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Polyamines Modulate Nitric Oxide Production and Cox-2 Gene Expression in Respons

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发表于 2009-3-5 00:01 |显示全部帖子
作者:Geuranne S. Tjabringaa, Peter S. Vezeridisa, Behrouz Zandieh-Doulabia, Marco N. Heldera,b, Paul I.J.M. Wuismanb, Jenneke Klein-Nulenda作者单位:aDepartment of Oral Cell Biology, Academic Center of Dentistry Amsterdam, Universiteit van Amsterdam, and Vrije Universiteit, andbDepartment of Orthopaedic Surgery, Vrije Universiteit University Medical Center, Amsterdam, The Netherlands + B/ _7 V- O3 c
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- ?  H5 H5 |- a, Z2 M          【摘要】+ Y" e, D1 b7 B7 n
      Jenneke Klein-Nulend, Ph.D., ACTA-Vrije Universiteit, Department of Oral Cell Biology, Van der Boechorststraat 7, NL-1081 BT Amsterdam, The Netherlands. Telephone:  31-20 444 8660; Fax:  31-20 444 8683; e-mail: j.kleinnulend@vumc.nl
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: k' z: J3 T7 ^, ?* GFor bone tissue engineering, it is important that mesenchymal stem cells (MSCs) display a bone cell-like response to mechanical loading. We have shown earlier that this response includes increased nitric oxide (NO) production and cyclooxygenase-2 (COX-2) gene expression, both of which are intimately involved in mechanical adaptation of bone. COX-2 gene expression is likely regulated by polyamines, which are organic cations implicated in cell proliferation and differentiation. This has led to the hypothesis that polyamines may play a role in the response of adipose tissue-derived MSCs (AT-MSCs) to mechanical loading. The aim of this study was to investigate whether genes involved in polyamine metabolism are regulated by mechanical loading and to study whether polyamines modulate mechanical loading-induced NO production and COX-2 gene expression in human AT-MSCs. Human AT-MSCs displayed a bone cell-like response to mechanical loading applied by pulsating fluid flow (PFF), as demonstrated by increased NO production and increased gene expression of COX-2. Furthermore, PFF increased gene expression of spermidine/spermine N (1)-acetyltransferase, which is involved in polyamine catabolism, suggesting that mechanical loading modulates polyamine levels. Finally, the polyamine spermine was shown to inhibit both PFF-induced NO production and COX-2 gene expression, suggesting that polyamines modulate the response of human AT-MSCs to mechanical loading. In conclusion, this is the first study implicating polyamines in the response of human AT-MSCs to mechanical loading, creating opportunities for the use of polyamines in tissue engineering approaches targeting skeletal defects.
9 [, Z. [7 w. _3 G* u          【关键词】 Polyamines Adipose tissue-derived mesenchymal stem cells Mechanical loading Fluid shear stress Fluid flow Nitric oxide Spermidine/spermine N ()-acetyltransferase Cyclooxygenase- Bone cells$ h. y& b5 P1 o4 U5 @% z- o# w* N
                  INTRODUCTION
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6 |' ]8 g1 k0 `. w7 D9 F/ yMesenchymal stem cells (MSCs) constitute a promising tool for tissue engineering approaches. These stem cells are characterized by self-renewal capacity, long life span, and the potential to differentiate into several lineages, including bone, cartilage, muscle, and fat .7 v! s1 z- D. |" L- C% {) ]7 Z
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For bone tissue engineering it is crucial that MSCs differentiate into osteoblast-like cells and display a bone cell-like response to mechanical loading . These mechanosensitive AT-MSCs may constitute an interesting target for bone tissue engineering approaches.
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: S" k: J: Z2 T. a, x; [2 ^0 k$ \Interestingly, polyamines, which are organic cations derived from amino acids present in all mammalian cell types, have also been implicated in the regulation of COX-2 gene expression , polyamines may also play a role in the response of AT-MSCs to mechanical loading.
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9 o6 |, t  Z. [6 {1 n- UTo our knowledge, no studies have reported on the role of polyamines in the response of human AT-MSCs to mechanical loading. We hypothesize that genes involved in polyamine metabolism are mechanoresponsive in human AT-MSCs and that polyamines modulate the response of human AT-MSCs to mechanical loading. To test this hypothesis, human AT-MSCs were subjected to PFF, and the effect of PFF on gene expression of SSAT, which is involved in polyamine catabolism, was determined. Furthermore, we studied whether mechanical loading induced a bone cell-like response in human AT-MSCs, including increased NO production and COX-2 gene expression. Finally, we investigated whether polyamines modulate this mechanical loading-induced response in human AT-MSCs.
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MATERIALS AND METHODS- S5 s( \" b& T' o3 V, _& U7 H

) q) |" O3 W% |% w$ RResection and Tumescent Liposuction( f  b% F; ?1 h' B' E8 z

. \' o6 U6 M6 Y8 h6 pHuman subcutaneous adipose tissue samples were obtained from five female donors (mean age 36 years, range 34¨C38) as waste material after elective surgery and donated upon informed consent of the patients by the Departments of Plastic Surgery from various clinics in The Netherlands. Resection and tumescent liposuction were used to obtain adipose tissue from the abdomen and hip. For resection, the material was cut out en-block under general anesthesia. For tumescent liposuction, a hollow blunt-tipped cannula was introduced into the subcutaneous space through a small incision. To minimize blood loss and tissue contamination by peripheral blood cells prior to aspiration, the vasoconstrictive agents epinephrine (1 mg/ml; Centrafarm B.V., Etten-Leur, The Netherlands, http://www.centrafarm.nl), prilocaïne HCl (1.5%; Pharmacy, Vrije Universiteit  University Medical Center, Amsterdam, The Netherlands, http://www.vumc.nl/english), and kenacort-A10 (10 mg/l; Bristol-Myers Squibb, New York, http://www.bms.com) were added to a 0.65% NaCl saline solution (Baxter, Utrecht, The Netherlands, http://www.baxter.com), which was infused into the adipose compartment. The adipose tissue was mechanically disrupted by moving the cannula through the adipose compartment, and the liposuction material was aspirated by gentle suction.' X8 E" N8 M( g1 f4 t' h

