned to assay a number of bound cells. The percentage of bound cells was calculated as a ratio between the fluorescence of washed and unwashed wells after subtraction of the background fluorescence from both values. At least six wells were used for each experimental condition. At least three independent experiments were conducted for each treatment.
Adhesion of hMSCs to collagen-coated or tissue culture plates was studied using a 96-well collagen I coated clear-bottom black plate (BD Biosciences, Franklin Lakes, NJ, USA) and a 96-well cell culture clear-bottom black plate, respectively. Immobilization of vWF was achieved by incubation of plates with a solution of vWF (0 to 8 μg/ml) in HBSS for four hours. The adhesion of hMSCs to the plates was assayed before and after vWF immobilization. In order to remove unbound vWF wells were washed with HBSS before the adhesion assay. Immobilization of vWF on collagen I coated and tissue culture plates was monitored by ELISA. Plates were treated with vWF as described above, washed three times with the wash buffer from ELISA development kit (R&D Systems) and incubated with peroxidase-conjugated rabbit polyclonal anti-human vWF antibody (Dako North America, Inc., Carpinteria, CA, USA) according to the manufacturer's recommendations. ELISA was developed and the optical densities were measured at 450 nm with a 595 nm reference wavelength in a POLARstar OPTIMA microplate reader (BMG Labtech Inc., Cary, NC, USA). Immobilization of vWF was determined using eight measurements per each experimental condition.
In experiments on the inhibition of p38 MAPK and ERK-1,2 HUVECs were pre-incubated with inhibitors of protein kinases for 45 minutes and then stimulated with vWF. Activity of p38 MAPK was inhibited with SB203580. SB202474, a chemical analog of SB203580, was used as a negative control. Phosphorylation and activation of ERK-1,2 was inhibited with U0126 or PD98059.
Flow cytometric analysis of antigen expression on the surface of hMSCs and HUVECs
Analysis of E-selectin, VCAM1 and ICAM1 expression on the surface of HUVECs and integrin αvβ3 and GPIbα expression on the surface of hMSCs was conducted using flow cytometry. Cells were dissociated and resuspended in the flow cytometry buffer consisting of 2% bovine serum albumin (Sigma) and 0.1% sodium azide (Sigma-Aldrich, St. Louis, MO, USA) in Dulbecco's phosphate buffered saline (PBS; Sigma). HUVECs were dissociated using Hank's based enzyme free cell dissociation solution (Millipore, Billerica, MA, USA). HMSCs were dissociated with trypsin-EDTA solution (Lonza). Cells (2 × 105 cells) were stained with corresponding fluorochrome-conjugated monoclonal antibodies (BD Biosciences) for 30 minutes at room temperature according to the manufacturer's recommendations. After incubation with antibodies, cells were washed with 5 ml of the flow cytometry buffer and resuspended in the flow cytometry buffer containing 1% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA). Background staining was assessed by incubation of cells with mouse fluorochrome- and isotype-matching immunoglobulins. Flow cytometric analysis was performed by acquiring 5,000 events on a FACScan flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Data were processed with a CellQuest™software package supplied by instrument manufacturer (BD Biosciences, Franklin Lakes, NJ, USA). The cellular debris was assessed on the basis of forward and right angle scattering analysis and excluded from further analysis by a CellQuest™software package (BD Biosciences, Franklin Lakes, NJ, USA).
Human phospho-MAPK array
Analysis of protein kinase phosphorylation in HUVECs treated with vWF was conducted using the human phospho-MAPK array kit (R&D Systems). Confluent HUVECs grown on a 100 mm tissue culture plate were washed twice with HBSS and treated with 4 μg/ml vWF in HBSS for 0 to 35 minutes. After the treatment cells were washed with HBSS and lysed with the manufacturer supplied buffer and protein, phosphorylation was developed according to the manufacturer's recommendations. Phosphorylation of protein kinases was detected by exposure of phospho-MAPK array to X-ray film (Kodak, Rochester, NY, USA). All arrays from the same experiment were processed simultaneously and exposed to the same X-ray film.
Western blot analysis of p38 MAPK and ERK-1,2 phosphorylation
Confluent HUVECs grown on a 100 mm tissue culture plate were washed twice with HBSS, treated with 4 μg/ml vWF in HBSS for 0 to 5 minutes and lysed with 1 ml of lysis buffer containing 0.025 M Tris-HCl, pH 7.4, 0.15 M NaCl, 5 mM EDTA, 1% Triton X-100, 0.5% Nonidet P-40, and a set of protease inhibitors (Roche Applied Science, Indianapolis, IN, USA) and phosphatase inhibitors (cocktails type 1 and 2) (Sigma) for 15 minutes at 4°C. Extract was cleared by centrifugation at 15,000 × g at 4°C for 30 minutes. Proteins (25 μg) were separated in Bis-Tris 10% Criterion gel (Bio-Rad, Hercules, CA, USA) using XT MOPS running buffer (Bio-Rad) and transferred to a nitrocellulose membrane (Bio-Rad). Western blot was performed using polyclonal antibodies against phosphorylated p38 MAPK (Thr180/Tyr182), total p38 MAPK, phosphorylated ERK-1,2 (Thr202/Tyr204) and total ERK-1,2 (Cell Signaling Technology, Danvers, MA, USA). Western blot was developed with Rabbit TrueBlot HRP-labeled anti-rabbit antibody (eBioscience, San Diego, CA, USA) and ECL Western blotting detection reagents (GE Healthcare UK Limited, Pittsburgh, PA, USA).
