Reduced expression of platelet endothelial cell adhesion molecule-1 in bone marrow cells in mice after skeletal unloading.
One week of tail suspension significantly decreased the expression of PECAM-1 in mouse tibial bone marrow cells but not those of a number of other vascular factors. Anti-PECAM-1 antibody suppressed both ALP+ CFU-f formation and ALP production under co-culture of the osteoblastic cell line and the PECAM-1+ endothelial cell line. This study suggests that the reduced ALP activity after skeletal unloading is related to downregulation of PECAM-1 expression in bone marrow cells in mice.


BACKGROUND
Vascular factors play a role in bone development and regeneration. We tested the hypothesis that skeletal unloading reduces osteogenic potential by inhibiting the molecules related to angiogenesis and/or vasculogenesis in bone marrow cells.


METHODS
Eight-week-old male mice were assigned to three groups after acclimatization for 1 week: ground control (GC), tail suspension (TS), and reloading after 7-day TS (RL). Bilateral tibial and humeral samples were used for analyses. MC3T3-E1, a mouse osteoblastic cell line, and EOMA and ISOS-1, mouse endothelial cell lines, were also used.


RESULTS
Flow cytometric analysis revealed that 7-day TS significantly decreased the expression of platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31) in tibial bone marrow cells, but not those of angiopoietin-1, angiopoietin-2, Flk-1 (vascular endothelial growth factor receptor-2), and vascular endothelial cadherin. The expression of PECAM-1 in tibial marrow cells was reduced at day 3 of TS to 80% and still showed significantly low levels at day 7 of TS to 72% of that at the respective days of GC. This decreased expression of PECAM-1 after 7-day TS showed the GC level at 5-day reloading after 7-day TS. However, the expression of PECAM-1 in humeral marrow cells (internal bone marrow control) after TS and RL remained unchanged and equivalent to that of GC. The expression level of PECAM-1 mRNA was significantly lower at day 7 of TS to 62% of that in GC. Double labeling analyses revealed that PECAM-1+ cells mostly consisted of endothelial cells and partially of granulocytes. In bone marrow cell cultures, the formation of alkaline phosphatase (ALP)+ colony forming units-fibroblastic was significantly reduced in the presence of anti-PECAM-1 antibody in the medium compared with the presence of immunoglobulin G (0.025 times as much as ALP production with immunoglobulin G). ALP production by cultured MC3T3-E1 was enhanced in combination with PECAM-1+ EOMA (1.8 times as much as ALP production by MC3T3-E1 alone), but not in combination with PECAM-1- ISOS-1. Anti-PECAM-1 antibody inhibited the increase in ALP production under co-culture with EOMA.


CONCLUSIONS
Our data show that the reduced ALP activity after skeletal unloading is closely correlated with reduced expression of PECAM-1 in bone marrow cells. We speculate that the loss of osteogenic potential after skeletal unloading is caused by the suppression of PECAM-1 signaling on endothelial cellular surface.
Sakuma-Zenke
Miyuki
M
Department of Orthopaedic Surgery, University of Occupational and Environmental Health, Iseigaoka, Kitakyushu, Japan. myukisa@cocoa.ocn.ne.jp
Sakai
Akinori
A
Nakayamada
Shingo
S
Kunugita
Naoki
N
Tabata
Takahiro
T
Uchida
Soshi
S
Tanaka
Shinya
S
Mori
Toshiharu
T
Nakai
Kenichiro
K
Tanaka
Yoshiya
Y
Nakamura
Toshitaka
T
D016475:3T3 Cells; D000469:Alkaline Phosphatase / Q000378:metabolism; D042683:Angiopoietin-1 / Q000096:biosynthesis; D042702:Angiopoietin-2 / Q000096:biosynthesis; D000818:Animals; D001792:Blood Platelets / Q000378:metabolism*; D001835:Body Weight; D001846:Bone Development; D001854:Bone Marrow Cells / Q000166:cytology*; D018920:Coculture Techniques; D015536:Down-Regulation; D042783:Endothelial Cells / Q000166:cytology; D004730:Endothelium, Vascular / Q000378:metabolism; D005434:Flow Cytometry; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018919:Neovascularization, Physiologic; D010006:Osteoblasts / Q000378:metabolism; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000096:biosynthesis*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells; D013696:Temperature; D013977:Tibia / Q000166:cytology / Q000378:metabolism; D013997:Time Factors; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000096:biosynthesis
D042683:Angiopoietin-1; D042702:Angiopoietin-2; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D040301:Vascular Endothelial Growth Factor Receptor-2; D000469:Alkaline Phosphatase