6 J" `" o; s( wAT-MSC Isolation" L+ S$ B. w* j9 ^5 u/ n

/ \' D2 ]' X% ~To isolate the stromal vascular fraction, resected adipose tissue was chopped into small pieces, and both the lipoaspirate and the chopped resected adipose tissue were washed extensively with phosphate-buffered saline (PBS). Adipose tissue was digested with 0.1% collagenase A (Roche Diagnostics GmbH, Mannheim, Germany, http://www.roche.de) in PBS containing 1% bovine serum albumin (BSA) for 45 minutes at 37¡ãC under intermittent shaking. After washing with Dulbecco's modified Eagle's medium (DMEM-glucose; Cambrex Bio Science Verviers S.p.r.l., Verviers, Belgium, http://www.cambrex.com) containing 10% fetal bovine serum (FBS; HyClone, Logan, UT, http://www.hyclone.com) and centrifugation for 10 minutes at 600g, the cell pellet was resuspended in PBS and passed through a 200-µm mesh (Beldico S.A., Marche-en-Famenne, Belgium, http://www.beldico.be) to remove debris. Subsequently, cells were subjected to ficoll density centrifugation (lymphoprep,  = 1.077 g/ml, osmolarity 280 ¡À 15 mOsm; Axis-Shield, Oslo, Norway, http://www.axis-shield.com) to remove contaminating erythrocytes. The cell-containing interface was harvested and washed with DMEM containing 10% FBS. Viability of the cells was assessed by the trypan blue exclusion assay using light microscopy. Three to 10 x 106 cells were resuspended in a mixture of DMEM containing 10% FBS and Cryoprotective medium (1:1; Freezing Medium; Cambrex Bio Science Verviers S.p.r.l.), frozen using a Kryosave (HCI Cryogenics BV, Hedel, The Netherlands, http://www.linde-gascryoservices.com), and stored in liquid nitrogen.& {! A6 ~$ l  d5 e( @+ u" F

  ?: p& y- y$ P& l- K" a( yAT-MSC Culture) e/ X+ \, N" C/ B  }
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Upon thawing, human AT-MSCs were cultured in culture medium consisting of DMEM (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) supplemented with 10% FBS (HyClone), 500 µg/ml streptomycin sulfate (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), 600 µg/ml penicillin (Sigma-Aldrich), 50 µg/ml gentamycin (Invitrogen), and 2.5 µg/ml fungizone (Invitrogen). After reaching confluency, cells were passaged using 0.25% trypsin and 0.1% EDTA in PBS.& Q* T) c0 `$ N

