Prostacyclin suppresses twist expression in the presence of indomethacin in bone marrow-derived mesenchymal stromal cells.
BACKGROUND
Iloprost, a stable prostacyclin I2 analogue, seems to have an osteoblast-protective potential, whereas indomethacin suppresses new bone formation. The aim of this study was to investigate human bone marrow stromal cell (BMSC) proliferation and differentiation towards the osteoblastic lineage by administration of indomethacin and/or iloprost.


METHODS
Human bone marrow cells were obtained from 3 different donors (A=26 yrs/m; B=25 yrs/f, C=35 yrs/m) via vacuum aspiration of the iliac crest followed by density gradient centrifugation and flow cytometry with defined antigens (CD105+/73+/45-/14-). The cells were seeded and incubated as follows: without additives (Group 0; donor A/B/C), with 10(-7) M iloprost only (Group 0+ilo; A/B), with indomethacin only in concentrations of 10(-6) M (Group 1, A), 10(-5) M (Group 2, B), 10(-4) M (Group 3, A/B), and together with 10(-7) M iloprost (Groups 4-6, A/B/C). On Day 10 and 28, UV/Vis spectrometric and immunocytochemical assays (4 samples per group and donor) were performed to investigate cell proliferation (cell count measurement) and differentiation towards the osteoblastic lineage (CD34-, CD45-, CD105+, type 1 collagen (Col1), osteocalcin (OC), alkaline phosphatase (ALP), Runx2, Twist, specific ALP-activity).


RESULTS
Indomethacin alone suppressed BMSC differentiation towards the osteoblastic lineage by downregulation of Runx2, Col1, and ALP. In combination with indomethacin, iloprost increased cell proliferation and differentiation and it completely suppressed Twist expression at Day 10 and 28. Iloprost alone did not promote cell proliferation, but moderately enhanced Runx2 and Twist expression. However, the proliferative effects and the specific ALP-activity varied donor-dependently.


CONCLUSIONS
Iloprost partially antagonized the suppressing effects of indomethacin on BMSC differentiation towards the osteoblast lineage. It enhanced the expression of Runx2 and, only in the presence of indomethacin, it completely suppressed Twist. Thus, in the treatment of avascular osteonecrosis or painful bone marrow edema, the undesirable effects of indomethacin might be counterbalanced by iloprost.
Department of Orthopedics, Medical Faculty, University of Düsseldorf, Düsseldorf, Germany.
Oliver
O
Kemper
Department of Orthopedics, Medical Faculty, University of Düsseldorf, Düsseldorf, Germany.
Monika
M
Herten
Institute for Transplantation Diagnostics and Cell Therapy, University Düsseldorf, Düsseldorf, Germany.
Johannes
J
Fischer
Department of Orthopaedics, University Duisburg-Essen, Essen, Germany.
Marcel
M
Haversath
Department of Orthopedics, University Duisburg-Essen, Essen, Germany.
Sascha
S
Beck
Department of Orthopaedics, University Hospital of Duisburg-Essen, Essen, Germany.
Tim
T
Classen
Department of Orthopaedics, University Hospital of Duisburg-Essen, Essen, Germany.
Sebastian
S
Warwas
Department of Orthopaedics, University Hospital of Duisburg-Essen, Essen, Germany.
Tjark
T
Tassemeier
Department of Orthopedics, University Duisburg-Essen, Essen, Germany.
Stefan
S
Landgraeber
Department of Orthopedics, Medical Faculty, University Düsseldorf, Düsseldorf, Germany.
Sabine
S
Lensing-Höhn
Department of Orthopedics, Medical Faculty, University of Düsseldorf, Düsseldorf, Germany.
Rüdiger
R
Krauspe
Department of Orthopedics, University Duisburg-Essen, Essen, Germany.
Marcus
M
Jäger
D000328:Adult; D000469:Alkaline Phosphatase / Q000378:metabolism; D015703:Antigens, CD / Q000378:metabolism; D001854:Bone Marrow Cells / Q000166:cytology*; D002452:Cell Count; D002454:Cell Differentiation / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D024042:Collagen Type I / Q000378:metabolism; D050659:Core Binding Factor Alpha 1 Subunit / Q000378:metabolism; D011464:Epoprostenol / Q000008:administration & dosage / Q000494:pharmacology*; D005260:Female; D006801:Humans; D016285:Iloprost / Q000494:pharmacology; D007150:Immunohistochemistry; D007213:Indomethacin / Q000008:administration & dosage / Q000494:pharmacology*; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008297:Male; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism*; D015675:Osteocalcin / Q000378:metabolism; D051879:Twist-Related Protein 1 / Q000378:metabolism*
D015703:Antigens, CD; D024042:Collagen Type I; D050659:Core Binding Factor Alpha 1 Subunit; D009687:Nuclear Proteins; C493709:RUNX2 protein, human; C103173:TWIST1 protein, human; D051879:Twist-Related Protein 1; D015675:Osteocalcin; D011464:Epoprostenol; D007770:L-Lactate Dehydrogenase; D000469:Alkaline Phosphatase; D016285:Iloprost; D007213:Indomethacin