Cell-based ELISA for p38 MAPK and ERK-1,2 phosphorylation in HUVECs treated with vWF
Phosphorylation of p38 MAPK (Thr180/Tyr182) and ERK-1,2 (Thr202/Tyr204) was assayed using corresponding cell-base ELISA kits (R&D Systems). Confluent HUVECs grown on a 96-well cell culture clear-bottom black plate were washed twice with HBSS and treated with 0 to 6 μg/ml vWF in HBSS for four hours. After the treatment, cells were washed with HBSS, fixed with 4% paraformaldehyde in phosphate-buffered saline for 30 minutes and stained according to the manufacturer's recommendations. Fluorescence of total protein kinase at 450 nm and phosphorylated protein kinase at 600 nm were acquired in a POLARstar OPTIMA microplate reader. Background fluorescence was estimated as recommended by the manufacturer from the control wells stained with corresponding secondary antibody and the relative ratio of the fluorescence of phosphorylated protein kinase to the fluorescence of total protein kinase was calculated. At least six wells were used for each experimental condition.
Activity assays of p38 MAPK and ERK-1,2
Activities of p38 MAPK and ERK-1,2 in HUVEC lysate were assayed using the p38 and p44/42 MAPK assay kits (Cell Signaling Technology). Confluent HUVECs grown on a six-well cell culture plate were washed twice with HBSS and treated with 0 to 6 μg/ml vWF in HBSS for four hours. After the treatment cells were washed with HBSS and lysed with the provided buffer according to the manufacturer's recommendations. Phosphorylated p38 MAPK (Thr180/Tyr182) and ERK-1,2 (Thr202/Tyr204) were immunoprecipitated from HUVEC lysates of equal volume (1 ml) and protein concentrations (1.5 mg/ml) with corresponding anti-phospho-p38 MAPK (Thr180/Tyr182) and anti-phospho ERK-1,2 (Thr202/Tyr204) antibodies supplied by the manufacturer. Enzymatic activities of immunoprecipitated protein kinases were assayed using recombinant ATF-2 protein as a substrate for p38 MAPK and recombinant Elk-1 protein as a substrate for ERK-1,2. Phosphorylation of ATF-2 and Elk-1 proteins was detected by Western blots. For this, reaction mixtures (35 μl) were separated in Bis-Tris 10% Criterion gel using XT MOPS running buffer and transferred to a nitrocellulose mem-brane. Western blot was performed using anti-phospho-ATF-2 (Thr71) or anti-phospho-Elk-1 (Ser383) antibodies. Immunoreactive bands were visualized using affinity purified HRP-labeled goat anti-rabbit F(ab')2 fragment antibody (Kirkegaard and Perry Laboratories, Gaithersburg, Maryland, USA) and ECL Western blotting detection reagents (GE Healthcare UK Limited).
Affymetrix DNA microarray analysis
RNA was extracted from HUVECs using the RNeasy kit (Qiagen, Germantown, MD, USA), and analysis of gene expression in HUVECs was performed on Affymetrix Human Genome U133 Plus 2.0 array (Santa Clara, CA, USA) according to the manufacturer's recommendations. Raw microarray data were processed with the affy package of the Bioconductor project using MAS 5.0 algorithm and subjected to t-test.
Analysis of DNA content using Quanti-iT PicoGreen dsDNA reagent
HUVECs were treated with vWF and inhibitors of protein kinases as described above and washed with HBSS. To prepare the cell lysate 100 μl of cell lysis solution (0.2% v/v Triton X-100, 10 mM Tris (ph 7.0), 1 mM EDTA) was added to each well (96-well plate), and the plate was process through a total of two freeze at -80°C/thaw at room temperature cycles. After a final thaw 100 μl of the aqueous working solution of Quant-iT PicoGreen dsDNA reagent (Invitrogen) prepared according the manufacturer's instructions was added to each well. Fluorescence was measured using a Polarstar OPTIMA microplate reader (BMG Labtech Inc.) at excitation/emission wavelengths of 485/520 nm. DNA standard curve was generated using dsDNA standard provided with the Picogreen Assay kit and used for determining the DNA concentration of the samples.
Confluent monolayer of HUVECs on Lab-Tek II chamber CC2 glass slides (Nalge Nunc International, Rochester, NY, USA) was treated with 4 μg/ml vWF in HBSS for four hours and the adhesion assay with hMSCs was conducted as described in the hMSC adhesion assay section. Cells were fixed with 5% paraformaldehyde, permeabilized with 0.1% Triton x100 in phosphate buffered saline, blocked with 5 mg/ml bovine serum albumin in PBS for one hour and stained with 1 μg/ml AF488-conjugated CD31 antibody (BD Pharmingen, Franklin Lakes, NJ, USA), the specific antigen marker of HUVECs, and 1 μg/ml PE-conjugated CD90 antibody (BD Pharmingen), the specific antigen marker of hMSCs, for four hours. After staining cells was washed with PBS and images were acquired on Olympus FluoView FV1000 confocal microscope (Olympus America Inc. Center Valley, PA, USA).