, G! q4 P6 w9 vHuman AT-MSC Characterization
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Human AT-MSCs at passage one were analyzed by fluorescence-activated cell sorting (FACS) for the expression of the MSC surface markers CD105/endoglin and CD166/activated leukocyte cell adhesion molecule (ALCAM). Cells were washed in washing buffer (PBS/0.1% BSA) and blocked for 10 minutes with washing buffer containing 5% goat serum and 0.1% BSA. After washing, cells were incubated for 15 minutes with phycoerythrin-labeled anti-CD166/ALCAM (BD Biosciences, PharMingen, San Diego, http://www.bdbiosciences.com) monoclonal antibodies. To analyze CD105/endoglin expression, cells were incubated with anti-CD105/ALCAM monoclonal antibodies (Abcam plc, Cambridge, UK, http://www.abcam.com), washed in washing buffer, and incubated with a fluorescein isothiocyanate-labeled secondary antibody (Biotrend, Cologne, Germany, http://www.biotrend.com). Control cells were incubated with the same concentration isotype-matched control antibodies of irrelevant specificity (Dako Denmark A/S, Glostrup, Denmark, http://www.dako.dk). After washing in washing buffer, expression of the indicated markers was assessed with a FACScan (BD Biosciences, PharMingen).
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Pulsating Fluid Flow" g$ k8 j2 x) @# B  o+ K
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Human AT-MSCs (up to passage three) were seeded on poly-lysine-coated (50 ug/ml; poly(L-lysine) hydrobromide, molecular weight 15¨C30 x 104; Sigma-Aldrich) glass slides (size 2.5 x 6.5 cm) at 2 x 105 cells per glass slide and cultured overnight in a Petri dish. The next day, cells were subjected to 1 hour of PFF as described previously , in culture medium containing only 2% FBS. Briefly, PFF was generated by pumping 13 ml of medium through a parallel-plate flow chamber containing the AT-MSCs. The cells were subjected to a 5-Hz pulse with a mean shear stress of 0.6 Pa, a pulse amplitude of 0.3 Pa, and a peak shear stress rate of 8.4 Pa/second. Static control cultures were kept in a Petri dish under conditions similar to those of the experimental cultures (i.e., 37¡ãC in a humidified atmosphere of 5% CO2 in air). To study the effect of polyamines on the response of human AT-MSCs to PFF, cells were preincubated for 30 minutes and subjected to 1 hour of PFF or static control treatment in medium containing 2% FBS with or without the polyamine spermine at 1 or 10 µM. Medium samples for measurement of NO concentrations were taken at 0, 10, 30, and 60 minutes of PFF or static control treatment. RNA was isolated from both PFF-treated and static control cells using Trizol reagent (Invitrogen) either directly or after postincubation without PFF for the indicated time periods in fresh medium. Subsequently, gene expression of SSAT, COX-1, COX-2, runx-2, osteopontin, and PMF-1 was determined.5 G& I: p7 q2 k2 e
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Nitric Oxide) ]1 n0 U, q" ~9 z
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NO production was measured as nitrite (NO2¨C) accumulation in conditioned medium using Griess reagent containing 1% sulfanilamide, 0.1% naphtylethelene-diamine-dihydrochloride, and 2.5 M H3PO4. Serial NaNO2 dilutions in nonconditioned medium were used as standard curve. The absorbance was measured at 540 nm with a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, http://www.bio-rad.com).
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9 b: l; q* ~% d2 gReal-Time Polymerase Chain Reaction
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Total RNA was extracted using Trizol reagent (Invitrogen) according to the manufacturer's instructions. To increase RNA yield, 5 µg of glycogen (Roche Diagnostics GmbH) was added to RNA in isopropanol prior to centrifugation. Total RNA (500¨C750 ng) was reverse-transcribed using 250 U/ml transcriptor reverse transcriptase (Roche Diagnostics GmbH), 0.08 A260 units random primers (Roche Diagnostics GmbH), and 1 mM each dNTP (Invitrogen) in transcriptor reverse transcriptase reaction buffer for 30 minutes at 55¡ãC followed by 5 minutes of inactivation of reverse transcriptase at 85¡ãC. The cDNA was diluted (10x), and 2 µl of cDNA was used per reaction for real-time polymerase chain reaction (PCR) using a SYBRGreen reaction kit for 18S (3 mM MgCl2) or Fast start plus SYBRGreen kit (both from Roche Diagnostics GmbH) for SSAT, COX-1, COX-2, runx-2, osteopontin, and PMF-1 in a LightCycler (Roche Diagnostics GmbH). To avoid contamination with genomic DNA, intron-spanning primers (except for 18S) were designed (Table 1) using Clone manager suite software program, version 6 (Scientific & Educational Software, Cary, NC, http://www.scied.com), and used at a concentration of 1 µM.
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, g+ N$ B+ J% B2 E, m/ v8 f9 lTable 1. Primers used for real-time polymerase chain reaction0 F8 W- ^  q! R
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PCR conditions were as follows. For 18S: 1 minute of preincubation at 95¡ãC, followed by 30 cycles of amplification at 95¡ãC for 5 seconds, 56¡ãC for 10 seconds, 72¡ãC for 15 seconds, and 82¡ãC for 5 seconds, followed by melting curve analysis; for SSAT: 10 minutes of preincubation at 95¡ãC, followed by 35 cycles of amplification at 95¡ãC for 5 seconds, 57¡ãC for 5 seconds, 72¡ãC for 15 seconds, and 82¡ãC for 5 seconds followed by melting curve analysis; for COX-1: 10 minutes of preincubation at 95¡ãC, followed by 35 cycles of amplification at 95¡ãC for 10 seconds, 57¡ãC for 8 seconds, 72¡ãC for 10 seconds, and 82¡ãC for 5 seconds, followed by melting curve analysis; for COX-2: 10 minutes of preincubation at 95¡ãC, followed by 40 cycles of amplification at 95¡ãC for 5 seconds, 57¡ãC for 10 seconds, 72¡ãC for 15 seconds, and 82¡ãC for 5 seconds followed by melting curve analysis; for runx-2: 10 minutes of preincubation at 95¡ãC, followed by 35 cycles of amplification at 95¡ãC for 2 seconds, 55¡ãC for 8 seconds, 72¡ãC for 10 seconds, and 82¡ãC for 5 seconds, followed by melting curve analysis; for osteopontin: 10 minutes of preincubation at 95¡ãC, followed by 35 cycles of amplification at 95¡ãC for 2 seconds, 57¡ãC for 8 seconds, 72¡ãC for 10 seconds, and 82¡ãC for 5 seconds, followed by melting curve analysis; for PMF-1: 7 minutes of preincubation at 95¡ãC, followed by 44 cycles of amplification at 95¡ãC for 10 seconds, 58¡ãC for 5 seconds, 72¡ãC for 10 seconds, and 85¡ãC for 5 seconds, followed by melting curve analysis.
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With the LightCycler software, the crossing points were assessed and plotted versus the serial dilution of known concentrations of the standards derived from each gene. PCR efficiency (E) was obtained by using the formula E = 10¨C1/slope. Data were used only if the PCR efficiency calculated was between 1.85 and 2.0.* G8 j, X- e, L
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Data analysis was performed using the LightCycler software. After normalization for 18S housekeeping gene expression, relative target gene expression was determined.( C( F! y* q. {8 _

