Simvastatin suppresses dexamethasone-induced secretion of plasminogen activator inhibitor-1 in human bone marrow adipocytes

Bone marrow fluid was obtained from the femoral necks of 40 patients (6 men, 34 women; age range 52-81 years) undergoing hip joint replacement surgery. Patients with diabetes mellitus, rheumatoid arthritis, and metabolic bone disorders and those with a history of glucocorticoid therapy were excluded. We divided samples into three or four groups, including the control, dexamethasone and simvastatin groups. Each experiment was performed with samples from 6 or 7 patients (n = 6 or 7). The energy status in each experiment is as follow; Figure 1A, B: mean BMI, 24.9; mean age, 69.3 years, Figure 2A, B: mean BMI, 23.3; mean age, 64.8 years, Figure 3A, B: mean BMI, 24.4; mean age, 66.5 years, Figure 4: mean BMI, 23.7; mean age, 60.3 years. BMI and age were analyzed statistically, but no significant trends were noted. The study protocol was approved by the Institutional Ethics Review Board and informed consent was obtained from each patient before surgery.

Bone marrow fluid was mixed with 20 mL of Dulbecco's modified Eagle's medium (DMEM: Gibco BRL, Grand Island, NY) and treated with 0.1% collagenase A (Sigma Chemical Co., St. Louis, MO) for 1 h at 37°C. Digested cells were then centrifuged at 200 × g for 5 min, and the adipocyte layer was carefully aspirated from the upper lipid phase [12, 19, 24]. This procedure includes bone marrow collection and complete cell digestion by collagenase treatment, and because only adipocytes float in the medium, they can be isolated by centrifugation and aspiration of the floating layer. To purify the isolated adipocytes, cells were filtered through a 200-μm diameter nylon mesh, and washed three times with fresh medium. Adipocytes were counted, and 5×10⁶ cells were then suspended in 2 mL serum-free DMEM in 15-mL Falcon tubes and subjected to suspension culture in the presence of 1 μM dexamethasome (Wako Pure Chemical Industry, Osaka, Japan) or 10 μM simvastatin (Toronto Research Chemicals, Toronto ON, Canada) under 5% CO₂ at 37°C. Samples of primary adipose cells and conditioned medium were taken at various times over the course of 48 h.

PAI-1 and adiponectin mRNA expression was measured by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR). Total RNA was extracted from adipocytes using an RNeasy Mini Kit 250 (Qiagen, Chatsworth, CA) according to the manufacturer's instructions. Total RNA (100 ng) was reverse-transcribed with Taqman One Step PCR Master Mix Reagents kit (Applied Biosystems, Foster City, CA). Sequences of PCR primers were PAI-1 (SERPINE1, Hs00167155_m1), adiponectin (ADIPOQ, Hs00605917_m1), and GAPDH (GAPDH, Hs99999905_m1) (Taqman Gene Expression Assay, Applied Biosystems; product name, product number, respectively). PCR assays were conducted using the ABI PRISM 7000 Sequence Detector System (Applied Biosystems) according to the manufacturer's instructions. Expression of each mRNA was estimated after adjustment for GAPDH mRNA expression.

PAI-1 and adiponectin levels in culture medium were quantified by enzyme-linked immunosorbent assay (ELISA). The protein levels of total PAI-1 and adiponectin were determined using an LPIA-PAI test (Mitsubishi Chemical Yatron, Tokyo, Japan) and a human adiponectin ELISA kit (Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan), respectively.

PAI-1 and adiponectin mRNA expression in bone marrow adipocytes were examined by RT-PCR. PAI-1 mRNA expression was significantly up-regulated by 388% (P = 0.002) with dexamethasone and down-regulated by 45% (P = 0.002) with simvastatin, as compared to control levels (Figure 1A), while adiponectin mRNA expression was 95% with dexamethasone, and 125% with simvastatin (Figure 1B).

A time-course study of PAI-1 mRNA expression with 1 μM dexamethasone or 10 μM simvastatin was performed. Significant increases in PAI-1 mRNA expression were observed using dexamethasone. Peak PAI-1 mRNA expression was 490% (P = 0.001), as compared to control levels, at 12 h (Figure 2A), and was down-regulated by 34% (P = 0.001) at 48 h with simvastatin (Figure 2B).

Total PAI-1 and adiponectin levels in culture medium were measured by ELISA after 36 h of suspension culture. Dexamethasone increased total PAI-1 secretion by 166% (P = 0.001) and simvastatin decreased total PAI-1 secretion by 64% (P = 0.002), as compared to control levels, while pre-treatment with simvastatin reversed dexamethasone-induced total PAI-1 secretion by 89% (P = 0.109) (Figure 3A). Dexamethasone and simvastatin slightly, but not significantly, increased adiponectin secretion. Pre-treatment with 10 μM simvastatin following 1 μM dexamethasone slightly increased adiponectin secretion by 110% (P = 0.249), as compared to control levels (Figure 3B).

A time-course study of total PAI-1 secretion with dexamethasone or simvastatin was conducted and the total PAI-1 in culture medium was measured by ELISA. We divided bone marrow adipocytes into four groups, and total PAI-1 protein secretion was determined by ELISA at 12, 18, 24 and 36 h. The four groups were: untreated controls; treatment with 1 μM dexamethasone at 12 h; treatment with 10 μM simvastatin at 0 h; and treatment with 10 μM simvastatin at 0 h, followed by 1 μM dexamethasone at 12 h. At 24 h and 36 h, dexamethasone increased total PAI-1 secretion by 136% (58.3 ng/mL: P = 0.002) and 167% (101.5 ng/mL: P = 0.001), respectively, as compared to control levels. At 24 h and 36 h, simvastatin decreased total PAI-1 secretion by 56.1% (24 ng/mL: P = 0.002) and 68.3% (41.5 ng/mL: P = 0.002), respectively, as compared to control levels. Simvastatin pretreatment following dexamethasone showed no significant changes at 24 h and 36 h (Figure 4).