0 R$ q: n$ w. T5 e% mCell Viability Assay7 H* u; t; C! s% S6 a  ~, t7 H

) a! L9 z9 P1 s: F+ jCells were seeded in 96-well plates and incubated for 90 minutes with various concentrations of spermine in culture medium containing 2% serum. After postincubation for 3 hours, 1 day, or 4 days in culture medium containing 10% serum, medium was replaced with medium containing (tetrazolium salt ; Roche Diagnostics GmbH) reagent (1:10). The assay is based on a reaction between mitochondrial dehydrogenase released from viable cells and tetrazolium salt of WST-1. Absorbance at 450 nm was measured at different time periods, and cell viability was determined as the percentage of absorption as compared with cells not stimulated with spermine.' J" u: h, z7 V0 ?! N
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Statistical Analysis9 j* w! |" Z! u7 G& D; V* M! T1 D

) f" ]" A8 L: O' \Data on NO production were obtained from four separate experiments using four different donors, data on real-time PCR data were obtained from four to eight separate experiments using four different donors, and data on NO production and gene expression after spermine treatment were obtained from three separate experiments using three different donors. Data are expressed as mean ¡À SEM, and statistical analysis was performed using the Student's t test.4 n1 |" @% d2 M( d  ?
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RESULTS6 ^9 Q$ R# O  M7 e$ I% ?- C6 b
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Human AT-MSCs were characterized by FACS analysis using monoclonal antibodies directed against the MSC markers CD105/endoglin and CD166/ALCAM. Approximately 58% of human AT-MSCs at passage 1 were positive for CD105/endoglin (Fig. 1A), and 59% of the human AT-MSCs were positive for CD166/ALCAM (Fig. 1B). The expression of these stem cell markers in human AT-MSCs was similar at passage 3 and at passage 1.
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) b3 `2 @# \5 r* H3 U: [- VFigure 1. Flow cytometric analysis of human adipose tissue-derived mesenchymal stem cells (AT-MSCs). After one passage of culturing, human AT-MSCs were stained with monoclonal antibodies directed against either (A) CD105/endoglin (MSC marker) or (B) CD166/ALCAM (activated leukocyte cell adhesion molecule, MSC marker). An isotype-matched monoclonal antibody served as a control, which is indicated as gray area. The signals are shown as white area.
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7 P% Q6 o7 v; c! K% `% K) cFor bone tissue engineering applications, it is important that human AT-MSCs differentiate along the osteogenic pathway and display a bone cell-like response to mechanical stimuli. This response to mechanical loading includes increases in NO production and in COX-2 gene expression, which mediates prostaglandin production by bone cells and is intimately involved in the response of bone tissue to mechanical loading. The effect of mechanical loading on NO production by human AT-MSCs was studied by subjecting cells to 1 hour of PFF or keeping cells under static control conditions. NO production was significantly increased (by threefold) after 60 minutes of PFF treatment as compared with the static control cultures (Fig. 2).9 {  j+ k3 E' v0 z0 K

/ o4 J" O- m/ U! G% V  GFigure 2. Effect of pulsating fluid flow (PFF) on nitric oxide (NO) production by human adipose tissue-derived mesenchymal stem cells. Cells were subjected to PFF (closed circles) for 1 hour or kept under static control conditions (open circles), and NO production in medium samples taken at various time points was determined. Data are expressed as mean ¡À SEM. Statistical analysis was performed using the Student's t test. Asterisk indicates significant effect of PFF, p
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To study the effect of mechanical loading on COX-2 gene expression in human AT-MSCs, cells were subjected to 1 hour of PFF or kept under static control conditions. After postincubation for various time periods, gene expression of COX-2 was determined. PFF increased COX-2 gene expression by sixfold after 3 hours of postincubation and by fivefold after 6 hours of postincubation as compared with the static control cultures (Fig. 3A). In contrast to COX-2 gene expression, that of COX-1 was not affected by PFF (Fig. 3B). Prostaglandin production by human AT-MSCs was not affected during the 1 hour of PFF treatment as compared with static control treatment (data not shown). This may be explained by the time point of measurement of prostaglandin production which precedes upregulation of COX-2.5 O# V# W, I8 ^8 c9 Z$ d. U
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Figure 3. Response of human adipose tissue-derived mesenchymal stem cells to PFF. Cells were subjected to PFF for 1 hour or kept under static control conditions, and RNA was isolated after various postincubation periods. Gene expression of COX-2 (A), COX-1 (B), and SSAT (C) was determined by real-time polymerase chain reaction, and data were normalized to 18S gene expression. Data are presented as the ratio of PFF-treated control over static control and as mean ¡À SEM. Statistical analysis was performed using the Student's t test. Asterisk indicates significant effect of PFF, p
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% f9 a! l# c8 W# w1 e6 CIn addition to increasing COX-2 gene expression, 1 hour of PFF increased gene expression of runx-2, which is a transcription factor involved in early stages of osteogenic differentiation, 3 hours after PFF treatment by 1.3-fold (data not shown). In contrast to runx-2 gene expression, that of osteopontin, a bone matrix molecule involved in later stages of osteogenic differentiation, was not significantly affected by PFF within the time frame of the experiment (data not shown).1 a3 L; K, @" U9 r6 i: i5 y
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To study whether mechanical loading regulates expression of polyamine-related genes, the effect of PFF on gene expression of SSAT, which regulates polyamine catabolism, was determined (Fig. 3C). Cells were subjected to PFF for 1 hour or kept under static control conditions, and gene expression of SSAT was determined by real-time PCR at various postincubation periods after PFF treatment. One hour of PFF treatment increased SSAT gene expression at 3 hours, but not immediately after PFF treatment, nor after 6 hours of postincubation. In addition, the effect of PFF on gene expression of PMF-1, which regulates SSAT gene expression, was determined. One hour of PFF did not affect PMF-1 gene expression at 3 hours after PFF treatment (data not shown). To test polyamine toxicity, the effect of polyamines on cell viability of human AT-MSCs was studied (Fig. 4A). Cells were treated for 90 minutes with different concentrations of spermine, and cell viability was determined after various postincubation periods. Spermine (0.1¨C1,000 µM) did not affect cell viability 3 hours after treatment. After 1 and 4 days of postincubation, only high concentrations of spermine (1,000 µM) affected cell viability. Therefore, in the following experiments, spermine was used at nontoxic concentrations of 1 and 10 µM.
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: Z( v! A: r) c2 k$ L/ QFigure 4. Effect of the polyamine spermine on the response of human AT-MSCs to PFF. To determine the effect of spermine on cell viability, cells were seeded in 96-well plates and incubated for 90 minutes with various concentrations of spermine. After postincubation for 3 hours (closed squares), 1 day (open circles), or 4 days (closed circles), medium was replaced with medium containing WST reagent (1:10). (A): Cell viability was determined by measuring absorbance at 450 nm, and data are presented as the percentage of absorption as compared with cells not stimulated with spermine. To determine the effect of spermine on PFF-induced NO production and COX-2 gene expression, human AT-MSCs were preincubated for 30 minutes and then subjected to PFF for 1 hour or kept under static control conditions (stat) in medium with or without the polyamine spermine. (B): NO production in medium samples taken after 60 minutes PFF was determined. (C): COX-2 gene expression was determined 3 hours after PFF treatment. Data on COX-2 gene expression are presented as ratio of PFF-treated over time-matched static control, whereby the static control was set at 1. Data are presented as mean ¡À SEM, and statistical analysis was performed using the Student's t test. Asterisk indicates significant effect of spermine on PFF-induced NO production and COX-2 gene expression, p
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0 o' V/ j  u/ N$ q' r- M# bTo study whether polyamines modulate PFF-induced NO production and COX-2 gene expression, human AT-MSCs were preincubated in medium with or without the polyamine spermine (1 or 10 µM) for 30 minutes and subsequently subjected to PFF for 1 hour in medium with or without spermine (1 or 10 µM). Spermine was shown to decrease PFF-induced NO production (Fig. 4B). Furthermore, spermine decreased PFF-induced COX-2 (Fig. 4C) gene expression as measured at 3 hours after PFF treatment. Spermine did not affect COX-2 gene expression in static control cells (data not shown).
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To study the effect of polyamines on gene expression of SSAT and PMF-1, human AT-MSCs were preincubated in medium with or without the polyamine spermine (1 or 10 µM) for 30 minutes and subsequently subjected to PFF or kept under static control conditions for 1 hour in medium with or without spermine (1 or 10 µM). In static control cultures, spermine at both 1 and 10 µM increased SSAT gene expression (Fig. 5A), whereas PMF-1 gene expression was increased by 1 µM spermine (Fig. 5B). In flow-treated cells, spermine did not affect gene expression of SSAT and PMF-1 (data not shown).0 T7 c) N- H) b9 i( O
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Figure 5. Effect of the polyamine spermine on gene expression of SSAT (A) and PMF-1 (B) in static control cells. Human adipose tissue-derived mesenchymal stem cells were incubated for 90 minutes in medium with the polyamine spermine. After 3 hours of incubation in fresh medium without spermine, gene expression of SSAT and PMF-1 was determined. Data are presented as ratio of spermine-treated over nontreated control cells, whereby the nontreated control was set at 1. Data are presented as mean ¡À SEM, and statistical analysis was performed using the Student's t test. Asterisk indicates significant effect of spermine on SSAT and PMF-1 gene expression in static control cells, p 0 s0 K9 J7 v4 v3 U3 T

5 z  a0 H, q( c3 A: i' O. wDISCUSSION
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In the present study, we demonstrate that gene expression of SSAT, which regulates polyamine catabolism, is responsive to PFF, suggesting that mechanical loading regulates polyamine levels in human AT-MSCs. Furthermore, polyamines were found to decrease PFF-induced NO production and COX-2 gene expression in human AT-MSCs, suggesting that polyamines modulate the response of human AT-MSCs to mechanical loading (Fig. 6). Our findings, for the first time, implicate polyamines in the response of AT-MSCs to mechanical loading, which may open opportunities for the use of polyamines in bone tissue engineering approaches using AT-MSCs.1 `  A( w* M$ Z2 k) Z- y4 M

1 \; G& _0 W4 Q6 u/ [3 FFigure 6. Proposed model of the response of human AT-MSCs to mechanical loading. In this model, mechanical loading increases NO production and gene expression of COX-2, both of which are implicated in the mechanical adaptation of bone. In addition, gene expression of runx-2, which is a transcription factor expressed during early stages of osteogenic differentiation, is increased. Furthermore, mechanical loading increases gene expression of the polyamine-related gene SSAT, resulting in regulation of polyamine levels. Regulation of polyamine levels subsequently results in modulation of the response of human AT-MSCs to mechanical loading. Finally, spermine also modulates SSAT gene expression, resulting in regulation of polyamine levels. Abbreviations: AT-MSC, adipose tissue-derived mesenchymal stem cell; COX, cyclooxygenase; NO, nitric oxide; SSAT, spermidine/spermine N (1)-acetyltransferase.0 K. ~) k" \  @; b+ U
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We found that PFF increases gene expression of SSAT, which regulates polyamine catabolism. Furthermore, gene expression of PMF-1, which regulates SSAT gene expression, was not affected 3 hours after PFF treatment. This suggests that PMF-1 is not involved in the response of human AT-MSCs to mechanical loading or that modulation of PMF-1 gene expression by PFF occurs at a time point different from that of SSAT. Intracellular levels of polyamines are regulated by a complex network of factors that affect polyamine biosynthesis and catabolism via different enzymatic steps . Therefore, the finding that PFF increases SSAT gene expression in human AT-MSCs suggests that PFF decreases polyamine levels in these cells. However, PFF may also initially increase polyamine levels. To restore polyamine homeostasis in the cells, a feedback mechanism may be initiated resulting in increased SSAT gene expression. Interestingly, spermine was shown to increase gene expression of both SSAT and PMF-1 in static control cultures, suggesting that polyamine homeostasis is regulated via a feedback mechanism.- `& t- y) V" ?6 Q' o
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Polyamines are involved in a variety of cellular processes, including proliferation and differentiation, and several studies have implicated a role for polyamines in bone growth and development . In the present study, we demonstrate that polyamines also modulate PFF-induced NO production and COX-2 gene expression. This suggests that in addition to regulating differentiation of AT-MSCs, polyamines may modulate the response of human AT-MSCs to mechanical loading.
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For bone tissue engineering, it is important that MSCs differentiate into osteoblast-like cells and display a bone cell-like response to mechanical loading. Several studies have demonstrated that MSCs are mechanosensitive . In the present study, we demonstrate that human AT-MSCs are also responsive to mechanical loading by PFF, even without stimulation of osteogenic differentiation, as demonstrated by increased NO production and COX-2 gene expression. However, NO production was increased relatively late in human AT-MSCs (after 60 minutes of PFF treatment) as compared with goat-derived AT-MSCs that had been stimulated in their osteogenic differentiation (after 5 minutes of PFF treatment) and mature bone cells. Therefore, we can only speculate that the unstimulated AT-MSCs display a bone cell-like response to mechanical loading, which is delayed as compared with osteogenically stimulated AT-MSCs and mature bone cells. In addition, future studies are needed to determine whether human AT-MSCs that are stimulated in their osteogenic differentiation show an earlier NO response to PFF as compared with unstimulated human AT-MSCs.
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In addition to regulation of NO production and COX-2 gene expression, we demonstrate that gene expression of runx-2, which is a bone-related transcription factor expressed during early stages of osteogenic differentiation, is increased by PFF. Different studies have demonstrated that mechanical loading affects osteogenic differentiation of MSCs .
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$ f; d" Y, O4 L% ^- \% m3 @* pCONCLUSION
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4 b! c5 Z" r% M+ sThis is the first study demonstrating that PFF regulates expression of the polyamine-related gene SSAT in human AT-MSCs. Furthermore, polyamines modulate PFF-induced NO production and COX-2 gene expression. This study suggests that mechanical loading may regulate polyamine levels in human AT-MSCs and that polyamines modulate the response of human AT-MSCs to mechanical loading. For bone tissue engineering, it is important that MSCs differentiate into bone-like cells and display a bone cell-like response to mechanical loading. Our finding that polyamines modulate the response of human AT-MSCs to mechanical loading creates opportunities for the use of polyamines in tissue engineering approaches targeting skeletal defects.0 Z& A8 M- O/ l& O

  {3 l; p/ W8 }$ u9 ^4 ?  e% m6 g/ RDISCLOSURES
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The authors indicate no potential conflicts of interest.$ {" N* |8 V2 A5 e

( }% d6 p. g  X" ~ACKNOWLEDGMENTS0 S) U* O4 g+ F5 o  `- ^! S0 G; w
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This study was supported by the VU interfaculty research initiative MOVE. The work of Peter Vezeridis was supported by a grant from the U.S. Fulbright Fellowship Program. The Technology Foundation STW supported the work of Behrouz Zandieh-Doulabi (STW grant no. VPG.5935). We thank Gerrit Jan Schuurhuis and Guus Westra (Department of Hematology, VU University Medical Center) for help in FACS analysis, Florine van Milligen (Department of Pathology, VU University Medical Center) for kindly donating human AT-MSCs, and Ruud Bank for help in polyamine analysis.
2 O4 _- A  Y: W% P+ L0 E) ]          【参考文献】
- @: G' s4 \5 V& a
5 d2 u+ R' F! E: `# ]! }) g( W  D/ ~2 A
Pittenger MF, Mackay AM, Beck SC et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999;284:143¨C147.. x- A. J: o. g' I/ G: @

8 F8 N' p+ v& }. f' x$ ZZuk PA, Zhu M, Ashjian P et al. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell 2002;13:4279¨C4295.) a9 j' s: B0 @
6 w" Q2 \$ i$ E% Z: Q8 H* v7 e% [
Zuk PA, Zhu M, Mizuno H et al. Multilineage cells from human adipose tissue: Implications for cell-based therapies. Tissue Eng 2001;7:211¨C228.- h3 Q: j( u/ \% a

: U% u3 C, r. J& \1 a: ~/ qOedayrajsingh Varma MJ, Helder MN, Klein-Nulend J et al. Adipose tissue-derived mesenchymal stem cell yield and growth characteristics are affected by the tissue-harvesting procedure. Cytotherapy 2006;8:166¨C177.
5 s! t) v4 C6 O) P, U( @% _5 N& d
Knippenberg M, Helder MN, Zandieh-Doulabi B et al. Adipose tissue-derived mesenchymal stem cells acquire bone cell-like responsiveness to fluid shear stress upon osteogenic stimulation. Tissue Eng 2005;11:1780¨C1788.8 Y" \& V% Q) c3 h; r3 ]

/ B, X+ E! {* w! j0 OTurner CH, Forwood MR, Otter MW. Mechanotransduction in bone: Do bone cells act as sensors of fluid flow? FASEB J 1994;8:875¨C878.! n8 m- m! ^( n7 K4 q. H
# S+ K, z) R( [2 F4 y
Piekarski K, Munro M. Transport mechanism operating between blood supply and osteocytes in long bones. Nature 1977;269:80¨C82./ x- X% O) }) ?" l6 b, o: B, B

3 s0 X7 n# z$ WKnothe Tate ML, Knothe U. An ex vivo model to study transport processes and fluid flow in loaded bone. J Biomech 2000;33:247¨C254.# U  _# W7 z* S8 V
0 l' z" o; B  V
Cowin SC, Weinbaum S. Strain amplification in the bone mechanosensory system. Am J Med Sci 1998;316:184¨C188.! o8 b' `; P- e3 m1 Q: Z
' M& H( Z, z& v7 W- l# }
Klein-Nulend J, van der Plas A, Semeins CM et al. Sensitivity of osteocytes to biomechanical stress in vitro. FASEB J 1995;9:441¨C445.
: ?+ x* F( |" J, {0 Z1 {( r0 j; l5 k- [2 c8 `
Burger EH, Klein-Nulend J. Mechanotransduction in bone¨Crole of the lacuno-canalicular network. FASEB J 1999;13 (suppl):S101¨CS112.+ X+ R7 R# g$ d$ W6 t" b1 b* y) h

0 d1 b7 m) r7 ?; k+ c9 G1 S1 Q& N. w. hAjubi NE, Klein-Nulend J, Nijweide PJ et al. Pulsating fluid flow increases prostaglandin production by cultured chicken osteocytes¨Ca cytoskeleton-dependent process. Biochem Biophys Res Commun 1996;225:62¨C68.9 \7 `) `& Y& n% L: k. I; t
& E2 C* Q2 q9 l+ Z. h; p% ?; G
Klein-Nulend J, Semeins CM, Ajubi NE et al. Pulsating fluid flow increases nitric oxide (NO) synthesis by osteocytes but not periosteal fibroblasts¨Ccorrelation with prostaglandin upregulation. Biochem Biophys Res Commun 1995;217:640¨C648.$ c, [7 J3 C! [* p1 d9 N5 u

) g% d. e2 r, M) ]Forwood MR. Inducible cyclo-oxygenase (COX-2) mediates the induction of bone formation by mechanical loading in vivo. J Bone Miner Res 1996;11:1688¨C1693.
. x; G/ X5 M2 M% V" F
" l; W5 w! \- _4 o) P- i+ {, L; O5 |" FChow JW, Fox SW, Lean JM et al. Role of nitric oxide and prostaglandins in mechanically induced bone formation. J Bone Miner Res 1998;13:1039¨C1044./ K9 w/ j# g* n' ~. R  G4 \

$ V* M7 k/ P# j( a) c7 HSmith WL, DeWitt DL, Garavito RM. Cyclooxygenases: Structural, cellular, and molecular biology. Annu Rev Biochem 2000;69:145¨C182.
) o$ f/ p% y1 L! Q7 t1 J9 g# c
/ z0 @; |) c# B/ L0 E# L8 EBakker AD, Klein-Nulend J, Burger EH. Mechanotransduction in bone cells proceeds via activation of COX-2, but not COX-1. Biochem Biophys Res Commun 2003;305:677¨C683.
! W  b9 g4 }* |$ ]8 f8 D+ Q. g
# l% B$ F. C/ e" b5 b* Y  dSmith WL, Garavito RM, DeWitt DL. Prostaglandin endoperoxide H synthases (cyclooxygenases)-1 and -2. J Biol Chem 1996;271:33157¨C33160.$ d$ ^- O3 j5 h$ k) m

( f- m( M2 C. e5 Y  lJoldersma M, Burger EH, Semeins CM et al. Mechanical stress induces COX-2 mRNA expression in bone cells from elderly women. J Biomech 2000;33:53¨C61.
+ D! H( r" g+ C0 n/ t5 M' p- h. T9 _4 B
Westbroek I, Ajubi NE, Alblas MJ et al. Differential stimulation of prostaglandin G/H synthase-2 in osteocytes and other osteogenic cells by pulsating fluid flow. Biochem Biophys Res Commun 2000;268:414¨C419.
/ S+ w  c2 n8 {" ~3 O" I3 L1 T& z0 U& i) m( |
Parker MT, Gerner EW. Polyamine-mediated post-transcriptional regulation of COX-2. Biochimie 2002;84:815¨C819.
( _4 W) G7 E4 p& D2 [
& U; k6 P6 e+ O* t1 }/ R$ GTurchanowa L, Dauletbaev N, Milovic V et al. Nonsteroidal anti-inflammatory drugs stimulate spermidine/spermine acetyltransferase and deplete polyamine content in colon cancer cells. Eur J Clin Invest 2001;31:887¨C893." k8 l7 W+ A8 R3 ]0 J2 w
4 c5 }7 D. R5 U- B( c9 M( l
Wallace HM, Fraser AV, Hughes A. A perspective of polyamine metabolism. Biochem J 2003;376:1¨C14.
8 b4 |8 s9 ?6 x) |1 S
$ N. J5 S; d" P9 _- x8 oPendeville H, Carpino N, Marine JC et al. The ornithine decarboxylase gene is essential for cell survival during early murine development. Mol Cell Biol 2001;21:6549¨C6558.
8 A& S  d0 _/ F9 h5 y9 l7 g
; r, c+ u% t' A8 _: SLux GD, Marton LJ, Baylin SB. Ornithine decarboxylase is important in intestinal mucosal maturation and recovery from injury in rats. Science 1980;210:195¨C198.
( x9 q9 l! w! q+ H: `/ m$ O  y% I+ W0 Q" i0 _; h5 B/ A$ `
Heby O. Role of polyamines in the control of cell proliferation and differentiation. Differentiation 1981;19:1¨C20.5 y" ~6 n& H; L) G1 u8 S6 Z" i

( O+ x$ q  s7 D8 R6 @) hGerner EW, Meyskens FL Jr. Polyamines and cancer: Old molecules, new understanding. Nat Rev Cancer 2004;4:781¨C792.6 V! M4 X$ ]) W" }' m+ F" [5 P! j
# [- X' o+ \3 C  g* U
Vittur F, Lunazzi G, Moro L et al. A possible role for polyamines in cartilage in the mechanism of calcification. Biochim Biophys Acta 1986;881:38¨C45.8 f- M2 ~" v* b  \. Z5 g6 [$ [

( l9 l5 Z" S" d8 J5 ~$ CTakano T, Takigawa M, Suzuki F. Role of polyamines in expression of the differentiated phenotype of chondrocytes: Effect of DL-alpha-hydrazino-delta-aminovaleric acid (DL-HAVA), an inhibitor of ornithine decarboxylase, on chondrocytes treated with parathyroid hormone. J Biochem (Tokyo) 1983;93:591¨C598.
) I, Y5 L5 A& L! ]+ S4 m& j7 e$ |
Takano T, Takigawa M, Suzuki F. Role of polyamines in expression of the differentiated phenotype of chondrocytes in culture. Med Biol 1981;59:423¨C427.
. f# [1 U$ j# z+ R7 G
# I# Q, C5 j' J, @+ u' y/ q# X- TZandieh-Doulabi B, Knippenberg M, Klein-Nulend J et al. 1,25-dihydroxyvitamin D3 modulates the expression of genes involved in polyamine catabolism in adipose tissue-derived mesenchymal stem cells. Transactions ORS 2005;30:959.. n; j: P( s" r3 U9 F# w

+ n+ k/ s0 O" ^( }% H  Q" i. t3 wWang Y, Devereux W, Stewart TM et al. Characterization of the interaction between the transcription factors human polyamine modulated factor (PMF-1) and NF-E2-related factor 2 (Nrf-2) in the transcriptional regulation of the spermidine/spermine N1-acetyltransferase (SSAT) gene. Biochem J 2001;355:45¨C49.
: H( I( R- h1 u3 L" j: m
- Y- b8 z0 Z4 D, I8 DWang Y, Devereux W, Stewart TM et al. Cloning and characterization of human polyamine-modulated factor-1, a transcriptional cofactor that regulates the transcription of the spermidine/spermine N(1)-acetyltransferase gene. J Biol Chem 1999;274:22095¨C22101.
1 {+ u6 Q8 J$ J9 X2 D/ P! {/ X" p) S. m5 F
Matsui-Yuasa I, Otani S, Morisawa S et al. Induction of spermidine/spermine N1-acetyltransferase by parathyroid hormone in rabbit costal chondrocytes in culture. J Biochem (Tokyo) 1985;97:387¨C390.  j" L) |0 V9 @* f: R( j
5 R8 E. i: |5 N
Thomas GP, El Haj AJ. Bone marrow stromal cells are load responsive in vitro. Calcif Tissue Int 1996;58:101¨C108.# h1 j5 |2 Z9 e* ~" I! i/ F
, g  f" m# e5 k
Yoshikawa T, Peel SA, Gladstone JR et al. Biochemical analysis of the response in rat bone marrow cell cultures to mechanical stimulation. Biomed Mater Eng 1997;7:369¨C377.1 E2 E) e# i3 N) X5 P% u- y+ Q

7 {* ^0 z8 @/ qZhang R, Supowit SC, Klein GL et al. Rat tail suspension reduces messenger RNA level for growth factors and osteopontin and decreases the osteoblastic differentiation of bone marrow stromal cells. J Bone Miner Res 1995;10:415¨C423." b2 h, F# x5 J1 E) ?& Z/ x

* ~, {% E! c4 ^" Z. [$ \7 d  N' }Simmons CA, Matlis S, Thornton AJ et al. Cyclic strain enhances matrix mineralization by adult human mesenchymal stem cells via the extracellular signal-regulated kinase (ERK1/2) signaling pathway. J Biomech 2003;36:1087¨C1096.
$ L# s- y/ ^6 Z4 c+ s
! }' b6 ?; q5 r% c/ D! sKreke MR, Huckle WR, Goldstein AS. Fluid flow stimulates expression of osteopontin and bone sialoprotein by bone marrow stromal cells in a temporally dependent manner. Bone 2005;36:1047¨C1055.
/ p+ R+ {  ^+ o5 X4 E4 L8 N" A/ ?9 T' P- }4 z( `2 i$ b
Jessop HL, Rawlinson SC, Pitsillides AA et al. Mechanical strain and fluid movement both activate extracellular regulated kinase (ERK) in osteoblast-like cells but via different signaling pathways. Bone 2002;31:186¨C194.

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